Molecular Approaches to Drug

Abuse Research

Volume III: Recent Advances and 

Emerging Strategies

Editor:

Theresa N.H. Lee, Ph.D.

NIDA Research Monograph 161

1996

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

National Institutes of Health

National Institute on Drug Abuse

Division of Basic Research

5600 Fishers Lane

Rockville, MD 20857
ACKNOWLEDGMENT

This monograph is based on the papers from a technical

review on "Molecular Approaches to Drug Abuse Research"

held on March 22-23, 1994. The review meeting was

sponsored by the National Institute on Drug Abuse.

COPYRIGHT STATUS

The National Institute on Drug Abuse has obtained

permission from the copyright holders to reproduce certain

previously published material as noted in the text. Further

reproduction of this copyrighted material is permitted only

as part of a reprinting of the entire publication or chapter. 

For any other use, the copyright holder's permission is

required. All other material in this volume except quoted

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and may be used or reproduced without permission from the

Institute or the authors. Citation of the source is

appreciated.

Opinions expressed in this volume are those of the authors

and do not necessarily reflect the opinions or official policy

of the National Institute on Drug Abuse or any other part of

the U.S. Department of Health and Human Services.

The U.S. Government does not endorse or favor any specific

commercial product or company. Trade, proprietary, or

company names appearing in this publication are used only

because they are considered essential in the context of the

studies reported herein.

National Institute on Drug Abuse

NIH Publication No. 96-4022

Printed 1996

NIDA Research Monographs are indexed in the Index Medicus. 

They are selectively included in the coverage of American

Statistics Index, BioSciences Information Service, Chemical

Abstracts, Current Contents, Psychological Abstracts, and

Psychopharmacology Abstracts.

ii
Table of Contents

Click on page or subject to go to page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Theresa N.H. Lee

Immunological Approaches to Nicotine Receptors . . . . . . . . . . . 3

James W. Patrick, Shawn Neff, Kelly Dineley, 

and David Char

The Dopamine D4 Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Hubert H.M. Van Tol

Behavioral Characterization of Mice Packing the 

5-HT1B Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Sylvie Ramboz, Frédéric Saudou, Djamel Aït Amara,

Catherine Belzung, Andrée Dierich, Marianne LeMeur, 

Louis Segu, René Misslin, Marie-Christine Buhot, 

and René Hen

Mechanisms Regulating Proenkephalin Gene Expression:

Contributions of Transgenic Models . . . . . . . . . . . . . . . . . . . . . . 59

Steven E. Hyman and David Borsook

Human Opioid Receptors: Chromosomal Mapping and

mRNA Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Karen Miotto, Daniel Kaufman, Benito Anton, 

Duane E. Keith, Jr., and Chris J. Evans

Molecular Biology of Opioid Receptors . . . . . . . . . . . . . . . . . . . 83

Karen Raynor, Haeyoung Kong, Susan Law, Jennifer

Heerding, Melanie Tallent, Fredrick Livingston, John Hines,

and Terry Reisine

Mu Opioid Receptors: Cellular Action and Tolerance

Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Anton Mestek, Yan Chen, and Lei Yu

Cloning and Characterization of Multiple Opioid Receptors . . . . . . . . 127

Huda Akil, Fan Meng, Alfred Mansour, Robert Thompson,

Guo-Xi Xie, and Stanley Watson

iii
Regulation of Acute and Chronic Opioid Receptor Functions

by OBCAM, a Cell Adhesion-Like Molecule . . . . . . . . . . . . . . . 141

Horace H. Loh and Andrew P. Smith

Discrete Structural Domains and Cell-Specific Expression

Determine Functional Selectivity of the Dopamine and

Norepinephrine Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Kari J. Buck, Dominique Lorang, and Susan G. Amara

Drug Interactions with Vesicular Amine Transport . . . . . . . . . . 176

Doris Peter, Juan Jimenez, Yongjian Liu, Andrew Merickel,

David Krantz, and Robert H. Edwards

Strategies for Identifying Genes Underlying

Drug Abuse Susceptibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

John C. Crabbe, Kari J. Buck, Pamela Metten, and 

John K. Belknap

iv
1

Introduction: Molecular

Approaches to Drug Abuse

Research

Theresa N.H. Lee

The Extramural Molecular Biology and Genetics Program was

launched in 1988 in the Division of Basic Research of the National

Institute on Drug Abuse (NIDA) to encourage and support

investigator-initiated basic research to employ molecular approaches

to drug abuse research on all abused substances. In the past 6 years,

the program has reached many milestones and achieved numerous

successes. The progress made prior to 1991 was best documented in

NIDA Research Monograph Series volumes 111 and 126 based on the

proceedings of the first and second conferences on "Molecular

Approaches to Drug Abuse Research" held in 1989 and 1991,

respectively. The third conference on "Molecular Approaches to

Drug Abuse Research" was held on March 22 and 23, 1994, at the

Bethesda, MD, campus of the National Institutes of Health. This

conference intended to capture the recent major advances in this field

and to look at the future directions of the program.

The Molecular Biology and Genetics Program has indeed come a

long way since 1988. NIDA not only has persuaded an

unprecedented number of outstanding scientists to join the extramural

scientific community engaging in drug abuse research, but recently

celebrated the molecular cloning of the opioid receptors that had

eluded numerous dedicated drug abuse researchers for more than 15

years. With the completion of the tedious task of gene cloning of all

the pertinent receptors, transporters, channels, and regulatory proteins,

a new era has begun. Using these powerful tools, researchers can now

employ approaches previously unimaginable and concentrate their

efforts and resources on elucidating the basic mechanism of each

abused substance as well as the molecular and cellular mechanisms

underlying tolerance, dependence, and withdrawal to generate better

strategies for effective treatment, education, and prevention.

This conference was organized into three technical sessions:

transgenic/ knockout animal models and other genetic approaches,

studies on three families of transporters, and a whole session devoted

to the rapidly progressing field of opioid receptors. Thirteen

accomplished scientists presented their cutting-edge research, which
2

included many of the major breakthroughs in the field. Due to the

ceiling on the number of speakers to invite for the conference, only

one example from each category or superfamily could be included

rather than encompassing all the recent accomplishments. The

proceedings of this conference are presented in the following chapters

of this monograph. By the time this monograph is published,

virtually all the speakers at this conference will be members of the

NIDA extramural scientific community.

Under the able leadership of Dr. James Patrick, significant consensus

among the speakers has also been reached during the discussion

session of this conference. Some of the highlights are: (1) refining

tools for producing transgenic/knockout animals, especially

conditional or tissue-specific knockouts; (2) developing effective

expression systems for drug receptors; (3) expanding research on the

polymorphism and diversity of drug receptors, transporters, and other

relevant proteins; (4) developing clinically useful, safe opioid drugs

employing the knowledge obtained from recent opioid receptor

advances; and (5) using proper animal models to systematically screen

nongenetic factors for clues leading to treatment of addiction.

AUTHOR

Theresa N.H. Lee, Ph.D.

Program Director

Molecular Biology and Genetics Program

Division of Basic Research

National Institute on Drug Abuse

Parklawn Building, Room 10A-19

5600 Fishers Lane

Rockville, MD 20857
3

Immunological Approaches to

Nicotine Receptors

James W. Patrick, Shawn Neff, Kelly Dineley, and David

Char

Nicotine is a drug whose abuse results in approximately 400,000

deaths per year in the United States alone (Surgeon General 1988). It

is by many accounts the most addictive drug available without

prescription; nearly 40 percent of those currently addicted have tried

to stop using the drug (Schellingt 1992). Although many are

successful in overcoming their dependence, 80 percent of those who

stop using return to the drug within 24 months (Schellingt 1992).

New drug replacement protocols are increasing the success rate, but

pharmacokinetic problems of dosage, route, and side effects remain.

Clearly, new drugs and new protocols that would help overcome

addiction to nicotine would be valuable and might reduce the loss of

life that results from its use.

Drugs of abuse such as nicotine exert both short-term and long-term

effects through their interactions with specific molecular targets such

as receptors, ion channels, and transporters. A powerful approach to

the study of these drugs has been the identification and study of their

specific binding sites and mechanisms of action. This approach has

the straight-forward and valid rationale that if one understands the

mechanism of action of a drug one can eventually understand and

reduce its potential for abuse. In addition, if one understands the

binding site one can perhaps design drugs with great receptor

specificity and lower abuse potential, which could be used to break

dependence.

Early biochemical data supported this idea that the sites of action of

drugs were homogeneous. However, the idea of a single class of sites

for each drug essentially precluded the idea of designing ligands that

might distin-guish between binding sites associated with abuse and

binding sites for the same drug that might not be associated with

abuse. Although the apparent homogeneity of binding sites

seemingly facilitated biochemical and binding studies, it moved the

authors’ explanations of drug action from the drug and its

interactions with its receptor to some more complex, interactive, but

ill-defined aspect of the nervous system. The potential for critical

analysis of drug function was essentially limited to those few cases in

which the drug-binding sites were restricted to one or a few specific
4

brain nuclei or cell types. Only under these conditions could one

associate the actions of a drug with the function of specific central

nervous system (CNS) nuclei.

It is now known that the diversity of drug/receptor interactions far

exceeds scientists’ earlier estimates. The diversity is found at two

levels. First, binding sites previously thought to be homogeneous are

now known to be heterogenous (for reviews see Hollenberg 1991 and

Scholfield et al. 1990). For example, nicotine was once thought to

bind to a homogeneous class of binding sites in brain; it is now known

that there are many different oligomeric receptors that bind and are

activated by nicotine (for a review see Luetje et al. 1990b). Drugs that

bind to gamma aminobutyric acid (GABA) receptors in brain extracts

are now known to distinguish between dozens of functionally

different GABA receptors (Stephenson and Duggan 1991). Second,

drugs bind to specific identified receptors that were not previously

thought to bear the appropriate binding sites. Curare, a drug that was

once thought to bind exclusively to, and be diagnostic for, nicotinic

acetylcholine receptors, is now known to have its highest affinities for

serotonin (5-HT3) receptors (Andres et al. 1991). Phencyclidines are

no longer thought to be specific for any receptor but appear to block

a wide variety of ligand-gated ion channels (Amador and Dani 1991).

Even glycine is no longer considered specific for glycine receptors,

but is known to be required for activation of one class of the

glutamate receptors (Johnson and Ascher 1987). Both the diversity of

receptor types and the cross-modality of drug/receptor interactions is

likely to become more significant as more is learned.

The consequences of drug use are also more diverse than anticipated.

It was once thought that drug use produced changes in the number of

specific binding sites, changes in the efficacy of the drug inactivation

mechanisms, or perhaps a change in the abundance of an endogenous

ligand. The diversity of drug receptors now forces a consideration of

changes in the actual structure of the receptor molecules or changes in

the distribution of these molecules on the surface of a neuron. One

must now consider the possibility that drugs of abuse have long-term

effects that result from the expression of specific genes that are

activated as a consequence of the action of the drug on one of its

many different receptors. Dopamine modulation of progesteroneregulated gene expression is an important example because it may tie

either abuse potential or drug need to variations in normal

physiological parameters such as endocrine function (Power et al.

1991a, 1991b). Finally, the potential for long-term activitydependent modification of synaptic function and the roles these
5

mechanisms play both in the adult and during development is

appreciated more fully. This offers potential for under-standing and

perhaps ameliorating some adverse effects of drugs on the fetus

during development.

There have been many elegant studies of nicotine binding sites in the

CNS, both in extracts and in brain slices (Baneerjee et al. 1990; Clarke

et al. 1985; Lippiello and Fernandes 1986; Marks et al. 1986;

Martino-Barrows and Kellar 1987; Pabreza et al. 1991; Raja et al.

1988; Wonnacott 1987). These studies show that both high and low

affinity binding sites are abundant, and that they are widely

distributed in the brain. A key contribution from these studies is the

observation that the number of binding sites changes with chronic

exposure to nicotine (Marks and Collins 1983; Norbert et al. 1983;

Schwartz and Kellar 1983). In addition, the toxin alphabungarotoxin, which played a major role in the impressive progress

made on understanding the nicotinic acetyl-choline receptor present

at the neuromuscular junction (Fambrough 1976), also binds to brain

membrane preparations and to cells known to contain neuronal

nicotinic acetylcholine receptors (Brown and Fumagalli 1977;

Carbonetto et al. 1978; Duggan et al. 1976; Jacob and Berg 1983;

Patrick and Stallcup 1977a, 1977b). Although more is known about

this toxin-binding component (Couturier et al. 1990; Schoepfer et al.

1990; Seguela et al. 1992), its role on neurons is still unknown.

Antibodies against the Torpedo nicotinic acetylcholine receptor

recognize proteins in the CNS and have been used to determine the

distribution of the antigenic determinants in the chicken brain

(Lindstrom et al. 1987). They have also been used to purify the

molecules that carry the antigenic deter-minants (Whiting and

Lindstrom 1986) and one subunit of these oligo-meric molecules

binds the affinity labeling reagent MBTA (Whiting and Lindstrom

1987). There is also good evidence in chick ciliary ganglion neurons

that at least one of the antibodies recognizes a functional receptor

(Smith et al. 1986). It is also the case that the amino terminal

sequence of one of the proteins purified on the basis of its

immunological cross-reactivity corresponds to the sequence of a

functional neuronal nicotinic receptor deduced from an expressible

cDNA clone (Whiting et al. 1987).

An alternative approach to understanding nicotine function in the

CNS was based on the idea that nucleic acids encoding muscle

nicotinic acetyl-choline receptor subunits would hybridize, at low

stringency, to nucleic acids encoding neuronal nicotinic acetylcholine
6

receptor subunits. This work resulted first in a clone encoding an

alpha subunit expressed in the PC12 cell line (Boulter et al. 1986) and

subsequently in the isolation of clones encoding nine members of the

rat neuronal nicotinic acetylcholine receptor subunit gene family

(Boutler et al. 1990; Deneris et al. 1987, 1988; Duvoisin et al. 1989;

Goldman et al. 1987; Lamar et al. 1990; Wada et al. 1988) and a rat

alpha-bungarotoxin binding component (Seguela et al. 1992). Other

labs have isolated clones encoding chicken nicotinic receptor subunits

(Nef et al. 1987, 1988) and two chicken alpha-bungarotoxin binding

components (Couturier et al. 1990; Shoepfer et al. 1990).

Nine clones define nine genes that are included in the neuronal

nicotinic acetylcholine receptor gene family on the basis of their

similarities in sequence. These genes are expressed in unique but

overlapping sets of anatomical loci in the brain. Using in situ

hybridization the combination of neuronal nicotinic receptor genes

expressed in over 500 brain loci has been determined (Miller and

Patrick 1992; Wada et al. 1989, 1990).

Some but not all of these genes encode subunits that participate in the

formation of oligomeric nicotine-gated ion channels. Oocyte

expression studies have established four functional alpha subunits

(alpha2, alpha3, alpha4, and alpha7) and two functional beta subunits

(beta2 and beta4) (Boulter et al. 1987; Deneris et al. 1987; Duvoisin

et al. 1989). Expression of functional nicotine-gated ion channels in

Xenopus oocytes results when DNA encoding beta2 or beta4 is

injected in pairwise combination with DNA encoding either alpha2,

alpha3, alpha4, or when alpha7 is expressed alone. Other members of

the gene family, alpha5, alpha6, alpha8, and beta3, have not yet to

been shown to participate in the formation of functional ligand-gated

ion channels.

Different combinations of alpha and beta subunits produce receptors

with strikingly different responses to agonists and antagonists.

Receptor sensitivities to agonists and antagonists are dependent upon

the subunit composition of the receptor, and both the alpha and the

beta subunits confer pharmacological and electrophysiological

characteristics to these receptors (Boulter et al. 1987; Duvoisin et al.

1989; Luetje and Patrick 1991; Luetje et al. 1990a, 1993; Papke et al.

1989). These receptor-specific characteristics are not simply

quantitative differences in agonist sensitivity but are qualitative in

nature. A ligand that is an agonist on a receptor composed of one

combination of subunits can be an antagonist on a receptor composed

of another combination of subunits (Leutje and Patrick 1991). There
7

are also striking quantitative differences. Nicotine differs by about

one hundredfold in its ability to activate certain receptor

combinations. These ligands, therefore, provide structural backbones

for chemical modifications that would enhance their ability to

discriminate between receptor subtypes. In principle, the potential to

make subtype-specific agonists and antagonists exists. Such drugs,

coupled with knowledge of the known anatomical location of

expression of individual subunits in the CNS, will provide a strong

rational basis for whole-animal experiments designed to dissect out

the roles that different nicotinic receptors may play in behavioral

responses to nicotine.

The neuronal nicotinic receptors differ from one another and from

the muscle-type nicotinic receptor in two additional ways. They have

a substantial permeability to calcium ions (Mulle et al. 1992; Seguela

et al. 1992; Vernino et al. 1991) and their function is regulated by

calcium ions acting on the outside of the cell (Vernino et al. 1991).

The hetero-oligomeric receptors comprised of both alpha and beta

subunits have permeabilities to calcium that are about five times that

of the muscle receptor and about one-fifth that of the N-methyl-daspartate (NMDA) receptor. The homooligomeric alpha7 receptor

has a permeability to calcium that is greater than that of the NMDA

receptor (Seguela et al. 1992). This suggests that these nicotine

receptors play an important role in regulating calcium-dependent

cytoplasmic processes and may contribute to activity-dependent

neurotoxic cell death. Furthermore, the activation of these receptors

by nicotine is regulated by external calcium over physiologically

relevant concentrations (Vernino et al. 1991). This regulation by

external calcium provides yet another mechanism for control of

receptor function. Clearly, these studies on the pharmacology,

permeability, and regulation of the various nicotine receptors provide

an important foundation on which to build scientists’ understanding

of the cellular basis of the behavioral and addictive effects of nicotine.

Exploitation of the diversity of these receptors requires reagents to

identify specific combinations of receptor subunits in the brain, tools

to quantitate these receptors in specific brain loci, techniques to

localize receptors on the neuronal cell surface, and mechanisms for

testing specific hypotheses that might account for the nicotineinduced regulation of nicotine binding sites. The following

paragraphs describe progress in expressing the subunits of neuronal

nicotinic receptors in bacterial expression systems, the production of

antisera, and the selection and testing of subunit-specific antibodies.
8

Nine cDNA clones that encode subunits of the neuronal nicotinic

acetyl-choline receptors expressed in rat brain have been cloned and

sequenced. These cDNA clones provided the primary structure for

these proteins and produced functional receptors in single-cell

expression systems such as the Xenopus oocyte. Although these

clones are extremely valuable they do not, in and of themselves,

readily provide access to the in vivo function of the proteins they

encode. Neither do they provide biochemical quantities of protein or

even biochemical access to the proteins expressed in the CNS.

Therefore, they must be used in an expression system to generate

reason-able quantities of protein corresponding to each of the nine

cDNA clones that encode the members of this gene family. The pET

expression system has several advantages (Rosenberg et al. 1987;

Studier et al. 1990). It is available in three reading frames and the

amount of sequence added to the expressed protein can be both

known and controlled. Most important, the system is well designed to

protect the inserted coding sequences from selective processes such as

deletion until synthesis is initiated. The system is also known to

produce large quantities of protein.

Choice of the region of the subunit to be used for antibody

production is extremely important and involves several considerations.

It would obviously be easier to obtain subunit-specific antibodies if

nonconserved regions were used. This would also increase the

likelihood of obtaining a good immune response as those sequences

not conserved among subunits are generally not conserved across

species. The disadvantage to this approach is that the obvious

nonconserved sequences are in the cyto-plasmic domains of the

receptor and are thus not accessible to the anti-bodies applied to the

outside of the cell. Antibodies to this domain will not stain living

cells. More importantly, antibodies to this domain cannot be used to

distinguish between the receptors on the cell surface and the precursor

receptors, targeted for the cell surface, which exist in vesicles inside

the cell. This is because the cytoplasmic domains of the cell surface

receptors and their precursors are in the same topological space (i.e.,

the cytoplasm). However, antibodies to extracellular domains will

stain living cells and can be used to distinguish between surface and

precursor receptors. The disadvantage of the extracellular domains is

that they are very conserved and it is difficult to obtain subunitspecific antibodies.

Antibodies were made against the extracellular domain of each

subunit and peptides were used to generate specificity as described

below. This approach allows production of polyclonal antibodies
9

against the extra-cellular domain of the protein and affinity

purification of antibodies that are specific for each subunit. In

addition, it also provides tools for a quantitative mechanism for

assessing both the specificity of the anti-bodies and the abundance of

the antigen in intact tissue.

Figure 1 shows the regions of the subunits used as immunogens and

the sequences chosen for peptide synthesis. Figure 2 shows the

sequences of the peptides used to select the subunit-specific antibodies

from the poly-clonal sera. It is important that the peptides used to

acquire specificity be derived from homologous regions of each

subunit so the peptides can be used to demonstrate nonreactivity with

peptides derived from the other subunits. Peptides corresponding to

this specific region of the protein were chosen for several reasons.

First, it is extracellular and the specific sequence offers substantial

variability between subunits. Second, it is a region known to promote

antibody synthesis in the case of the muscle nicotinic receptor

(Bellone et al. 1989; Tzartos et al. 1988) and antibodies directed

against this sequence are known to react with the native protein

(Tzartos and Lindstrom 1980). Each of the indicated constructs

except alpha7 were made and checked for proper insertion and

reading frame by sequencing through the ligation sites. Bacteria were

transformed with the pET vector containing the indicated insert and

induced to make protein. Induced bacteria were harvested and the

protein purified by extraction of the inclusion bodies out of detergent

solutions. The coomassie-stained gel in figure 3 shows the bands

corresponding to each subunit except alpha7.

The authors’ approach to generation of specific antisera by making

anti-bodies first against the protein expressed in bacteria and then by

acquiring specificity through affinity purification is not the usual one.

Many laboratories have tried to make antibodies against peptides or to

make monoclonal antibodies against bacterially expressed protein.

The anti-bodies against the peptides frequently recognize the amino

or carboxy-terminal sequences and thus fail to recognize the native

protein. Although there are many successful applications of each of

these approaches, the production of antibodies against the very similar

extracellular domains of neurotransmitter receptors has been difficult.

The results in figure 4 show the reaction of anti-beta2 antibody with

bacterially expressed proteins and the specificity attained following

purification with peptide. The affinity-purified antibody has lost its

cross-reactivity to beta4 and retained activity on beta2. The authors

have obtained the same results with antibodies directed against each of

the other subunits.
10
11
12
13

The authors used affinity chromatography on peptide columns to

prepare a subunit-specific antibody for each of the subunits and tested

the product on all of the peptides. The data in figure 5 show a slot

blot of peptide coupled to ovalbumin and reacted with each of the

antibodies (and with an antibody directed against the intracellular

domain of alpha7). The results show reactivity with the appropriate

peptide and nonreactivity with the inappropriate peptides. These

results show that the affinity-purified antibodies distinguish between

peptides but do not address the issue of whether they react with native
14

protein. These antibodies have been used to detect neuronal nicotinic

receptors on the surface of transiently expressed COS cells indicating

that they do recognize the native protein (Neff et al. 1995).

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AUTHORS

James W. Patrick, Ph.D.

Professor and Head

Shawn Neff, B.A., M.S.

Research Technician

Kelly Dineley, B.A., M.S.

Graduate Student

David Char, M.D., Ph.D.

Research Associate

Division of Neuroscience

Baylor College of Medicine

One Baylor Plaza, 5603

Houston, TX 77030
20

The Dopamine D4 Receptor

Hubert H.M. Van Tol

INTRODUCTION

The observation that receptors which mediate their signals through

heterotrimeric guanosine triphosphate (GTP)-binding proteins (Gproteins) share a considerable amount of sequence similarity has

resulted in the rapid molecular characterization of this receptor

superfamily. Moreover, it became clear that several receptor families

consisted of more receptor subtypes than originally anticipated from

pharmacological studies. The dopamine (DA) receptor family, which

was originally thought to comprise two receptor types (D1 and D2),

includes to date five different DA receptor genes (Bunzow et al. 1988;

Dearry et al. 1990; Sokoloff et al. 1990; Sunahara et al. 1990, 1991;

Tiberi et al. 1991; Van Tol et al. 1991; Zhou et al. 1990). The major

difference between the D1-like receptors D1 and D5/D1B lies in their

distribution, but pharmacologically and functionally these two

receptors are almost indistinguishable (Sunahara et al. 1991; Tiberi et

al. 1991). The D2-like receptor family includes two alternatively

spliced forms of the D2 receptor (Dal Toso et al. 1989; Giros et al.

1989; Grandy et al. 1989; Monsma et al. 1989), and the D3 (Sokoloff

et al. 1990) and D4 (Van Tol et al. 1991) receptors. Apart from clear

differences in distribution profiles, the D2-like receptors also display

pharmacological differences. The cloned D2 receptor is able to

inhibit adenylyl cyclase (Albert et al. 1990), and is also able to

activate several other signal transduction pathways (Elsholtz et al.

1991; Kanterman et al. 1991; Vallar et al. 1990). Moreover, the two

alternatively spliced forms of the D2 receptor are not identical in their

functional activity (Hayes et al. 1992; Montmayeur et al. 1993).

Because of the increased complexity of the DA receptor system, the

role of this system in self-rewarding behaviors has become potentially

more complex and might need a reevaluation of the roles played by

the individual receptor systems. The observation that D3, D4, and

D5/D1b receptors are relatively more concentrated in the limbic

system than the D1 and D2 receptors suggests a role for these newly

recognized receptors in the etiology of addiction. This is illustrated

by the observation that dopamine agonists with higher potencies for

D3 receptors seem to be relatively more effective in decreasing

cocaine self-administration in rats (Caine and Koob 1993).
21

MOLECULAR CHARACTERIZATION OF THE D4 RECEPTOR

After the initial cloning of the D1 and D2 receptors, it was speculated

that there were several other DA receptor types. These speculations

were based on several observations, particularly that dopamine

receptors from different tissues or brain areas displayed

pharmacological and functional profiles that were not in agreement

with the predominant D1 and D2 receptor types described for brain

tissue (Anderson et al. 1990; Nisoli et al. 1992; Sokoloff et al. 1984).

The search for novel DA receptor subtypes was based on the

presumed homology between these receptor subtypes with the D1 and

D2 receptors. The search was begun by scanning several tissues and

cell lines for the presence of ribonucleic acid (RNA) species that

would hybridize to a D2 receptor probe encoding the putative

transmembrane regions VI and VII under lower stringency conditions,

but not under high stringency conditions. Using this approach, D2

positive hybridizing RNA species (but not D2 itself) were identified in

mouse neuroblastoma NB41A3 and N4TG1, hamster kidney cells

BHK-21, and the human neuroepithelioma SK-N-MC. In the

subsequent screening of a cDNA library from the cell line SK-N-MC

the author succeeded in the isolation of a partial cDNA clone (750

bp) that displayed good homology with the D2 receptor and could

detect a 5.2 kb RNA species in the SK-N-MC cell line and rat brain

(Van Tol et al. 1991). Screening of several human and rat brain

cDNA libraries did not result in the isolation of full-length clones,

although several similar partial cDNAs were isolated. However,

genomic clones from human and rat were isolated that encoded the

entire coding region for this putative receptor (Asghari et al. 1994;

O'Malley et al. 1992; Van Tol et al. 1991) (figure 1).

Pharmacological analysis established that the isolated clones were

indeed novel DA receptor subtypes (see below), which were called the

DA D4 receptor (Van Tol et al. 1991). Based on the homology with

the D2 and D3 receptors, the entire coding sequence for the D4

receptor was identified in a 4.5 kb genomic DNA fragment.

The coding sequence is interspersed by three introns for which the

donor/acceptor splice junction sites were conserved at identical

positions as seen for introns in the D2 and D3 receptors. Introns 2

and 3 were positively identified by comparative analyses of the

genomic clones with
22
23

FIGURE 1. Genomic organization of the human D4.2 and rat D4

receptor genes. The coding regions are represented

by the blocks and the noncoding regions by the

connecting line. The repeat units alpha and zeta are

indicated as two white boxes in the coding block. The

position of the polyadenylation site is indicated with

An and an arrow. An Alu sequence has been

recognized within the first intron of the human gene

that is not present within the rat gene. The dotted line

within the first intron of the rat gene indicates where

the Alu sequence is inserted in the human gene. Apart

from the 48 bp repeat units in the third exon, three

other polymorphic markers are indicated: (1) a

polymorphic Smal site located immediately upstream

from the initiation codon (Petronis et al. 1994a,

1994b), (2) an insertion/deletion polymorphism of 4

amino acids in the first exon (Catalano et al. 1993),

and (3) a polymorphic G Mononucleotide repeat

within the first intron (Petronis et al. 1994a, 1994b).
24

the isolated partial D4 cDNA clones. The position of all three introns

was confirmed by exon trapping, a technique through which a fulllength D4 cDNA clone was also isolated (Van Tol et al. 1992).

Translation of the coding sequence into amino acid sequence revealed

that the isolated D4 receptor clones encode a polypeptide with seven

stretches of hydrophobic amino acids that could span the membrane.

These putative transmembrane spanning domains display the highest

amino acid sequence identity with the putative transmembrane regions

of the D2 and D3 receptors, especially transmembrane domains 3, 6,

and 7,which have 60percent to 80 percent sequence similarity to the

homologous domains in the D2 and D3 receptors. The overall

sequence similarity between the D4 and the D2 and D3 receptors is

about 40percent. The sequence similarity is least preserved in the

intra- and extracellular loops and tails, which supports the notion that

the transmembrane domains form the ligand-binding pocket, as has

been shown for adrenergic receptors. This is also supported by the

fact that an Asp residue in transmembrane 3 and two Ser residues in

transmembrane5, which have been identified as crucial amino acids

for catecholamine binding in several catecholaminergic receptors, are

conserved in the D4 receptor (Strader et al. 1988, 1989). In analogy

to the models created for other G-protein-coupled receptors, the

amino terminus is located extracellularly and contains one putative Nlinked glycosylation site. The carboxy terminus ends in a Cys, as is

seen for D2 and D3 receptors, and can serve as a potential substrate

for palmitoylation (O'Dowd et al. 1989). Within the third cytoplasmic

loop of the human D4 receptor there are only a limited number of

residues that potentially can be used for phosphorylation, in contrast

to the rat D4 receptor where there are several more such sites (Asghari

et al. 1994; O'Malley et al. 1992; Van Tol et al. 1991). An unusual

structural feature of the human D4 receptor is the presence of a

polymorphic 16 amino acid repeat sequence in the third cytoplasmic

loop, which is not found in the isolated rat gene (see below; figure 1).

The location of this repeat sequence corresponds to the location of the

alternatively spliced 29 amino acid sequence of the D2 receptor.

Through the cloning of several D4 genes and cDNAs it became clear that

a region within the third cytoplasmic loop of the human D4 receptor was

polymorphic (Van Tol et al. 1992). Subsequent detailed analyses of this

region by Southern blot, polymerase chain reaction, and sequence

analysis demonstrated that the polymorphic sequence occurs as a 48 bp
25

tandem repeat of 2 to 10 repeat units (figures 1 and 2). The different

polymorphic repeat variants of the D4 are classified according to the

number of repeats D4.2 to D4.10. Thus far 18 different repeat units have

been identified which display over 90 percent sequence similarity among

each other, and which have been identified by different Greek letters

(Lichter et al. 1993). Although the first and last repeat units are always

the so-called alpha and zeta units, respectively (there is one exception: an

allele has been identified in which the last unit codes for a xi unit that is

identical in amino acid sequence to the zeta unit), the other units can be

found in any position between the alpha and zeta units. This has resulted

in the identification of 27 different alleles in over 200 analyzed chromosomes (Asghari et al. 1994; Lichter et al. 1993; Van Tol et al. 1992)

(figure 2).

In Northern blot analysis of cells transfected with the human D4 gene,

several D4 cDNA variants indicate that the repeat sequence is not

spliced out of the D4 gene, but is part of the coding sequence. This is

supported by the fact that there are no sequences in or surrounding

the repeat, which could be used as splice donor and acceptor sites.

Moreover, exon trapping failed to excise this sequence. Thus the 27

variant alleles would actually code for 20 different D4 receptor

variants. The putative amino acid sequence for the different repeat

regions demonstrate a high Pro content in this region (30 percent to

40 percent). Recently, such Pro-rich segments have been identified as

potential SH3-binding domains (Ren et al. 1993; Yu et al. 1994). On

the other hand, it might be postulated that the variation in the third

cytoplasmic loop sequence might have consequences for the

specificity and/or efficacy in signal transduction as seen for the two

alternatively spliced forms of the D2 receptor. Thus far, there is only

limited evidence to support this speculation (Asghari et al., in press).

PHARMACOLOGY

The most extensive pharmacological characterization of the D4

receptor has been done by transient expression of this receptor in

COS-7 cells. The pharmacological characterization of receptors

derived from the expression of the entire gene or cDNA in COS-7

does not show any differences, except for the fact that higher levels of

expression can be obtained with the cDNA than with the gene cloned

into identical expression vectors. Transient expression of the D4

receptor in COS-7
26
27

cells resulted in the detection of concentration-dependent and

saturable [3H]spiperone binding with an affinity dissociation constant

of approxi-mately 100 picomolars (pM). The [3H]spiperone binding

could be competed with dopamine (inhibition constant (Ki)

approximately 27nano-molars (nM)), but less efficiently by

norepinephrine and serotonin (Ki>2micromolars (μM). Dopamine

competition of [3H]spiperone binding revealed the presence of a high

affinity site with a dissociation constant of 500 to 1,000 pM and a low

affinity site of 10 to 50 nM. Inclusion of 200μM Gpp[NH]p in the

binding buffer resulted in the conversion of the biphasic dopamine

competition curves to a single affinity site of approximately 10 to 50

nM (Asghari et al. 1994; Van Tol et al. 1991, 1992). These binding

data suggest functional coupling of the D4 receptor in COS-7 cells.

Dopamine D4 receptor binding characteristics have been determined

for several D2 agonists and antagonists. This revealed a D4

pharmacological profile that has several similarities with the D2

receptor; however, some remarkable differences have also been

detected (figure 3). Probably the most striking is the relatively high

affinity of the atypical neuroleptic clozapine for the D4 receptor (Ki

10 to 20 nM) as compared to the D2 receptor (Ki 100 to 200 nM),

and the poor affinity of the D2 antagonist raclopride for the D4

receptor (1 to 2 μM) (Asghari et al. 1994; Van Tol et al. 1991, 1992).

Furthermore, the D4 receptor demonstrates stereo-selectivity for (+)-

and (-)-butaclamol, several (+)- and (-) aporphines, (+)and (-) PHNO,

(+) and (-) quinpirole, but not for (+) and (-)-sulpiride (Seeman and

Van Tol 1993).

Thus far, no compound has been reported that is selective for the D4

receptor. Although clozapine seems to be tenfold more selective for

the D4 receptor as compared to other dopamine receptors, the

muscarinic, 5-HT2, and 5-HT7 receptors have similar affinities for

clozapine as the D4 receptor. However, by making use of the

differential affinities of the benzamides [3H]emonapride and

[3H]raclopride for the different D2-like receptors, the density of D4-

like sites in brain tissue can be indirectly determined (Seeman et al.

1993). Scatchard analysis with [3H]emonapride will detect and allow

determination of the total density of all D2-like receptors (D2, D3,

D4), while such analysis with [3H]raclopride will only detect and

reveal the density of the D2, D3 receptor pool. Therefore, the

difference in maximal density detected by both radioligands in

scatchard analysis will reflect the density of a receptor pool that is D4-

like.
28
29

their affinity for several D4 receptor variants. Moreover, for all

variants tested, two similar affinity states for dopamine binding were

observed that were sensitive to the inclusion of Gpp[NH]p. Under

standard DA receptor binding conditions, no major differences have

been observed in pharmacological characteristics between variants of

the D4 receptor. However, small differences in the sensitivity for

sodium chloride on clozapine binding have been observed between

D4 variants (Asghari et al. 1994; Van Tol et al. 1992). As yet, it is

unclear whether these observed differences are indicative of

differences in receptor function. Genetic association studies have thus

far not indicated a good correlation between differences in clozapine

responsiveness in the patient population and the different D4 isoforms

(Shaikh et al. 1993). However, inclusion of four different

polymorphic markers for the D4 receptor gene (figure 1) in these

analysis gives some ability to predict clozapine responsiveness

(Kennedy, personal communication, July 1994).

COUPLING TO ADENYLYL CYCLASE

In order to determine whether the D4 receptor possesses the ability to

block adenylyl cyclase activity, the D4.2 variant was cloned by

homologous recombination into vaccinia virus. This recombinant

vaccinia virus could infect several cell types which would, upon

infection, express D4 receptors that display a pharmacological profile

identical to that described previously. Approximately 2 to 3 days

after infection, cells express D4 receptor levels, as determined by

[3H]spiperone binding, of approximately 1 pmol/mg protein.

Functional analyses of GH4C1 cells, mouse fibroblast L cells, and Rat1 cells demonstrated that all these cells, upon infection with recombinant D4 vaccinia virus, could block adenylyl cyclase activity by

dopa-mine (Bouvier et al. 1993). Detailed analyses of infected Rat-1

cells demonstrated that dopamine could block forskolin-stimulated

adenylyl cyclase activity and increased intracellular cyclic adenosine

monophos-phate (cAMP) levels with a 50 percent effective

concentration (EC50) of about 10 nM. This activity could be blocked

by spiperone and clozapine, but not by raclopride, which was in

agreement with the affinities of these drugs for the D4 receptor

(Bouvier et al., in press).

As well, several forms of the human D4 receptor were stably

transfected into Chinese hamster ovary cells (CHO-K1). These cell

lines express the dopamine D4 receptor at concentrations of

approximately 200 to 400 fmol/mg protein. Stimulation of these cells
30

by various concentrations of dopamine did not significantly change

the intracellular cAMP levels, compared to nontransfected cells and

nonstimulated cells. However, in the D4-expressing cell lines, DA

could reduce forskolin-induced intracellular cAMP increases by up to

90 percent (Asghari et al., in press). This reduction was concentration

dependent with an EC50 of approxi-mately 15 nM (figure 4).

Comparative analysis of various cell lines expressing the D4.2, D4.4,

or D4.7 variants failed to show any major differences in either

efficacy or EC50 of DA to block adenylyl cyclase activity, although

the potency of DA for D4.7 was slightly reduced (Asghari et al., in

press). The D2 antagonists emonapride, haloperidol, and clozapine

could inhibit the dopamine-induced cAMP changes in forskolinstimulated cells in a concentration-dependent manner and with a rank

order that was in close agreement with the rank order seen for the

affinities of these compounds for the D4 receptor. Raclopride was not

able to block dopamine's activity at the D4 receptor at concentrations

less than 5 μM, which is expected considering the low affinity of this

ligand for the D4 receptor. The D4.2, D4.4, and D4.7 all displayed

similar functional profiles for these four D2 antagonists.

EXPRESSION OF THE D4 GENE

Dopamine D4 receptor messenger RNA (mRNA) has been detected in

various brain regions from humans, monkey, and rat. Northern blot

analysis of several dissected brain regions has shown a distribution for

this receptor that is dissimilar to the D2 and D3 receptors. A regional

distribution study in monkey brain showed relatively high levels for

D4 mRNA in frontal cortex, amygdala, midbrain, and medulla (Van

Tol et al. 1991). Lower levels were detected in striatum and

hippocampus. Although D4 mRNA is detectable by Northern blot

analysis in poly A+ enriched RNA preparations, D2 mRNA can be

monitored easily by the same methodology in total RNA preparations,

suggesting two orders of magnitude difference in the density of both

RNAs (Bunzow et al. 1988). This is confirmed by the relatively low

signal that is detected by in situ hybridization. In situ hybridization

data basically confirm the locali-zation seen by Northern analysis,

although cellular localization is more detailed and thus revealed the

presence of D4 mRNA at relatively high levels in dentate gyrus

(Mansour et al. 1991; Meador-Woodruff et al. 1991, 1994; O'Malley

et al. 1992). Interestingly, D4 receptor mRNA has also been detected

in relatively high levels in rat heart (O'Malley et al. 1992) and retina

(Cohen et al. 1992).
31

Alternatively, the author attempted to monitor the location of D4

binding sites. By using the differential binding characteristics of

[3H]raclopride and [3H]emonapride for D2, D3, and D4 receptors

(see above), D4-like binding sites were detected in caudate putamen of

human and rat but not in frontal cortex. This can be interpreted to

indicate that a large number of the D4 mRNA containing cortical

neurons have their projections with D4 sites in noncortical areas,

including caudate putamen. Less likely is that this RNA is not

translated into a functional protein. Preliminary data using in vitro

autoradiography of [3H]emonapride in the presence of an excess of

unlabeled raclopride ( > 100 nM) confirms the location of these

raclopride "insensitive" [3H]emonapride binding sites seen by

scatchard analysis, and also revealed several D4-like binding sites in

other rat brain areas, including entorhinal cortex, colliculi, and central
32

gray area (Nobrega, personal communication, June 1994). Whether

these sites are genuine D4 receptors awaits further characterization.

Little is known as yet about whether, and how, D4 receptor density is

regulated. Experiments in which rats have been treated for a

prolonged period with haloperidol indicate a twofold increase in both

D4 mRNA and D4 binding sites, as defined by emonapride and

raclopride, in caudate putamen (Schoots et al. 1995). Interestingly,

the number of dopamine D4-like binding sites is also considerably

increased in postmortem caudate putamen tissue of schizophrenics

(Seeman et al. 1993). In the schizophrenic tissues the D4 receptor

levels were elevated approximately sixfold over control tissues.

Although the majority of the tested tissues originated from

schizophrenic individuals who were treated with neuroleptics, similar

increases were also seen in the drug-naive individuals, while no

significant changes were measured in tissues obtained from patients

with Huntington's disease who were treated with neuroleptics.

Furthermore, as described above, rats treated with neuro-leptics only

showed a twofold change in receptor levels, suggesting that at least a

certain proportion of the increased receptor density must be attributed

to the disease. On the other hand, the relation of the D4 receptor to

schizophrenia is unclear. Genetic linkage and association studies of

schizophrenia and bipolar affective disorder with several polymorphic

markers for the D4 receptor have not supported this receptor as the

primary cause for these disorders (Barr et al. 1993; Macciardi et al.

1994). Similar results have been obtained for the D2 and D3

receptors (Kennedy 1994). This, however, does not rule out any of

these receptors as therapeutic targets to control these disorders, since

drugs with different D2-like binding profiles and clinical profiles, like

raclopride, haloperidol, and clozapine are all effectively used in the

treatment of these diseases.

CONCLUDING REMARKS

The role of the dopamine D4 receptor in the DA system with respect

to addiction is unclear. Although the DA system has been clearly

established as a modifier of self-reward behavior, the recognition of

several new DA receptor genes warrants a reevaluation of several

components of the system. The absence of a wide variety of agonists

and antagonists that are highly selective for the different receptors has

prevented the evaluation of the functional role of these receptors in

drug addiction. From studies done by Caine and Koob (1993) it is

clear that different DA receptors might contribute differently to self-
33

reward behavior. With respect to the D4 receptor, one published study

indicates a higher frequency of the occurence of the alleles D4.3 and

D4.6 in alcoholism (George et al. 1993); however, another study

failed to demonstrate such an association (Adamson et al. 1995).

To establish unequivocally that the D4 receptor is not a genetic factor

contributing to addiction, other paradigms might also have to be

tested. As for the roles of the D4 receptor and other newly

recognized DA receptors in addiction behavior, researchers are largely

ignorant about the different behavioral functions of these receptors.

Although the development of highly specific D4 agonists and

antagonists might be of great value, alternative approaches should be

investigated. As shown for the 5-HT1B receptor (Hen et al., this

volume), the use of DA receptor gene "knockout" mice might be of

great value for unraveling the functional roles of the dopamine

receptors for different behavioral paradigms.

One of the most interesting molecular features of the human D4

receptor is probably the extensive polymorphic repeat sequence in the

coding sequence of this receptor. The identification of at least 27

different haplo-types for the repeat encoding 20 putative different D4

receptor proteins has revealed another level of receptor diversity

which is thus far unprecedented within the G-protein-coupled receptor

superfamily. Until now it was recognized that receptor diversity was

generated by the existence of several genes coding for different

subtypes or by alternative splicing. Large structural polymorphisms

as described for the D4 receptor might account for differences in

drug responsiveness and susceptibility to neuropsychiatric disorders,

including addiction. It would be of interest to see whether such

extensive interindividual differences in coding sequence also exist for

other proteins, or whether this is a unique feature of the D4 receptor.

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AUTHOR

Hubert H.M. Van Tol, Ph.D.

Associate Professor

Departments of Psychiatry and Pharmacology

University of Toronto

and

Clarke Institute of Psychiatry

Laboratory of Molecular Neurobiology

250 College Street

Toronto, Ontario M5T 1R8

CANADA
39

Behavioral Characterization of Mice

Packing the 5-HT1B Receptor

Sylvie Ramboz, Frédéric Saudou, Djamel Aït Amara,

Catherine Belzung, Andrée Dierich, Marianne LeMeur,

Louis Segu, RenéMisslin, Marie-Christine Buhot,

and René Hen

INTRODUCTION

Serotonin (5-hydroxytryptamine, 5-HT) is a neuromodulator that is

involved in a number of mood disorders such as depression, anxiety,

and impulsive violence. In an attempt to dissect the contribution of

individual 5-HT receptor subtypes to behavior, the authors have

generated, by homologous recombination, mutant mice lacking the 5-

HT1B receptor. These mice did not exhibit any obvious

developmental or behavioral defect; however, the hyperlocomotor

effect of the 5-HT1A/5-HT1B agonist RU24969 was completely

absent in mutant mice, indicating that this effect is mediated by 5-

HT1B receptors. Moreover, when confronted with an intruder,

isolated mutant mice attacked the intruder faster and more intensely

than wild-type mice, suggesting an involvement of 5-HT1B receptors

in the modulation of aggressive behavior. These data might be related

to the fact that a class of 5-HT1 agonists, termed serenics, have

antiaggressive properties, and with the findings that certain impulsive

aggressive behaviors are associated with deficits in central serotonin.

5-HT is a biogenic amine that is involved in a wide range of

physiological functions including sleep, appetite, pain perception,

sexual activity, memory, and mood control (for a review see Wilkinson

and Dourish 1991). A central serotonin deficit has been associated

with behaviors such as suicidality, impulsive violence, depression, and

alcoholism (Eichelmann 1992; Roy et al. 1991), and serotonergic

drugs are used in the treatment of a number of pathological states

including migraine, depression, and anxiety (Sleight et al. 1991). The

multiple actions of serotonin are mediated by the interaction of this

amine with at least 14 receptors (for a review see Saudou and Hen, in

press), most of which belong to the guanosine triphosphate binding

protein (G-protein)-coupled receptor family.
40

The purpose of this study was to determine the contribution of one of

these receptors, the 5-HT1B subtype, to the various behavioral

responses elicited by serotonin and serotonergic drugs. The 5-HT1B

receptor, which is the rodent homolog of the human 5-HT1Db

receptor, is expressed in a variety of brain regions, including motor

control centers such as the basal ganglia, as well as structures involved

in mood control such as the central gray, hippocampus, and raphe

nuclei (Boschert et al. 1993; Bruinvels et al. 1993; Maroteaux et al.

1992). Pharmacological studies using poorly specific agonists have

suggested that activation of 5-HT1B receptors might lead to an

increase in anxiety and locomotion (Griebel et al. 1990; Pellow et al.

1987) and to a decrease in food intake, sexual activity, and aggressive

behavior (Fernandez-Guasti et al. 1992; Kennett et al. 1987; Koe et al.

1992; Olivier et al. 1987). In particular, a class of 5-HT1 agonists

have been termed serenics because of their antiaggressive properties in

several rodent aggression models (Flannelly et al. 1985; Mos et al.

1992; Olivier et al. 1986, 1989). However, it is not clear to what

extent the effects of the serenics are mediated by 5-HT1B receptors

because these drugs also activate 5-HT1A receptors and possibly some

of the recently discovered 5-HT receptors. In addition, the

consequences of a blockade of 5-HT1B receptors, or of their human

counterpart the 5-HT1Db receptors, are unknown, since there are no

specific antagonists for these receptors.

In order to study the function of the 5-HT1B receptor, the authors

have generated by homologous recombination homozygous mutant

mice lacking both copies of the gene encoding this receptor. These

mice are viable and fertile, and they were analyzed for a variety of

behaviors that are thought to be modulated by 5-HT1B receptors such

as locomotion, anxiety, and aggression.

5-HT1B RECEPTOR GENE TARGETING

The 5-HT1B receptor gene was disrupted by homologous

recombination (Capecchi 1989; figure 1). The JA construct consisted

of 6.0 kilobase-pairs (kb) of genomic sequence in which part of the

5-HT1B coding sequence was replaced by a neomycin

phosphotransferase gene (neo) under the control of the GTI-II

enhancer (Lufkin et al. 1991; figure 1A). In the JB construct, the

neocassette was inserted in the coding sequence of the 5-HT1B gene

(figure 1B). The two linearized targeting vectors were electroporated

into D3 embryonic stem (ES) cells and G418 resistant colonies were

screened by Southern blotting (figure1). The EX400 probe identified
41

positive clones with the expected 10 and 8.5 kb Kpn I fragments for

the JA and JB constructs respectively (figure 1). Four positive clones

were obtained with both constructs yielding a targeting frequency of

1/15 (JA) and 1/12 (JB) (table 1). Southern analyses using Xba I

digests and the E2A1 probe or the neo probe confirmed that accurate

targeting occurred and that no additional integration took place (data

not shown). Cells from the positives clones JA7 and JB13 were

microinjected into 3.5-day C57BL/6 mouse blastocysts. The two

clones gave rise to highly chimeric mice that were bred with C57BL/6

females in order to test for germline transmission of the mutated 5-

HT1B receptor gene (table 1). The positive chimeras were bred with

females from the 129/Sv-ter inbred strain to obtain heterozygotes on

the 129/Sv-ter genetic background. Heterozygous mice were

phenotypically normal and fertile. Homozygous animals deriving

from both cell lines were generated by heterozygote crossings. In

243 JA offspring and 213 JB offspring, the expected 1:2:1 ratio of

wild-type, heterozygous, and homozygous mutant progeny was

observed. The homozygous mutants did not display any obvious

developmental or behavioral abnormality and were fertile. Although

the average lifespan has not yet been determined, no spon-taneous

deaths occurred during the first 12 months of life. All the analyses

presented here were performed on animals having a pure 129/Sv

genetic background. There were no differences between the mice

derived from the JA7 and JB13 targeted cell lines.

EFFECTIVENESS OF 5-HT1B RECEPTOR ABLATION

In order to ensure that disruption of the 5-HT1B receptor gene was

effective, the authors performed autoradiographic studies (figure 2)

on brains of wild-type, heterozygous, and homozygous mutants using

the radiolabeled ligand 3[125I]iodocyanopindolol ([125I]CYP).

When used in the presence of appropriate masking agents, this

radioligand binds specifically to the 5-HT1B receptor (Hoyer et al.

1985; Offord et al. 1988; Pazos and Palacios 1985). In wild-type

mice, [125I]CYP binding sites were found in the globus pallidus,

substantia nigra, cerebellar nuclei, subiculum, lateral geniculate

nucleus, central gray, and colliculi, while no specific binding was

observed in homozygous mutants (figure 2). These results

demonstrate that effective disruption of the 5-HT1B gene occurred

and that in these experimental conditions, [125I]CYP binding sites

correspond exclusively to 5-HT1B receptors. Heterozygous mice

displayed the same level of binding sites as wild-type mice (figure 2),

although they have only one functional allele of the 5-HT1B gene.
42
43

FIGURE 1. Homologous recombination at the 5-HT1B locus.

Schematic representation of the targeting event using JA

(A) and JB (B) constructs and Southern analysis of JA7

(A) and JB13 (B) mutant mice. The upper panels

correspond to the targeting vectors, genomic structure of

the 5-HT1B gene, and predicted structures of the mutated

alleles after homologous recombination. The black box

corresponds to the coding sequence of the 5-HT1B

receptor and the hatched box to the neocassette. Arrows

on the neocassette (purified from p 581 (Lufkin et al.

1991)) and on the 5-HT1B gene indicate the direction of

transcription (from left to right). The locations of the

probes E2A1 and EX400 used in Southern analysis are

shown. E2A1 and EX400 probes were used to screen

neomycin resistant clones after XbaI and KpnI digests,

respectively. Bottom, Southern blot analysis. Tail DNA

from wild-type, heterozygous, and homozygous JA7 (A)

and JB13 (B) mutant mice were cut by KpnI and

hybridized with the 3’ end EX400 probe.

KEY: E=EcoRI; B=BalI; X=XbaI; K=KpnI; V=EcoRV;

+/+=wild-type; +/-=heterozygous; and -/ =homozygous

mutant.
44
45

Locomotion

As shown in figure 2, the 5-HT1B receptor is localized in motor

control centers such as the globus pallidus, substantia nigra, and deep

cerebellar nuclei. Furthermore, pharmacological studies have

suggested an involvement of 5-HT1B receptors in the control of

locomotor activity (Green et al. 1984; Oberlander et al. 1986, 1987).

The activity of the mice in an open field was analyzed with a video

tracking device. No significant differences were detected between the

mutant mice and their wild-type littermates (figure 3). Administration

of the 5-HT1 agonist RU24969 stimulated locomotor activity in the

wild-type mice, while it had no effect in the mutants (figure 3). These

results indicate that the hyperlocomotor effect of RU 24969 is

mediated by 5-HT1B receptors.
46

TABLE 1. Homologous recombination of 5-HT1B gene. Analysis of

the recombination events and generation of homozygous mutant

mice.

Targeting Vector JA JB

Length of homology 4.5 kb 6 kb

Neomycin-resistant

colonies

62 49

Positives colonies 4 4

Positive colony used JA7 JB13

Chimeric males 23 2

Chimeric males analyzed 8 2

GLT chimeric males 5 2

Offspring Total 243 213

 from + / + 61 54

 heterozygote + / - 130 99

 crosses - / - 52 60

KEY: GLT = Germline transmission.

Anxiety

5-HT1 agonists such as RU 24969, eltoprazine, fluprazine, or 1-(3-

trifluromethylphenyl)piperazine (TFMPP) have been reported to

induce anxiogenic responses in rats and mice (Griebel et al. 1990;

Pellow et al. 1987). To evaluate the level of anxiety of the 5-HT1Bminus mice, the light/dark choice test (figure 4) was used. The time

spent in the lit compartment, as well as the number of transitions

between the dark and the lit compartments, have been considered as

indices of anxiety since they are increased by anxiolytic drugs. There

were no significant differences between mutants and their wild-type

littermates for either parameter, suggesting that the mutants have the

same level of anxiety as
47
48

the wild-type mice in this test (figure 4). In the wild-type mice, RU4969

increased significantly the number of transitions between the dark box

and the lit box, but it had no significant effect on the time spent in the lit

box (figure4). In the mutants, RU24969 did not modify these

parameters. The larger number of transitions displayed by the wild-type

mice are probably a result of the increase in locomotion induced by RU24969. The lack of response of the mutants demonstrates that the effect

of RU24969 is mediated by 5-HT1B receptors. In contrast with earlier

reports, RU 24969 had no anxiogenic effect. Such an effect might have

been masked in the present experiment by the hyperlocomotor effect of

this drug. A dose-response curve, as well as other anxiety tests, might

reveal an effect of RU24969.

Aggression

A class of 5-HT1 agonists including eltoprazine and fluprazine have been

termed serenics because of their antiaggressive properties (Flannelly et al.

1985; Mos et al. 1992; Olivier et al. 1986, 1989) and their effects have

been suggested to be mediated by 5-HT1B receptors. The authors

therefore investigated the aggressiveness of 5-HT1B-minus mice in a

classical aggression test. After an isolation period of 4 weeks, test mice

(resident) were analyzed for intermale aggression after introduction in

their cage of a wild-type mouse that had been reared in a group (intruder)

(figure 5). In this test, the latency of attack and the number of attacks

performed by the resident during a 3-minute period were used as

aggression indices. The mutant residents attacked the intruder faster than

the wild-type or heterozygous residents (figure 5A). Furthermore, the

number of attacks in the mutant group was significantly higher than in the

wild-type or heterozygote groups (figure 5B). In addition, the intensity

of attacks of the mutant residents was higher, as well as the number of tail

rattlings preceding the attacks (not shown). Similar results were obtained

in two tests performed 1 week apart. The level of aggressiveness was

higher in the second test with both the wild-type and the mutant animals,

in good agreement with previous reports showing that aggression

increases with fighting experience (Lagerspetz and Lagerspetz 1971). A

qualitative analysis of the attacks during the 3-minute test period revealed

additional marked differences between wild-type and mutant mice

(figure6). In the first test, 29 percent of the mutant residents attacked the

intruder within less than 10 seconds after introduction of the intruder in

the cage (impulsive attacks, figure 6A), while no wild-type or heterozygous mice attacked the intruder during that time interval. Conversely,

75 percent of the wild-type mice and only 21 percent of the mutants did
49
50
51

not attack during the 3-minute test. In the second test, the percentage

of mutants displaying impulsive attacks was even higher (46percent),

while still no wild-type animals performed such short latency attacks

(figure6B). These results indicate that the 5-HT1B-minus mice are

more aggressive than their wild-type or heterozygous littermates.

DISCUSSION

The authors have generated, by homologous recombination, mice

lacking the 5-HT1B receptor. Autoradiographic data demonstrated

the absence of 5-HT1B receptors in the homozygous mutants. Such

mice develop and live apparently normally. Preliminary histological

analyses of their central nervous systems did not reveal any obvious

defect (not shown). A number of behaviors were analyzed that were

thought to be modulated by activation of 5-HT1B receptors, such as

locomotion, anxiety, and aggression (Fernandez-Guasti et al. 1992;
52

Green et al. 1984; Griebel et al. 1990; Kennett et al. 1987; Koe et al.

1992; Oberlander et al. 1986, 1987; Olivier et al. 1987; Pellow et al.

1987). Surprisingly, in the authors’ test conditions, no differences

were detected in the levels of basal locomotor activity or anxiety in the

mutant mice. However, the hyperlocomotor effect of the 5-HT1

agonist RU 24969 was totally absent in the mutants, demonstrating

that this effect is mediated by 5-HT1B receptors. A decrease in

locomotor activity in the 5-HT1B-minus mice might therefore have

been expected. The absence of such a motor effect in these

experimental conditions suggests either that compensatory

mechanisms occurred during development or, alternatively, that in

normal “baseline” conditions the 5-HT1B receptor is not activated.

Preliminary results indicated that the levels of the 5-HT1A and 5-

HT1Da receptors were not altered in the mutants (not shown). The

levels of 5-HT and catecho-lamines as well as the levels of aminergic

receptors that are involved in motor control and which might have

compensated for the absence of the 5-HT1B receptor are currently

being analyzed. An alternative possibility, that 5-HT1B receptors are

activated in response to environmental changes such as stressful

situations, is appealing in light of the results obtained in the

aggression test. When the mutants are group housed they do not

appear to be more aggressive than grouped wild-type mice. However,

after a month of isolation and in the presence of an intruder, the

mutants are significantly more aggressive than the wild-type mice. In

male mice, isolation and the presence of a conspecific male intruder

have been shown to increase aggressive behavior (Lagerspetz and

Lagerspetz 1971). The authors’ results indicate that 5-HT1B-minus

mice are more responsive to the isolation and the stress or fear

generated by the intruder and suggest that 5-HT1B receptors might be

activated in stressful situations such as those encountered in this test.

The increased aggressiveness of 5-HT1B-minus mice is in good

agreement with the fact that a family of 5-HT1B agonists termed

serenics have anti-aggressive properties (Olivier et al. 1987). These

compounds were shown to decrease aggressive behavior in several

animal models including isolation-induced aggression in mice (Olivier

et al. 1989), resident-intruder aggression in rats (Flannelly et al. 1985;

Mos et al. 1992), and maternal aggression in rats (Mos et al. 1992).

The authors’ results suggest that the 5-HT1B receptor is at least in

part responsible for the antiaggressive properties of the serenics, but

do not rule out a participation of other receptors with a high affinity

for these compounds such as the 5-HT1A receptor. The effects of the

serenics in the 5-HT1B-minus mice are currently being tested in order
53

to determine whether 5-HT1B receptors are the preferential target of

these drugs.

Several studies have revealed an association between aggressive

behavior and a reduction in the activity of the serotonergic system. In

rodents and primates, aggressiveness is increased after inhibition of 5-

HT synthesis (Higley et al. 1992; Vergnes et al. 1986) or destruction

of serotonergic neurons (Molina et al. 1987). Mouse strains that

display increased aggressiveness have low brain 5-HT levels (Maas

1962). In humans, impulsive aggressive behaviors have been

associated with a deficit in central serotonin (Coccaro 1989).

Cerebrospinal fluid (CSF) concen-trations of the 5-HT metabolite 5-

hydroxyindole acetic acid (5-HIAA) is reduced in the brain of violent

offenders (Brown et al. 1979), arsonists (Virkkunen et al. 1987), and

people who committed violent suicide (Coccaro 1989; Mann et al.

1989). Interestingly, in one recent study, impulsive violent offenders

had low CSF 5-HIAA levels, while offenders who premeditated their

acts had high CSF 5-HIAA levels (Virkkunen et al. 1994). Similarly,

in studies of suicide victims, only those who committed violent

suicides exhibited low CSF 5-HIAA levels (Olivier et al. 1987; Pellow

et al. 1987). These findings suggest a link between low serotonin

levels and a lack of impulse control. The aggressive behavior

displayed by the 5-HT1B-minus mice might be considered impulsive,

since the mutants attacked much faster than the wild-type mice. In

light of these results it is tempting to speculate that low serotonergic

activity would result in a decreased activation of 5-HT1B receptors,

which might trigger aggressive behavior.

The 5-HT1B receptor is localized both presynaptically on

serotonergic terminals, where it inhibits the release of 5-HT, and

postsynaptically on other nerve endings, where it might inhibit the

release of various neuro-transmitters (for a review see Bruinvels et al.

1993). The antiaggressive effects of serenics are most likely mediated

by postsynaptic receptors since they are not affected by lesions of

serotonergic neurons (Sijbesma et al. 1991). Such postsynaptic

receptors might be localized in the central gray, a brain structure

involved in defensive behavior and response to fear (Fanselow 1991)

and containing moderate densities of 5-HT1B receptors (Boschert et

al. 1993; Bruinvels et al. 1993). Activation of the 5-HT1B receptor

might be a component of the adaptation to fearful stimuli.

Interestingly, several behavioral responses elicited by fear such as

those observed in a flight situation (increased locomotion and

decreased aggressiveness, sexual activity, and food intake) are also

induced by 5-HT1B agonists. These behaviors as well as the level of
54

stress hormones are currently being analyzed in order to determine

whether adaptation to stress or fear is altered in the absence of the 5-

HT1B receptor.

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ACKNOWLEDGMENTS

The authors thank M. Digelmann and E. Blondelle for ES culturing;

M.Duval and P. Mellul for blastocysts injection; P. Charles, F. Tixier,

and R.Matyas for animal care; E. Vogel for behavioral analyses; J.-L.-

Vonesch for image analysis; and B. Boulay and C. Lappi for

preparing the photographs and the manuscript. For helpful

discussions the authors thank J. Crabbe and N. Amlaiky, U. Boschert,

A. Ghavami, R.Grailhe, and J. Lucas. This work was supported by

grants from CNRS, INSERM, Ministère de la Recherche, Rhône

Poulenc Rorer, and Association pour la Recherche contre le Cancer.

AUTHORS

René Hen, Ph.D.

Associate Professor

Sylvie Ramboz, M.S.

Graduate Student

Center for Neurobiology and Behavior

Columbia University

722 West 168th Street

New York, NY 10032

Frédéric Saudou, Ph.D.

Scientist

Andrée Dierich, Ph.D.

Associate Professor
58

Marianne LeMeur, Ph.D.

Scientist

Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS

U184 de l’INSERM

Faculté de Médecine

11 rue Humann 67085 Strasbourg Cédex

France

Djamel Aït Amara, Ph.D.

Postdoctoral Student

Louis Segu, Ph.D.

Scientist

Marie-Christine Buhot, Ph.D.

Associate Professor

CNRS, GDR Neurosciences

Equipe Mémoire et Récepteurs Serotonine

BP71 13402 Marseille Cédex 20

France

Catherine Belzung, Ph.D.

Assistant Professor

Laboratoire d’Ethologie et de Psychophysiologie

Avenue Monge Parc Grandmont

37200 Tours

France

René Misslin, Ph.D.

Professor

Laboratoire de Psychophysiologie

7 rue de l’Université

67000 Strasbourg

France
59

Mechanisms Regulating

Proenkephalin Gene Expression:

Contributions of Transgenic Models

Steven E. Hyman and David Borsook

INTRODUCTION

Transgenic mouse models can contribute significantly to substance

abuse research in several ways. In this monograph, the use of gene

knockout mice is discussed separately (Hen, this volume). With the

caveat that developmental anomalies and gene compensation may

distort physiology or mask an interesting phenotype, gene knockout

mice are currently one of the most powerful tools available to study

the role of particular proteins in neural function. Transgenic mice

may also be engineered to express dominant negative mutations of

certain proteins; one possible advantage of this approach, where

possible, is that gene inactivation can be targeted to a particular brain

region. Transgenes can be inserted in a normal or a gene knockout

background to study the effects of a mutated gene or of

overexpression or ectopic expression of a particular gene.

Additionally, transgenic strategies can be used to deliver a foreign

protein to a site of interest.

In the research to be described here, a transgenic strategy to study

gene regulation in the brain was used. The authors have followed up

on previous studies of the human proenkephalin promoter that had

utilized transformed cell lines in order to facilitate studies of

transcriptional regulation by physiologically relevant stimuli in

neuronal cell types of interest.

PROENKEPHALIN GENE REGULATION

The proenkephalin gene encodes the precursor of the endogenous

opioid peptides met- and leu-enkephalin. Enkephalin peptides appear

to play roles in a variety of physiological processes including

descending analgesia, regulation of hormone release, the stress

response, and brain reward. Expression of the proenkephalin gene is

highly regulated by synaptic activity, cyclic adenosine

monophosphate (cAMP), and protein kinase C (PKC), with the
60

predominant effect on synthesis being transcriptional. For example,

in bovine adrenal chromaffin cells in primary culture, proenke-phalin

messenger ribonucleic acid (mRNA) levels have been shown to be

increased by nicotinic stimulation (Eiden et al. 1984), cAMP

(Schwartz et al. 1984; Kley et al. 1987), K+- or veratridine-induced

depolarization, calcium ionophores (Kley 1988; Kley et al. 1987), and

PKC (Kley 1988). In C6-glioma cells, proenkephalin mRNA levels

have been shown to be increased by ß-adrenergic receptor stimulation,

which increases intra-cellular cAMP (Yoshikawa and Sabol 1986). In

the brain, chronic treatment with dopamine type 2 (D2) receptor

antagonists, including haloperidol, increases enkephalin peptides and

proenkephalin mRNA in rat striatum (Romano et al. 1987; Sabol et al.

1983; Sivam et al. 1986; Tang et al. 1983). Lesions of the

nigrostriatal dopamine pathway, which block transmission via all

dopamine receptor types, also induce proenkephalin mRNA (Gerfen

et al. 1990, 1991; Jiang et al. 1990; Normand et al. 1988; Voorn et al.

1987; Young et al. 1986). Other neural stimuli that induce

proenkephalin mRNA include splanchnic nerve stimulation in the

adrenal gland (Fischer-Colbrie et al. 1988; Kanamatsu et al. 1986),

electrical stimulation and seizures in the hippocampus (Hong et al.

1980; Morris et al. 1988; White and Gall 1987; Xie et al. 1989;

Yoshikawa et al. 1985), nociceptive stimuli in the dorsal horn (Draisci

and Iadorola 1989), and stressors such as hypertonic saline or

naloxone-precipitated opiate withdrawal in the hypothalamus

(Lightman and Young 1987).

The authors’ group and others have studied the mechanisms by which

the human proenkephalin gene is regulated by cAMP and Ca2+ using

muta-genesis and transfection into transformed cell lines. It was

found that an enhancer comprised of three elements acting in

combination conferred second messenger activation upon the gene.

Within this enhancer, the deoxyribonucleic acid (DNA) regulatory

element that was shown to be absolutely required for regulation of the

proenkephalin gene by cAMP, depolarization, and Ca2+ is the

proenkephalin cAMP response element 2 (CRE-2) element that

contains the sequence TGCGTCA (Comb et al. 1988; Hyman et al.

1988, 1989; Nguyen et al. 1990). This element can bind both

activator protein 1 (AP-1) and CRE binding protein (CREB) in vitro

(Comb et al. 1988; Kobierski et al. 1991; Konradi et al. 1993;

Sonnenberg et al. 1989). To complicate matters, the proenkephalin

promoter can also be activated by both AP-1 proteins (Kobierski et al.

1991; Sonnenberg et al. 1989) and by CREB (Huggenvik et al. 1991)

in cotransfection studies. Both in vitro binding studies (whether by

electro-phoretic mobility shift or by footprinting) and cotransfection
61

studies provide information as to possible transcription factor-target

interactions, but they cannot determine what actually happens in

particular cell types in vivo.

Because gene regulation may differ markedly between transformed

cell lines and neurons, two approaches have been taken. The authors

have begun to study binding of transcription factors to key regulatory

elements in extracts made from neuronal cell types, rather than using

purified proteins from cell lines, and study of promoter function by

transfection into neurons in primary culture and by use of transgenic

mice has begun. The importance of transgenic models is underscored

by several issues. The proenkephalin gene is expressed in a highly

complex pattern in the brain, but appropriate cell culture models for

the multiple neuronal cell types expressing the gene are lacking.

Moreover, transformed cell lines cannot model the connectivity,

neurotransmitter receptors, networks of intracellular signal

transduction pathways, or the precise mixtures of transcription factors

that characterize the different types of enkepha-linergic neurons in

the brain. The use of transgenic animals permits investigation of

whether the same regulatory elements that appear to be utilized in cell

culture models are also utilized in vivo. The use of a betagalactosidase reporter gene in transgenic models has the additional

advantage of simplifying the approach to colocalization of the target

gene of interest with putative regulatory proteins because the

histochemical reaction used to detect beta-galactosidase activity is

highly compatible with immunohistochemistry (Borsook et al.

1994b). A final advantage is that there is no endogenous betagalactosidase activity in eukaryotic cells, permitting very sensitive

detection of proenkephalin gene expression in this transgenic model.

EXPRESSION AND REGULATION OF AN ENKEPHALIN-BETAGALACTOSIDASE FUSION GENE IN TRANSGENIC MICE

The three initial transgenic lines that were made contained a fusion

construct with 3 kilobases (kb) of human proenkephalin gene 5'

flanking sequence, the first exon and intron of the human

proenkephalin gene, the E.coli lac-Z transcription unit fused to the

second proenkephalin exon, and l.2kb of human proenkephalin 3'

flanking sequences (Borsook et al. 1992). All of the independent

lines of mice, ENK l.l, l.2, and l.3, expressed the fusion gene to some

degree. None of the mice had apparent morpho-logic or behavioral

abnormalities. No ectopic expression of the gene product was

observed when the beta-galactosidase expression was compared to the
62

endogenous proenkephalin mRNA by in situ hybridization (Borsook

et al. 1992, 1994a). However, none of the lines had complete

expression of the gene, suggesting that there might be strong

integration effects or that some additional regulatory sequences were

missing. All three of the lines had good fidelity of expression in the

reproductive system (Borsook et al. 1992), and all had some degree of

expression in the brain. The ENK l.2 line had expression only in

regions corresponding to the periaqueductal gray matter but nowhere

else in the brain, and was not further analyzed. ENK l.l and l.3 had

much fuller brain expression including the hypothalamus (Borsook et

al. 1994a). None of the lines, however, had the expected expression

of the transgene in the striatum.

Because proenkephalin gene expression was known to be highly

regulated by pharmacologic and physiologic stimuli in the

hypothalamus (Lightman and Young 1987), and because in the ENK

l.l and l.3 lines basal expression of the transgene in the hypothalamus

recapitulated the pattern of expression of the endogenous gene, a

detailed analysis of regulation of the transgene within the

hypothalamus was undertaken. All further analyses described here

were performed with the ENK l.l line.

REGULATION OF THE TRANSGENE IN THE HYPOTHALAMUS

The paraventricular nucleus (PVN) and supraoptic nucleus (SON) of

the hypothalamus are major sites of integration of the stress response.

The proenkephalin precursor and enkephalin peptides are found at

high levels within these nuclei, where they are thought to play an

important role in the control of release of hypothalamic stress

hormones including cortico-tropin releasing factor (CRF). It has

previously been shown in rat models that certain acute and chronic

stressors produce significant induction of proenkephalin gene

expression within these nuclei. For example, intra-peritoneal (IP)

injection of hypertonic saline and naloxone-precipitated opiate

withdrawal have been shown to produce rapid and significant

inductions of proenkephalin mRNA in the rat PVN (Lightman and

Young 1987). Multiple IP daily injections of hypertonic saline have

been shown to induce proenkephalin mRNA in the rat PVN even

more strongly (Young and Lightman 1992).

Basal expression of the transgene appears as small blue puncta within

cells, which may represent inclusion bodies; with induction, neurons

fill out with the blue beta-galactosidase reaction product (Borsook et
63

al. 1994a). By comparing histochemistry for the beta-galactosidase

reaction product with in situ hybridization for the endogenous

proenkephalin mRNA, the authors’ group found that in the ENK 1.1

line of transgenic mice, basal expression of the transgene and

induction by hypertonic saline injection (figure 1) and naloxoneprecipitated opiate withdrawal closely paralleled expression of the

mouse endogenous proenkephalin gene and the regulation previously

reported for the rat (Borsook et al. 1994a, 1994c; Lightman and

Young 1987). The transgene was also induced in the PVN by other

stressors, such as hypovolemia, cold swim, and lipopolysaccharide

injection. Since the endogenous gene and the transgene contain

similar genomic regulatory sequences but express entirely different

mRNAs, the observation that they are induced in parallel is consistent

with the hypothesis that stress activation of proenkephalin gene

expression in the hypothalamus occurs at the transcriptional level.

CREB INTERACTS WITH THE PROENKEPHALIN GENE IN THE

HYPOTHALAMUS

As described above, both AP-1 proteins and CREB can interact with

the proenkephalin gene in vitro. However, in cell extracts from rat

striatum (Konradi et al. 1993) and mouse hypothalamus (Borsook et

al. 1994b), the authors detected CREB, but not Fos binding to the

enkephalin CRE-2 element (TGCGTCA) using electrophoretic

mobility shift assays with antibody supershifts. CREB binding is

detected even under conditions such as acute stress, which give robust

AP-1 binding to a consensus AP-1 site (TGACTCA). The CRE-2 site

is contained within the regulatory sequences of the transgene, thus the

authors performed combined beta-galactosidase histochemistry with

immunocytochemistry for CREB to determine the localization of

CREB within the PVN with respect to cells expressing the transgene.

CREB protein was found in essentially all neurons within the PVN

(Borsook et al. 1994b). Moreover, no differences in total CREB

protein levels could be observed by immuno-histochemistry in

uninjected, normal saline-injected, or hypertonic saline-injected mice,

consistent with the idea that CREB is constitutively expressed in most

known cell types and is activated by phosphorylation (Gonzalez and

Montminy 1989). The authors then sought to determine whether

hypertonic saline stress induced phosphorylation of CREB on Ser133

in transgene-expressing neurons. The authors therefore colocalized

beta-galactosidase activity with phosphoCREB using an antiserum that

specifically detects CREB phosphorylated on Ser133 (Ginty et al.

1993).
64

Ten minutes after an injection of 0.15 molar (M) saline, only minimal

staining for phosphoCREB was observed in the PVN, indistinguishable

from an uninjected animal. Ten minutes after an injection of 1.5 M

saline, intense phosphoCREB staining was seen throughout the PVN,
65

including all cells observed to express the transgene (Borsook et al.

1994b). Mutational analyses are planned to determine whether the

CREB binding site is required for stress induction of the transgene

within the PVN.

MODULATION OF STRESS-REGULATED PROENKEPHALIN

GENE EXPRESSION BY OPIOIDS

In addition to facilitating studies of promoter function, the sensitivity

of the transgene also permitted detection of novel forms of regulation

of the proenkephalin gene within the hypothalamus. Since the

regulatory sequences within the transgene are known, studies of the

molecular mechanisms responsible for this regulation will be possible.

Two decades ago, researchers hypothesized that chronic morphine

treatment might result in agonist-induced feedback inhibition of

endogenous opioid biosynthesis and that this suppression of

endogenous opioid peptides might contribute to aspects of opioid

dependence and withdrawal (Hughes et al. 1975; Waterfield et al.

1976). Early studies designed to test this hypothesis found little

change in steady-state brain enkephalin peptide levels. However, these

studies did not address turnover rate or changes within restricted

populations of cells. More recently, opioid agonists have been shown

to decrease (Uhl et al. 1988) and opioid antagonists to increase

(Tempel et al. 1992) expression of proenkephalin mRNA or

enkephalin peptides in some cell types, suggesting a direct or indirect

opioid receptor-mediated effect on endogenous opioid gene

expression.

Using the authors’ mouse model, it was found that chronic morphine

administration dramatically suppresses transgene expression

compared with animals that received either placebo pellets or no

pellets. The chronic morphine regimen consisted of 8 milligram (mg)

morphine pellets inserted subcutaneously (SC) for 3 days, followed by

25 mg pellets for 4 days. Control animals received either placebo

pellets SC for 7 days or were unoperated. The suppression of

transgene expression by chronic morphine administration was to

almost unobservable levels, and was seen throughout the

hypothalamus (Borsook et al. 1994c). Animals injected with

morphine (10 mg/kg) or normal saline IP every 6 hours for 5 days

displayed a level of suppression of transgene expression that was

similar to the animals that received morphine pellets. There were no

differences between the placebo pellet and the unoperated conditions.
66

Chronic (7 days) administration of naltrexone pellets (10 mg or 25

mg) SC had minimal effects on transgene expression (Borsook et al.

1994c). While there was some slight increase in the intensity of

puncta, there was no filling of cells with the beta-galactosidase

reaction product, the indicator of substantial induction of the

transgene.

Surprisingly, administration of morphine (10 mg/kg IP) 4 hours prior

to a hypertonic saline stressor (acute condition) or administration of a

morphine pellet (8 mg) for 24 hours prior to a hypertonic saline

stressor (subacute condition) produced a marked enhancement of the

already substantial stress-induced increase in transgene expression

within the PVN and SON (Borsook et al. 1994c). In addition, regions

of the hypo-thalamus, in which transgene expression is not induced

by hypertonic saline stress alone, showed marked induction of the

transgene when stress followed morphine pretreatment. These areas

include the preoptic region, the nucleus circularis, the lateral

hypothalamus, the ventromedial nucleus, and the retrochiasmatic SON

(Borsook et al. 1994c). Thus, acute or sub-acute morphine

administration markedly sensitizes restricted populations of

proenkephalin-expressing hypothalamic neurons to stress. Chronic

opioid pretreatment (7 days) prior to hypertonic saline stress inhibits

this response, suggesting that desensitization has occurred.

Pretreatment with naltrexone inhibits stress induction of transgene

expression below the expected level observed in mice receiving only

hypertonic saline injections (Borsook et al. 1994c).

In this series of experiments, the authors’ transgenic model was used

to examine the effects of acute, subacute, and chronic opioid

administration on stress regulation of proenkephalin gene expression

within the hypo-thalamus. The advantages of the transgenic model

are that it is more sensitive and more readily quantified than in situ

hybridization and that genomic sequences responsible for the

observed regulation are known to be contained within the transgene,

making subsequent analysis of transcriptional mechanisms possible.

These results, which were confirmed by in situ hybridization for the

endogenous mRNA (Borsook et al. 1994c), are consistent with the

hypothesis that acute or subacute administration of morphine

sensitizes enkephalinergic hypothalamic neurons to stress. The use of

a morphine pellet in the subacute paradigm makes it unlikely that the

superinduction is due to mild, behaviorally unobservable withdrawal

acting along with the saline stress. Moreover, administration of
67

opioids chronically, using a paradigm that produced marked

dependence (as demonstrated by the possibility of producing

naloxone-precipitated withdrawal) not only suppressed basal

expression, but also inhibited stress-induced expression of the

transgene.

It was initially surprising that acute or subacute morphine

pretreatment prior to a hypertonic saline stressor superinduces

transgene expression. Morphine has principally inhibitory effects on

target neurons mediated by G-protein inhibitory (Gi)-linked opiate

receptors, especially mu receptors. As noted by Akil (this volume),

there are relatively few mu receptors expressed on neurons in the

PVN, although there are relatively high levels of kappa opioid

receptors. These observations make it unlikely that the effects of

morphine on PVN neurons are direct or cell autonomous, but the

effects likely involve intermediary circuitry.

SUMMARY

This chapter gives an overview of the utility of one type of transgenic

mouse model for substance abuse research, the use of transgenes in

which genomic regulatory sequences of interest are coupled to a

reporter gene. Using this mouse model, exploration of the

mechanisms regulating expression of the proenkephalin gene in the

mouse hypothalamus have begun. The resulting information

supplements experiments on gene regulation performed in cell lines

with information about regulation by pharmacologic and physiologic

stimuli of interest within the brain. With special reference to

mechanisms of opioid dependence, the particular model that has been

examined appears to be a useful tool to investigate mechanistic aspects

of the regulation of endogenous opioid genes by exogenous opioids.

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AUTHORS

Steven E. Hyman, M.D.

David Borsook, Ph.D., M.D.

Harvard Medical School

and

Laboratory of Molecular and Developmental Neuroscience

Massachusetts General Hospital, CNY-2

149 13th Street

Charlestown, MA 02129
72

Human Opioid Receptors:

Chromosomal Mapping and mRNA

Localization

Karen Miotto, Daniel Kaufman, Benito Anton,

Duane E. Keith,Jr., and Chris J. Evans

INTRODUCTION

The opioid system modulates complex processes such as

neuroendocrine function, analgesia, immunity, and cardiovascular

regulation. A challenge in the field of neuroscience has been the

molecular characterization of the opioid receptors. The recent

isolation and sequencing of the genes and cDNAs encoding the

opioid receptors permit studies of structure, function, and the cellular

localization of these receptors. This chapter describes recent studies

on the characterization and chromosomal mapping of the human

opioid receptor genes and the localization of human opioid receptor

messenger ribonucleic acid (mRNA) in the human brain.

MULTIPLE OPIOID RECEPTORS

Pharmacological studies using opioid peptides and alkaloid ligands

have identified several classes of opioid receptor including the mu (μ),

delta (d), and kappa (k) (Lord et al. 1977), (Martin et al. 1976). The

first opioid receptor cloned was the mouse d receptor reported

simultaneously by Evans and colleagues (1992) and Kieffer and

colleagues (1992). This receptor was isolated by ligand screening of

eukaryotic expression libraries. Subsequently the rat μ and d

receptors were isolated by low stringency hybridization using probes

derived from the d receptor cDNA (Chen et al. 1993; Fukuda et al.

1993). The mouse k receptor cDNA was isolated by polymerase

chain reaction (PCR) using degenerate oligo-nucleotide primers to the

structurally similar somatostatin receptor (Yasuda et al. 1993).

Multiple similar cDNA clones for the μ, d, and k opioid receptors

from several species have since been identified. Although subtypes

for all three classes of opioid receptors have been proposed based on

pharmacological studies, thus far only a single member from each

class of opioid receptor has been cloned (Mattia et al. 1992; Pasternak

1986; Xu et al. 1991; Zukin et al. 1988). The fact that only one gene
73

locus was identified for each class of opioid receptors suggests that the

subtypes identified by pharmacological studies may result from

posttranslational, splicing modifications or differential interactions

with associated proteins.

The cloning and sequencing of the opioid receptors also verified that

these receptors belonged to the family of guanosine triphosphate

binding protein (G-protein) receptors. G-protein receptors share

some structural features such as seven alpha-helical hydrophobic

domains that form the membrane spanning regions and methods of

signal transduction that involve activation of specific G-proteins

(Lameh et al. 1990; Strosberg 1991). It is the membrane-spanning

regions and cytoplasmic loops that share the greatest sequence

similarities between the classes of opioid receptors. The conservation

of these regions within the family of opioid receptor genes may

suggest overlapping functional and structural units of the opioid

receptors (Douglass et al. 1984; Hershey et al. 1991; Traut 1988).

Several investigators have recently identified an additional G-protein

receptor in rat (Bunzow et al. 1994; Chen et al. 1994; Fukuda et al.

1994; Wang et al. 1994a) and human brain (Keith et al. 1994;

Mollereau et al. 1994) that has a high homology to the opioid

receptor gene family. Although this receptor is not a μ, d, or k

subtype, it shares significant homology to these opioid receptors at the

amino acid level, particularly in the regions of the cytoplasmic loops

and transmembrane domains. The abundant distribution of this

receptor in structures of the hypothalamic-pitutary axis and cortical,

spinal, and limbic nuclei suggests a role in neuroendrocrine function

and the mediation of pain perception and analgesia (Bunzow et al.

1994; Keith et al. 1994; Wang et al. 1994a). Unfortunately, the lack

of identification of the endogenous ligand for this receptor has

delayed further characterization of the role of this relative of the

opioid receptor family.

MOLECULAR CLONING AND GENE STRUCTURE

The genes for the human μ, d, and k opioid receptors have been

isolated. The initial murine d opioid receptor cDNA was used by the

authors to screen a human genomic phage library to identify clones

containing the human genes. The authors subcloned μ, d, and k

receptor fragments and sequence analysis of the three revealed the

coding regions for the human opioid receptors. The human μ

receptor gene shares 95 percent amino acid identity with the mouse
74

and rat μ receptors (Wang et al. 1994b). Three introns interrupt the μ

receptor gene. The first two are located in the first and third

intercellular domains. A third intron is located close to the carboxy

tail of the receptor at amino acid position 397. Alternative splicing

has been postulated at this site, which may result in a different

carboxy terminus (Zimprich et al. 1994).

Analysis of the sequence of the human d gene fragment revealed 96-

percent amino acid identity over 120 bases between the human

genomic clone and the murine receptor cDNA (Selleri et al., in

process). Knapp and coworkers (1994) also cloned a human d opioid

receptor cDNA that encodes a 372 amino acid protein with 93 percent

amino acid identity to the mouse d receptor. Partial sequence analysis

by the authors of the human k gene showed a 100 percent DNA

homology with the human sequence reported by Mansson and

coworkers (1994). The introns in the murine k opioid receptor gene

correspond to amino acid positions 86 and 184 (Yasuda et al. 1993).

There appears to be conser-vation of these intronic positions in the

human and rodent k, d, and μ opioid receptor genes (Mansson et al.

1994).

The opioid receptors, rhodopsin, the dopamine type 2 (D2)-like

receptor, and the substance P receptors are a few among the many Gprotein receptors reported to have introns within the coding region

(Gingrich and Caron 1993; Hershey et al. 1991). In these receptors

the intronic division within the coding region is similar, occurring at

or near the membrane-spanning domain border. In each of the three

opioid receptors the introns are found just beyond the first

transmembrane spanning (TMS) region in the first intercellular loop

and just beyond the fourth TMS region in the second cytoplasmic

loop. The exception is the μ receptor, which also contains an

additional intron near the carboxy terminus. The position of the

introns in the human genes matches the position described for the

rodent opioid receptors genes, which suggests stong evolutionary

conservation of the intron/exon structure. The presence of introns

and their precise location may be useful in defining functional units

of the opioid receptors and in understanding the evolutionary

divergence of the G-protein receptor genes.

Genetic regulation of opioid receptors is of great interest due to the

role of the opioid system in syndromes believed to have heritable

components such as pain response, metabolism of opiates, and

addiction. Several groups of investigators are studying polymorphic

markers for the opioid receptors. Two different types of
75

polymorphisms have been identified for the human μ receptor: a

dinucleotide repeat and a restriction site polymorphism. PCR

technology coupled with the identification of variable nucleotide

repeats throughout the human genome has made it possible to rapidly

type a particular allele. Only a fragment of the authors' clone of the μ

receptor was found to contain a region containing a cytosineadenosine (CA) dinucleotide repeat. PCR amplification of this region

of DNA from 36 caucasian individuals reveals a significant degree of

polymorphism (Selleri et al., in process). Wang and coworkers

(1994b) also identified an Msp 1 polymorphism, which is under study

to identify allelic variants associated with disorders of the opioid

system.

CHROMOSOMAL LOCALIZATION OF OPIOID RECEPTORS

The chromosomal localization of the human μ, d, and k opioid

receptor genes was determined by direct fluorescent in situ

hybridization using the lambda phage DNA as probes to human

metaphase chromosome preparations. Using this technique, the d

receptor gene was mapped to chromosome 1p355-33, the k gene to

chromosome 8q11.23-21, and the μ gene to chromosome 6q25-26

(Selleri et al., in process). Other investigators have also mapped the

human opioid receptors to these locations (Befort et al. 1994; Wang et

al. 1994b; Yasuda et al. 1994).

Each of the opioid receptors map to a single locus that apparently is

not near receptors in which a mutation is suspected to cause any

known human genetic disease. The chromosomal locations of the

human μ, d, and k are in synteny with the reported position on mouse

chromosomes (Selleri et al., in process). Southern analysis of murine

opioid receptor genes in mouse neurogenetic mutants has not revealed

gross alterations in the opioid receptor DNA (Befort et al. 1994;

Kaufman et al. 1994). However, the information from polymorphic

and chromosome markers will contribute to understanding the genetic

regulation of the opioid receptors (Berrettini et al. 1994). Multiple

familial and acquired diseases have been linked to abnormalities in Gprotein receptors and the associated regulatory enzyme systems

(Clapham 1993; Emala et al. 1994). Since the cloning of the opioid

and other G-protein receptors has occurred quite recently, it is likely

that additional diseases will soon be found that are linked to mutations

or to regulatory alteration in these receptors.
76

LOCALIZATION OF OPIOID RECEPTOR mRNA

The field of opioid receptor biology has been advanced by

anatomical studies of receptor distribution. Radiographic analysis

with specific ligands revealed a unique neuroanatomical pattern for

the μ, d, and k opioid receptors. With the cloning and isolation of the

opioid receptors, direct analysis of the location of cells synthesizing

opioid receptors can be made. A comparison of the correspondence

between the binding data and the mRNA localization will provide

insights into the trafficking and neural localization of these receptors.

The authors examined the distribution of the opioid receptor mRNA

in the human prefrontal cortex and the striatum (Anton et al. 1994)

and found that the patterns of distribution and expression level of the

opioid receptor mRNA correlates well with the opioid receptor

binding sites reported by autoradiography (Quirion and Pilapil 1991).

In the prefrontal cortex, there is a high density of cells expressing μ

opioid receptor mRNA found in cortical layers II and IV-V, and a

moderate level of expression in layers I, III, and VI. Mu binding sites

are the most selective for interaction with morphine and naloxone and

they are also the most abundant and globally distributed opioid

receptor in the human brain. The μ binding sites are concentrated in

the superficial cortical laminae.

Delta opioid receptor mRNA was found in cells distributed homogeneously throughout all cortical laminae, although layers II-III and

V-VI contained more densely labeled cells. Overall, the distribution

of the d opioid receptor binding sites is similar to the μ receptor. In

contrast, the k opioid receptor mRNA in the human prefrontal cortex

is abundant in deep cortical laminae IV-VI. High levels of k opioid

autoradiographic binding sites have also been reported within deeper

cortical layers in this area, as well as other neocortical fields of human

brain (Maurer et al. 1983; Pilapil et al. 1987; Quirion and Pilapil

1991). This localization in the deeper cortical layers has been

postulated to induce the sedative properties of k agonists (Goodman

and Snyder 1982).

The caudate-putamen is a central structure for the flow of information

from the cortex to the ventral tegmental area and to the substantia

nigra. Circuits involving these brain areas have been implicated in

sensory-motor interaction, reward, and motivation. Moderate to high

levels of hybridization signals were found in human caudate-putamen

for μ, d, and k opioid receptor mRNAs (see table 1), although the

density of μ opioid
77

TABLE 1. Opioid receptor mRNA distribution in human brain.

Region Delta Mu Kappa

Prefrontal Cortex

Layers

 I 0 to low mod 0

 II mod high mod

 III high low mod

 IV mod mod high

 V high high high

 VI high high high

Striatum

Caudate nucleus

 (interno-medial) low to

mod*

high* mod to

high**

Putamen low to

mod*

high* high**

NOTE: 0, low, mod, and high indicate the relative density of cells

expressing receptor mRNA.

KEY:0 = undetectable; mod = moderate; * = diffuse; ** =

asymmetric cell clusters.

receptor was higher than for the d and k receptors. Cells containing

μopioid receptor mRNA were homogeneously distributed throughout

the human caudate-putamen. This homogeneous pattern of μ binding

sites at the striatal level was also seen in human autoradiographic

studies. These studies of the μ receptor highlight interesting species

differences in the localization of opioid receptors. In the caudateputamen, certain receptors and neurotransmitters preferentially bind

the patch or matrix compart-ments. In the rat, guinea pig, and

monkey the densest binding is observed for the μ receptor in the

patches, and this neuroanatomical segregation is not seen in the

human striatum (Herkenham and Pert 1981; Mansour et al. 1991).

Cells containing d and k opioid receptor mRNA were also found in

the human caudate-putamen, though the levels of the d receptor were

lower and more homogeneously distributed. A similar low density of

d autographic binding sites was reported by Pilapil and coworkers

(1987). Densely labeled k opioid receptor cells were grouped in

clusters predomi-nantly in the caudate. These clusters of cells
78

expressing k opioid receptor mRNA suggests the patchlike striatal

distribution. In support of these findings is the same striosomal

compartmentalization of k opioid receptor binding sites in human

caudate-putamen determined by the autoradio-graphic studies of

Quirion and Pilapil (1991). To confirm the striosomal localization of

k opioid receptor mRNA, additional histochemical studies are in

progress using the combination acetylcholinesterase histochemistry

and opioid receptor in situ hybridization in adjacent sections.

Prior to recent advances in the molecular characterization of the

opioid receptors, the definition of classes of opioid receptors was

made based on the order of receptor agonist and antagonist potency.

The cloning of the opioid receptors has clearly identified a single

gene for each of the human μ, d, and k opioid receptors. Conserved

introns with the coding regions of these receptors may represent

distinct functional domains. Conservation of the genomic structure

suggests that there are overlapping functional and structural units, and

perhaps evolution from a common ancestral gene. Conserved introns

with the coding regions of these receptors may represent distinct

functional domains. The question of subtypes remains, but the

identification of an orphan opioid receptor promises interesting

develop-ments on the horizon. The study of opioid receptor mRNA

in the human establishes the foundation for further analysis of the

precise distribution and mechanism of regulation of opioid receptor

expression. Ideally, the analysis of the chromosomal localization will

lead to the development of tools for the diagnosis and treatment of

clinical syndromes associated with the opioid system.

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ACKNOWLEDGMENTS

This study was funded by NIDA grant no. DA05010 and by a grant

from the W.M. Keck Foundation.

Tissue specimens were obtained from the National Neurological

Research Specimen Bank, VAMC Wadsworth Division, Los Angeles,

CA 90073, which is sponsored by NINDS/NIMH; the National

Multple Sclerosis Society; the Hereditary Disease Foundation; the

Comprehensive Epilepsy Program; the Tourette Syndrome

Association; the Dystonia Medical Research Foundation; and the

Veterans Health Service and Research Administration, Department of

Veterans Affairs.

AUTHORS

Karen Miotto, M.D.

Assistant Professor

Daniel Kaufman, Ph.D.
82

Assistant Professor

Benito Anton, M.D.

Research Scientist

Duane E. Keith, Jr., B.A.

Researcher

Chris J. Evans, Ph.D.

Professor

Department of Psychiatry and Biobehavioral Science

UCLA Neuropsychiatric Institute

760 Westwood Plaza

Los Angeles, CA 90024-1759
83

Molecular Biology of Opioid

Receptors

Karen Raynor, Haeyoung Kong, Susan Law, Jennifer

Heerding, Melanie Tallent, Fredrick Livingston, John

Hines, and Terry Reisine

INTRODUCTION

Morphine and its alkaloid derivatives are used extensively in the

treatment of pain (Gilman et al. 1990). These compounds are the

most potent class of analgesics used clinically. The high potency and

specificity of morphine suggest that it may bind to specific receptors

in the nervous system to induce its biological effects. In the early

1970s, several groups of researchers identified specific opioid

receptors in brain and peripheral tissues (Pert and Snyder 1973;

Simon 1991). While these receptors were highly sensitive to

morphine, morphine is not endoge-nously expressed in the body and

therefore could not be the endogenous ligand for these receptors.

This led to the search for the endogenous neurotransmitters at the

opiate receptors and, in the mid-1970s, several groups identified the

enkephalins (Hughes et al. 1975), dynorphins (Goldstein et al. 1979),

and beta-endorphin (Bradbury et al. 1975) as endogenous peptide

ligands for these receptors.

Both the enkephalins and dynorphins are neurotransmitters in the

brain involved in pain perception, cognitive functions, affective

behaviors, and locomotion, and they are involved in the central

control of certain endocrine functions such as water balance (Herz

1993; Jaffe and Martin 1990; Simon 1991). Both peptides are widely

distributed in the central nervous system (CNS) but localized to

discrete neuronal pathways (Khachaturian et al. 1983). Betaendorphin is expressed at much lower levels in the brain and only

synthesized in a few neuronal pathways in the CNS, in particular in

those originating from hypothalamic nuclei. As a result, the

enkephalins and dynorphins are considered the predominant central

opioid peptide transmitters.

The opioids induce their biological effects by interacting with three

major classes of receptors: the delta, kappa, and mu receptors (Herz

1993). Delta opioid receptors express low affinity for dynorphin, but
84

high sensitivity to enkephalin, indicating that enkephalin may be an

endogenous ligand for this receptor. In contrast, dynorphin A is very

potent at kappa receptors, whereas these receptors have low affinity

for enkephalin, suggesting that dynorphin A may be the endogenous

transmitter for the kappa receptor. The mu opioid receptor expresses

high affinity for enkephalin and the distribution of mu receptors and

enkephalin messenger ribonucleic acid (mRNA) are highly correlated,

suggesting that enkephalin may interact with mu receptors under

physiological conditions (Delfs et al. 1994). Beta-endorphin binds

potently to both the delta and mu receptor but has relatively lower

affinity at kappa receptors, suggesting that in peripheral tissues, where

endorphin is more abundant than enkephalins or dynorphin, betaendorphin may be an endogenous ligand for the delta and mu

receptors.

The pharmacological properties of the three opioid receptor classes

are distinct and can be clearly differentiated (Goldstein and Naidu

1989; Herz 1993; Lutz and Pfister 1992; Portoghese 1993; Schiller

1993; Simon 1991). Delta opioid receptors have high affinity for the

agonists deltorphin II, [D-penicillamine2, D-penicillamine5]

enkephalin (DPDPE),[D-Ser2,O-Leu5]-enkephaly-Thr (DSLET), and

the antagonist naltrindole. These compounds essentially do not bind

to the kappa or mu receptors. The kappa receptor has high affinity

for the agonists U50,488 and U69,593 and the antagonist norbinaltorphimine (nor-BNI). These agonists do not bind to the delta or

mu receptor and the antagonist is much less potent at these other

opioid receptors. Mu opioid receptors have high affinity for the

peptide agonist [D-Ala2-MePhe4, Gly-ol 5] enkephalin (DAMGO),

morphine and its derivatives, and the antagonists [D-Phe-Cys-Tyr-DTrp-Orn-Pen-Thr-NH2

] (CTOP) and naloxonazine. DAMGO and the

antagonists do not bind to delta or kappa receptors, and morphine and

its derivatives are much less potent at the delta or kappa receptors. All

three opioid receptors are sensitive to the antagonist naloxone,

although to varying degrees. Kappa and mu receptors have high

affinity for naloxone, while delta receptors have lower affinity for this

antagonist.

The development of pharmacological agonists and antagonists that

can distinguish the opioid receptors has facilitated investigations into

their distinct functional roles. Such agents were necessary because

most tissues express more than one opioid receptor type, which has

made it difficult to study each receptor individually to reveal any

unique biological actions. This problem has now also been overcome

by the recent cloning of the three opioid receptor classes, since each
85

receptor can now be expressed in a separate cell line and their

pharmacological, functional, and biochemical characteristics can be

studied independent of the other receptors.

Cloning of the Opioid Receptors

The first opioid receptor to be cloned was the delta receptor. Two

groups, Evans and colleagues (1992) and Kieffer and colleagues

(1992), simultaneously identified the delta receptor from cDNA

libraries derived from NG-108 neuroblastoma cells by expression

cloning. Using the fact that opioid and somatostatin receptors have

some similar pharmacological properties, Yasuda and associates

(1993) cloned the kappa receptor as well as the delta receptor by

screening a mouse brain cDNA library with probes selective for the

cloned somatostatin receptors. The probes were directed against

transmembrane spanning regions of the somatostatin receptors, which

are conserved between the somatostatin and opioid receptors.

Similarly, the rat kappa receptor cDNA has been cloned and its

predicted amino acid sequence is almost identical to the mouse

receptor (Meng et al. 1993; Minami et al. 1993). At approximately

the same time as the initial cloning of the mouse kappa receptor, Chen

and colleagues (1993) cloned the mu opioid receptor using probes

against conserved regions of the delta opioid receptor to screen a rat

brain cDNA library. This was possible because of the high degree of

amino acid sequence similarity between the delta and mu opioid

receptors, in particular in the transmembrane spanning regions, which

is where the probes of Chen and associates (1993) were directed.

Others (Thompson et al. 1993; Wang et al. 1993) have cloned the rat

mu opioid receptor with similar if not identical amino acid sequences

to those reported by Chen and colleagues (1993).

The predicted amino acid sequences of the three opioid receptors are

shown in figure 1. They are approximately 65 percent identical with

highest similarity in the transmembrane spanning regions and intracellular loops. Regions that diverge the most are the N- and C-termini

and the extracellular loops. All three receptors have the aspartatearginine-tyrosine (DRY) sequence and aspartates in the second and

third transmembrane spanning regions that are conserved among

guanosine triphosphate binding protein (G-protein)-linked receptors.

Each receptor has multiple potential glycosylation sites in its Nterminal region. It is likely that each receptor has carbohydrate

moieties associated with at least one of these sites since the size of the

native receptors identified from biochemical studies is considerably

larger than the size predicted
86
87

from the amino acid sequence of each cloned receptor. In fact,

studies to covalently cross-link the cloned delta receptor expressed in

CHO cells with 125I-beta-endorphin reveals the size of the receptor as

55 to 60 kilodaltons (kDa), which is larger than the predicted size of

41 kDa (Raynor and Reisine, unpublished observations).

Furthermore, the labeled receptor appears as a smear following SDSPAGE and autoradiography, which is consistent with the glycoprotein

nature of this receptor. Mutagenesis studies to knock out the glycosylation sites in these receptors may reveal the functional role of the

carbohydrates on these receptors.

The three opioid receptors have high similarity to somatostatin

receptors, particularly SSTR1, with approximately 40 percent amino

acid sequence similarity (Reisine and Bell 1993; Yasuda et al. 1993).

They have very low similarity with all other receptors. The similarity

with somatostatin receptors is consistent with the pharmacological

overlap of these receptors since some somatostatin analogs, including

the clinically employed peptide SMS-201-995, which binds with

moderate potency to mu opioid receptors (Maurer et al. 1982), and

the extremely potent and selective mu antagonist CTOP, were

developed using the structure of SMS-201-995 as its basis (Pelton et

al. 1985).

PHARMACOLOGICAL AND FUNCTIONAL PROPERTIES OF THE

CLONED OPIOID RECEPTORS

Following expression of the cloned receptors in COS-7, PC12, or CHO

cells, the pharmacological and functional properties of these receptors

were investigated (Kong et al. 1993a; Raynor et al. 1994a, 1994b;

Reisine and Bell 1993; Yasuda et al. 1993). The pharmacological

properties are described in table 1. The endogenous peptides, the

enkephalins, potently bound to the cloned delta and mu receptors.

Kappa receptors have very low affinity for these compounds. In

contrast, dynorphin A potently bound to the kappa receptor, but it was

less potent at the mu receptor and did not interact with the delta

receptor. Both the delta and mu receptors have high affinity for betaendorphin, whereas kappa receptors were relatively insensitive to this

peptide. The binding of beta-endorphin to the receptors was

dependent on the presence of the N-terminal tyrosine residue,

consistent with the opioid nature of the receptors. The affinities of the

cloned receptors for the endogenous peptide transmitters were similar

to those reported for the native opioid receptors.
88

TABLE 1. Binding potencies (Ki-nM) of ligands for the cloned k, d, and μ opioid receptors.

 Receptor

[3H]U-69,593

 Receptor

[3H]naltrindo

le

μ Receptor

[3H]DAMGO

Nonselective Compounds

Dynorphin A 0.5 >1,000 32

Leuenkephalin >1,000 4.0 3.4

Metenkephalin >1,000 1.7

0.65

ß-endorphin 52 1.0 1.0

des-Tyr1-ß endorphin >1,000 >1,000 >1,000

(-)naloxone 2.3 17

0.93

(+)naloxone >1,000 >1,000 >1,000

Levorphanol 6.5 5.0

0.086

Dextrorphan >1,000 >1,000 >1,000

(+)bremazocine 0.089 2.3

0.75

Ethylketocyclazocine 0.40 101 3.1

Etorphine 0.13 1.4

0.23

Pentazocine 7.2 31 5.7

Diprenorphine 0.017

0.23 0.072

ß-FNA 2.8 48

0.33

Naltrexone 3.9 149 1.0

Nalbuphine 39 >1,000 11

 Nalorphine 1.1 148

0.97
89

TABLE 1. Binding potencies (Ki-nM) of ligands for the cloned k, d, and μ opioid receptors 

(continued).

 Receptor

[3H]U-69,593

 Receptor

[3H]naltrindole μ Receptor

[3H]DAMGO

Mu-Selective

Compounds

CTOP >1,000 >1,000 0.18

Demorphin >1,000 >1,000 0.33

Methadone >1,000 >1,000 0.72

DAMGO >1,000 >1,000 2.0

PLO17 >1,000 >1,000 30

Morphiceptin >1,000 >1,000 56

Codeine >1,000 >1,000 79

Fentanyl 255 >1,000 0.39

Sufentanil 75 50 0.15

Lofentanil 5.9 5.5 0.68

Naloxonazine 11 8.6 0.054

Morphine 538 >1,000 14
90

TABLE 1. Binding potencies (Ki-nM) of ligands for the cloned k, d, and μ opioid receptors

(continued).

 Receptor

[3H]U-69,593

Receptor

[3H]naltrindole μ Receptor

[3H]DAMGO

Kappa-Selective

Compounds

NorBNI 0.027 65 2.2

Spiradoline 0.036 >1,000 21

U-50,488 0.12 >1,000

>1,000

U-69,593 0.59 >1,000 >1,000

ICI 204,488 0.71 >1,000 >1,000

Delta-Selective

Compounds

DPDPE >1,000 14 >1,000

D-Ala2-deltorphin II >1,000 3.3 >1,000

DSLET >1,000 4.8 39

BW 3734 17 0.013 26

DADL >1,000 0.74 16

SIOM >1,000 1.7 33

Naltrindole 66 0.02 64

NTB 13 0.013 12

BNTX 55 0.66 18
91

Binding to the cloned receptors was stereospecific. The opioid

antagonist (-) naloxone bound much more potently to the receptors

than the (+)isomer. Similar selectivity was observed with the agonist

levorphanol versus its inactive stereoisomer dextrorphan.

Kappa receptor selective agonists specifically bound to the cloned

kappa receptor. Furthermore, the antagonist nor-BNI is one

hundredfold selective for the kappa receptor. The pharmacological

profile of the kappa receptor is consistent with it being a kappa1

subtype defined previously in studies on the endogenously expressed

kappa receptors (Clark et al. 1989).

Mu receptor selective agonists and antagonists specifically bound to

the cloned mu receptor. Furthermore, many of the clinically used

opioids, such as morphine, methadone, codeine, and compounds with

high abuse potential such as fentanyl specifically bound to the cloned

mu receptor. This finding suggests that the therapeutic as well as the

side effects of physical dependence and respiratory depression

induced by these compounds are due exclusively to their interaction

with mu receptors.

A comparison of the potencies of a large series of opioids at binding

to the cloned kappa and mu receptors with their potencies at binding

to the endogenously expressed receptors reveals a very high

correlation. This indicates that the ligand specificities of the cloned

and endogenously expressed receptors are similar. This finding is of

interest considering that the artificial cellular environment of the cell

membrane where the cloned receptors are expressed would be

expected to be quite different from the environment of the

endogenously expressed receptor.

In contrast to the kappa and mu receptors, little similarity exists in the

pharmacological profile of the cloned delta receptor compared to the

endogenously expressed delta receptors. One clue that may explain

this disparity is the much higher affinity of the cloned receptor for the

antagonist NTB (the benzofuran analog of naltrindole) than the

antagonist 7-benzylidenenaltrexone (BNTX). These compounds have

been described as subtype selective, with BNTX selectively binding to

the delta1 receptor subtype and NTB selectively interacting with the

delta2 subtype (Portoghese 1993; Portoghese et al. 1993; Sofuglu et

al. 1991a, 1991b). Furthermore, the delta2 selective agonists DSLET

and deltorphin II are more potent at binding to the cloned receptor

than the delta1 selective agonist DPDPE. These findings suggest that

the cloned receptor may correspond to the delta2 receptor subtype.
92

Since the radioligands that have been used to examine native delta

receptors in brain do not distinguish the subtypes, the

pharmacological profile of the endogenously expressed delta receptor

may consist of a mixture of potencies at the two delta receptor

subtypes, whereas the findings on the cloned receptor may represent

the ligand selectivities of only one of the subtypes. If this is the case,

the findings suggest that either one more delta receptor subtype

remains to be cloned, or subtypes expressed endogenously in the

nervous system and peripheral tissues are created by differential

processing of a common gene product.

Following expression in cell lines, all three cloned opioid receptors

have been found to couple to pertussis toxin-sensitive G-proteins and

to mediate the inhibition of adenylyl cyclase activity by opioids (Chen

et al. 1993; Kong et al. 1993a; Reisine and Bell 1993; Yasuda et al.

1993). Furthermore, the cloned kappa receptor has been reported to

couple to a Ca++ channel and can mediate agonist inhibition of an Ntype Ca++ current (Tallent et al. 1993). Kappa receptors in the brain

have been reported to couple to N-type Ca++ currents in neurons

(Gross and MacDonald 1987; North 1993; Weisskopf et al. 1993),

and this interaction may be important for the ability of kappa agonists

and dynorphin to presynap-tically inhibit neurotransmitter release.

All three opioid receptors have been reported to couple to Ca++ and K+

channels and can mediate the modulation of ionic conductance

through these channels via pertussis toxin-sensitive G-proteins (North

1993).

Neurotransmitter receptors have been proposed to couple to Gproteins via their intracellular loops (Dohlman et al. 1988). The

intracellular loops of the opioid receptors are very similar, suggesting

that they may couple to similar G-proteins, consistent with the similar

effector systems they regulate. However, the C-termini of the

receptors differ significantly in amino acid sequence, and for some

neurotransmitter receptors the C-terminus has been shown to be

critical for G-protein and effector system coupling. This has been

most clearly shown in the case of the prostaglandin receptors (Breyer

et al. 1994; Namba et al. 1993) and one of the somatostatin receptors,

SSTR2 (Reisine et al. 1994), in which alternative splicing creates two

receptors with divergent C-termini and differing abilities to couple to

adenylyl cyclase. Direct studies to identify the G-proteins and

effector systems associated with each cloned opioid receptor will be

critical in establishing the cellular mechanism of action of opioids

acting via each receptor type.
93

Structure-Function Analysis of the Cloned Opioid Receptors

The cloning of the opioid receptors has now allowed for analysis of

the structural elements in the receptors involved in their functioning.

Many G-protein-linked receptors can be regulated by sodium ions

(Horstman et al. 1990; Kong et al. 1993b; Kosterlitz et al. 1988;

Limbird et al. 1982; Minuth and Jakobs 1986; Neve et al. 1990; Pert

and Snyder 1974). In particular, receptors that mediate agonist

inhibition of cyclic adenosine monophosphate (cAMP) formation can

have their affinity for agonists reduced in the presence of sodium

ions. Sodium ions were first reported to reduce the affinity of opioid

receptors for agonists (Kosterlitz et al. 1988; Pert and Snyder 1974).

To investigate the mechanisms by which Na+ regulates agonist binding

to alpha2-adrenergic receptors, Horstman and colleagues (1990)

mutated an aspartate in the second transmembrane spanning region of

the cloned alpha2-adrenergic 2a receptor to an asparagine and found

that Na + regulation of agonist binding was abolished. Similar results

were obtained with a mutation of the conserved aspartate in the second

transmembrane spanning region of the somatostatin receptor subtype

SSTR2 (Kong et al. 1993b). For both of these receptors few changes

in ligand binding properties were reported. These studies indicated

that a conserved aspartate in the second transmembrane spanning

region of several G-protein-linked receptors was necessary for Na+

regulation of agonist binding.

To investigate the mechanisms by which Na+ modulates opioid receptor

properties, Kong and associates (1993a) mutated aspartate 95 in the

cloned mouse delta opioid receptor to an asparagine (see figure 2), and

like the adrenergic and somatostatin receptors found that Na+ regulation

of agonist binding was lost, indicating that this residue was necessary for

the modulatory actions of Na+ on the delta receptor. However, in contrast

to the results reported with the other receptors, the ligand binding

properties of the mutant delta receptor were also altered. Whereas

antagonist binding to the mutant and wild-type receptors was similar,

selective delta receptor agonists had greatly diminished potencies at

binding to the mutant receptor. This finding suggests that selective

agonists and antagonists bind differently to the delta receptor, possibly by

interacting with distinct ligand binding domains. Furthermore, nonselective agonists that are able to potently bind to other opioid receptors

besides the delta receptor bound with similar affinities to the wild-type

and mutant delta receptor. This finding indicates that selective and

nonselective agonists bind differently to the delta opioid receptor. The

findings of these mutagenesis studies indicate that aspartate 95 is
94

necessary for Na+ regulation of agonist binding to the cloned delta

receptor. Furthermore, this amino acid residue appears critical for the

high affinity binding of selective agonists to the delta receptor, but is not

essential for the interaction of any other ligands with this receptor.

To further evaluate residues in the delta opioid receptor involved in ligand

binding, Livingston and associates (submitted) mutated aspartate 128 in

transmembrane spanning region 3 to an asparagine in the cloned mouse

delta receptor (see figure 2) and tested the effects of this mutation on

ligand binding. Previous studies on the beta-adrenergic receptor had

shown that mutation of the aspartate 113 in the third transmembrane

spanning region to an asparagine abolished high affinity antagonist

binding to the receptor and modestly diminished agonist binding (Strader

et al. 1987). It has been proposed that the aspartate provides a counterion

to the cation of beta-adrenergic ligands and serves to stabilize binding

through electrostatic interactions. Mutation of aspartate 128 of the delta

receptor to an asparagine abolished high affinity agonist binding to the
95

receptor but did not alter antagonist binding. This finding differs from

that reported on the beta-adrenergic receptor and indicates that agonists

and antagonists bind differently to the adrenergic and delta receptors.

Both aspartate 95 and 128 of the delta receptor are necessary for high

affinity binding of selective agonists. In contrast, only aspartate 128

is critical for nonselective agonist binding to the delta receptor. These

findings indicate that different types of agonists at the delta receptor

bind in fundamentally distinct manners. The aspartate 128 may serve

as the counterion to the cation provided by charged nitrogens in

opioid agonists. However, aspartate 95 contributes to the selectivity of

recognition of specific delta agonists.

Interestingly, neither mutation of aspartate 95 nor 128 affected

antagonist binding to the delta receptor. Antagonists also have

positively charged nitrogens necessary for high affinity binding to

opioid receptors. The lack of effect of mutation of the aspartates on

antagonist binding suggests that some other residues within the delta

receptor provide the negative counterion for antagonist binding. This

finding supports the hypothesis that agonists and antagonists bind to

the delta receptor via distinct molecular mechanisms, possibly to

distinct ligand binding domains.

To more directly identify the ligand binding domains of the kappa

and delta opioid receptors, Kong and associates (1994) have

generated chimeric kappa/delta opioid receptor by exchanging the Ntermini of each receptor and tested the mutant receptors for agonist

and antagonist binding (see figure 3). Comparison of the predicted

amino acid sequences of the kappa and delta opioid receptors reveals

that the N-terminus is the region of each receptor that differs the most

between each receptor and therefore is likely to contribute to

variations in the ligand selectivity. A kappa1-78/delta70-372 bound delta

selective agonists and antagonists in a similar manner as the wild-type

delta receptor. Similar results were obtained with a truncated delta

receptor lacking the N-terminal 70 residues. These findings indicate

that the N-terminus of the delta receptor is not needed for ligand

binding. It is likely that either the second and/or third extracellular

loops of the delta receptor contain the ligand binding domains since

these are the only other extracellular domains besides the N-terminus

that differ in amino acid sequence between the delta receptor and the

other opioid receptors. Mutagenesis studies to exchange these regions

between the opioid receptor should help to test this hypothesis.
96

In contrast to the results with the mutated delta opioid receptor, the

results of mutagenesis studies on the kappa receptor reveal that

agonists and antagonists bind to this receptor in a manner

fundamentally different from that found with any other

neurotransmitter receptor (see figure 3). Kappa selective antagonists

bound potently to the kappa1-78/delta70-372 chimera but kappa agonists

did not. Conversely, kappa selective agonists interacted with a delta1-

69/kappa79-380 chimera and a truncated kappa receptor lacking the Nterminal 78 amino acid residues. Kappa antagonists did not bind

potently to these latter mutant receptors. These findings indicate that

selective agonists and antagonists bind to clearly separable recognition

sites in the kappa receptor with antagonists interacting with sites within

the N-terminus and agonists binding to more C-terminal regions

probably in either the second and/or third extracellular loops. The

separation of the agonist and antagonist binding domains of the

kappa receptor suggests that the N-terminus must be able to fold upon

the receptor to allow the separate recognition sites to be in close

proximity to allow for competi-tion of agonist and antagonist
97

binding. The identification of the specific residues in the kappa

receptor involved in agonist and antagonist binding may facilitate the

further development of therapeutically useful opioids. This will be

particularly important in the case of the kappa receptor since kappa

agonists have minimal abuse potential and do not cause respiratory

depression, two major side effects of the use of mu receptor selective

agonists. In contrast, kappa agonists are effective analgesics and

useful diuretic agents.

Molecular Mechanisms of Tolerance Development to Opioids

A major limitation to the clinical use of opioids is the development of

tolerance (Koob and Bloom 1992; Koob et al. 1992). With tolerance,

increasing concentrations of an opioid are needed to maintain a

constant therapeutic effect. All three opioid receptor types undergo

tolerance development (Koob et al. 1992). The molecular basis of

opioid tolerance is not established, although it has been suggested that

tolerance development is related to opioid receptor desensitization

(Childers 1991; Loh and Smith 1990; Nestler 1993). To investigate

the molecular mechanisms of tolerance to kappa agonists, Raynor and

colleagues (1994) expressed the cloned kappa receptor in mouse

kidney fibroblast (COS) cells, treated the cells for increasing times

with various kappa agonists, and showed that the kappa receptors

desensitized. The desensitization was characterized by a reduced

affinity of the receptor for agonists, an uncoupling of the receptor

from G-proteins, and a diminished ability of the receptor to mediate

agonist inhibition of adenylyl cyclase activity. The desensitization

was reversible, blocked by opioid antagonists and induced in a

stereoselective manner by agonists. Furthermore, the enzyme betaadrenergic receptor kinase (BARK) (Benovic et al. 1989) appears to

be involved in kappa receptor desensitization. This was suggested by

experiments in which a BARK dominant negative mutant was

cotransfected into COS cells with the kappa receptor cDNA; in those

cells treatment with agonists did not cause receptor desensitization.

The BARK dominant negative mutant has been shown to block the

activity of wild-type BARK but can not catalyze the phosphorylation

of substrates due to a single point mutation in its catalytic domain

(Kim et al. 1993). These studies suggest that kappa agonists may

stimulate BARK activity to catalyze the phosphorylation of the kappa

receptor, thus causing an uncoupling of the receptor from G-proteins

and effector systems. The intracellular domains of the kappa receptor

have multiple potential phosphorylation sites that could act as

substrates for BARK, and mutagenesis studies may reveal which one
98

of those sites is phosphorylated during desensitization and is critical

for the desensitization of the kappa receptor.

Interestingly, the regulation of the three cloned opioid receptors by

agonists differs considerably. Agonist pretreatment of COS cells

expressing the cloned delta receptor results in rapid desensitization

and downregulation of the receptor within an hour of treatment. This

down-regulation of the delta receptor is not blocked by a BARK

dominant negative mutant suggesting that different cellular

mechanisms are involved in the regulation of the delta and kappa

receptors. Furthermore, after 4 hours of agonist pretreatment, kappa

receptors desensitize but do not downregulate, indicating that the

sensitivity of the kappa and delta receptors to downregulation differs.

This finding is of particular importance since the only intracellular

regions of the two receptors that differ in amino acid sequence and

that could represent domains of the receptors responsible for

variations in agonist regulation are the C-termini. Mutagenesis studies

to exchange the C-termini of the two receptors could test the

hypothesis that these regions are involved in the regulation of opioid

receptor sensitivity following agonist treatments and are critical for the

development of tolerance of these receptors.

Prolonged treatment (4 to 16 hours) of COS cells expressing the

cloned mu receptor did not desensitize nor downregulate this

receptor. Since all three receptors were expressed in the same COS

cell line, differences in sensi-tivity to agonist treatment cannot be

explained by variations in cellular environment. Instead, these

findings suggest that the three receptors are regulated differently at a

cellular level. As a result, the cellular basis of tolerance to the three

opioid receptors may be different. Tolerance to delta selective

agonists may involve the desensitization and downregulation of the

delta receptor. Tolerance to kappa selective agonists may involve the

slow desensitization of the receptor via a BARK mediated mechanism.

Tolerance to mu selective agonists such as morphine could involve

postreceptor events such as changes in intermediate early gene

expression or other cellular events. The fundamentally different

adaptive responses of the opioid receptors to prolonged agonist

treatment may reveal strategies to overcome tolerance development,

which is a major limiting factor in the continuous use of opioids in the

treatment of chronic pain.
99

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ACKNOWLEDGMENTS

This work was supported by grants from the National Institute of

Mental Health (grant nos. DA08451, MH45533, and MH 48518) and

the Scottish Rite Foundation.

AUTHORS

Karen Raynor, Ph.D.

Instructor

Haeyoung Kong, B.S.

Research Assistant

Susan F. Law, Ph.D.

Research Associate

Jennifer Heerding, M.S.

Research Assistant

Melanie Tallent, B.S.

Research Assistant

Fredrick Livingston, B.S.

Technician

John Hines, B.S.

Research Assistant

Terry Reisine, Ph.D.

Professor

Department of Pharmacology

University of Pennsylvania School of Medicine

103 John Morgan Building

36th Street and Hamilton Walk

Philadelphia, PA 19104
104

Mu Opioid Receptors: Cellular

Action and Tolerance Development

Anton Mestek, Yan Chen, and Lei Yu

INTRODUCTION

Opioids are some of the most effective pain-relieving drugs used in

the clinical management of pain (Gilman et al. 1990). In addition to

their analgesic effect, opioid peptides and alkaloids also affect a

number of physiological functions including hormone secretion,

neurotransmitter release, feeding, gastrointestinal motility, and

respiratory activity (Pasternak 1988). Extensive physiological,

behavioral, and pharma-cological studies have defined at least three

major types of opioid receptors designated mu, kappa, and delta

(Corbett et al. 1993; Goldstein 1987; Wood and Iyengar 1988).

Although there is substantial overlap in their tissue distribution and

pharmacological profiles, each opioid receptor type maintains a

unique pattern of expression while displaying characteristic binding

affinities for various subtype-selective ligands. The delta receptors

that bind the enkephalin peptides are expressed most predominantly

in the basal ganglia, striatum, and cerebral cortex (Mansour et al.

1988; Wood 1988). Although delta receptors have been implicated in

spinal analgesia (Porreca et al. 1984; Yaksh 1981), recently it has

been suggested that specific delta receptor subtypes may also be

involved in supraspinal analgesia (Pasternak 1993). The kappa

receptors are most highly expressed in cortex, striatum, and

hypothalamus (Mansour et al. 1987), with various subtypes identified

by autoradiography using subtype-selective ligands (Nock et al. 1988;

Unterwald et al. 1991). With the development of more highly

subtype-selective ligands, these receptors have been shown to mediate

both spinal and supraspinal analgesia (Pasternak 1993).

The mu receptor represents the third major class of opioid binding

sites. Named after morphine, the mu opioid receptor is the

physiological target of such potent analgesics as morphine and

fentanyl, as well as the endogenous opioid peptides, ß-endorphin,

enkephalins, and dynorphins (Wood and Iyengar 1988). Opioid

drugs with high abuse liability such as morphine, methadone, and

fentanyl all bind the mu receptor with high affinity. In addition,

heroin (diacetylmorphine), a semisynthetic derivative of morphine,

crosses the blood-brain barrier much more readily than morphine due
105

to its increased hydrophobicity. Once in the brain, heroin is rapidly

hydrolyzed to morphine, which acts at the mu opioid receptor and

results in a euphoric effect, thus conferring the reinforcing properties

of the drug and contributing to the development of addiction.

Because of the high affinity of these opioid narcotics at the mu

receptor, it is considered the main cellular mediator in the

development of tolerance (Loh et al. 1988) and opioid addiction (Di

Chiara and North 1992).

Analgesia and the development of opioid tolerance, dependence, and

addiction have been the subject of extensive studies (Collin and

Cesselin 1991). Several schemes, including receptor-mediated

modulation of membrane conductance, have been proposed for the

acute and chronic actions of opioids in the central nervous system

(Johnson and Fleming 1989). One scheme involves protein

phosphorylation by various kinases as a possible way to regulate

opioid-induced cellular processes. The molecular mechanism of such

regulation, however, has not been clearly delineated.

MOLECULAR CLONING OF MU OPIOID RECEPTORS

To begin exploring the molecular basis for opioid tolerance, the

authors sought to clone the cDNAs encoding mu opioid receptors.

Using a strategy of low-stringency hybridization for isolating opioid

receptors related to the mouse delta opioid receptor (Evans et al.

1992; Kieffer et al. 1992), the authors first reported the molecular

cloning of a mu opioid receptor from the rat brain (Chen et al.

1993a), and at the same time, isolated cDNAs encoding a kappa

opioid receptor (Chen et al. 1993b) and a novel member of the opioid

receptor gene family (Chen et al. 1994). Using the rat mu opioid

receptor as a hybridization probe, a cDNA for the human mu opioid

receptor has also been isolated (Mestek et al. 1995). The human and

rat mu opioid receptors are very homologous, with 94percent

similarity at the amino acid sequence level (figure 1). The mu opioid

receptors are also closely related to the delta and kappa opioid

receptors, with 62percent and 58 percent similarities to the rodent

delta (Evans et al. 1992; Fukuda et al. 1993; Kieffer et al. 1992) and

kappa (Chen et al. 1993b; Liet al. 1993; Meng et al. 1993; Minami et

al. 1993; Yasuda et al. 1993) opioid receptors, respectively. Figure 1

displays the alignment of the human mu opioid
106
107

receptor sequence against the rodent mu, kappa, and delta opioid

receptor sequences. The regions of greatest divergence among the

different types of opioid receptors include the N-and C-termini, the

fourth and sixth trans-membrane domains, and the second and third

extracellular loops. Several potential sites for posttranslational

modification are present (figure 1). The N-terminus contains five

potential N-linked glycosylation sites that remain conserved between the

human and rat mu opioid receptors. Aspartic acid residues that occur in

other guanosine triphosphate binding protein (G-protein)-coupled

receptors and have been shown to interact with the protonated amine

group of various ligands (Dohlman etal. 1991) are also present in the

putative transmembrane (TM) domains TM2 and TM3 of the mu

receptor, and two conserved cysteine residues believed to be involved in

disulfide bonding (Dixon et al. 1988) occur in the first and second extracellular loops. There are also potential phosphorylation sites for protein

kinase A (PKA) and protein kinase C (PKC), as well as the

multifunctional calcium/calmodulin-dependent protein kinase (type II

Ca2+/calmodulin-dependent kinase, CaM kinase). One of these sites,

conserved among all opioid receptor types, occurs in the third

intracellular loop. This region between TM5 and TM6 is often referred

to as the G-protein loop because of its importance in G-protein coupling

(Dohlman et al. 1991). The conser-vation of this site suggests that

phosphorylation may play a role in modula-ting signal transduction of

all the opioid receptors. There is also a cysteine residue in the Cterminus that is conserved among many G-protein-coupled receptors and

that may serve as a target for palmitoylation (Collins et al. 1991).

To characterize the pharmacological profile of the mu opioid receptors,

cDNA fragments containing the protein coding region of each of the mu

opioid receptors were subcloned into a mammalian expression vector

containing the human cytomegalovirus promoter. Using these plasmids,

the mu opioid receptor was either transiently expressed in COS-7 cells or

stably expressed in Chinese hamster ovary (CHO) cells. Membranes

from these cells were prepared and saturation binding was performed

using either [3H]diprenorphine, a nonselective opioid antagonist with

high affinity for all three types of opioid receptors, or [3H] [D-Ala2-Me

Phe4, Gly-ol5]-enkephalin (DAMGO), a mu-selective agonist. The mu

opioid receptors display high affinity binding to both [3H]diprenorphine

and [3H]DAMGO, with dissociation constant (Kd) values in the nanomolar (nM) range. Also, various unlabeled opioid ligands were used in

competitive displacement experiments. Several therapeutic opioid

ligands, including morphine, methadone, and fentanyl, all displayed nM

affinities at the mu opioid receptors, indicating that these highly potent

analgesic drugs are mu-selective. In addition to these exogenous opioids,
108

all three types of the endogenous opioid peptides, ß-endorphin, Met- and

Leu-enkephalins, and dynorphin A (1-17) displayed nM affinities at the

mu receptors, suggesting that the mu receptors may be activated by any

of these endogenous opioid peptides at physiological concentrations.

The high affinity of ß-endorphin at the mu receptors is expected, since it

has long been thought that ß-endorphin is the endogenous ligand for the

mu opioid receptor. Using the mu, delta, and kappa opioid receptor

clones isolated from rodents, binding studies performed in parallel have

demonstrated that enkephalins are also potent ligands at both the mu and

the delta opioid receptors (Raynor et al. 1994). The nM affinity of

dynorphin A (1-17) at the mu receptor is, however, somewhat

unexpected, since dynorphin A has the highest affinity at the kappa

opioid receptor (Goldstein 1987) and is thus considered a kappa agonist

(Chavkin et al. 1982). However, studies have shown that while dynorphin

A binds the kappa receptors with subnanomolar affinity, it does display

reasonably good binding at the mu opioid receptors, with affinity values

in the nM range (Goldstein 1987; Pasternak 1993). Thus, the possibility

exists that the mu receptor may interact with dynorphin A under

physiological conditions. Detailed studies of dynorphin A binding in the

human brain are very limited. There are some reports, however, using

membranes isolated from rat and bovine brain (Pasternak 1993). Using

guinea pig brain membranes, it has been shown that dynorphin A (1-17)

can displace radiolabeled PL-17, a mu-selective ligand, with a 5nM

affinity (Kawasaki et al. 1990). In another study, dynorphin A (1-17)

was found to displace radiolabeled DAMGO with 3 nM affinity (Vaughn

and Taylor 1989). The authors’ results further support the notion that

the mu opioid receptor may serve as a natural target for dynorphin A.

CELLULAR FUNCTION OF MU OPIOID RECEPTORS

The mu opioid receptor is known to exert two types of inhibitory effects

on a cell—reduction of the intracellular level of cyclic adenosine

monophosphate (cAMP) and inhibition of neuronal firing. Using the

cloned mu opioid receptors, the authors have begun to study the

molecular mechanisms for both of these processes.

The mu opioid receptors contain seven transmembrane hydrophobic

domains, a structural motif most commonly found in G-protein-coupled

receptors (Chen et al. 1993a). This suggests that the mu opioid receptors

may couple to heterotrimeric G-proteins to mediate intracellular signal

transduction. Previous studies using cell lines that constitutively express

endogenous opioid receptors also suggest that opioid receptors are
109

coupled to G-proteins (Carter and Medzihradsky 1993b; Childers 1991).

Upon activation of a G-protein, the alpha subunit of the G-protein

dissociates and displays an increase in its intrinsic GTPase activity. To

test whether the cloned mu opioid receptor couples to G-proteins,

GTPase activities from cell membranes expressing the mu receptor were

measured in the presence and absence of mu-selective ligands.

Treatment of CHO cells stably expressing the mu receptor with the muselective agonist DAMGO elevated the GTPase activity by 33percent.

This stimulation was blocked by the opioid antagonist naloxone. In

nontransfected parental CHO cells, on the other hand, GTPase activity

was not affected by DAMGO treatment. Because an increase in the low

affinity GTPase activity is indicative of G-protein activation, these results

suggest that the mu opioid receptor is functionally coupled to the Gproteins in these mammalian cells.

Since it has been reported that activation of the mu opioid receptor exerts

an inhibitory effect on adenylyl cyclase activity (Adams et al. 1991;

Childers 1991; Cox 1993; Frey and Kebabian 1984; Sharma et al.

1975a), the authors next sought to examine the effect of mu opioid

receptor activation on the intracellular levels of cAMP. In nontransfected

COS-7 cells, treatment with the mu-selective ligands had no significant

effects, while DAMGO stimulation of cells expressing the mu opioid

receptor reduced the intracellular cAMP levels significantly. This effect

on adenylyl cyclase activity was blocked by the mu-selective antagonist

naloxonazine, indicating that the inhibitory effect was mediated through

the activation of the mu opioid receptor and that the receptor was

functionally coupled to the inhibition of adenylyl cyclase activity.

Studies in cell lines expressing endogenous opioid receptors

demonstrated that chronic treatment with opioid agonists may modulate

the coupling between opioid receptors and adenylyl cyclase (Carter and

Medzihradsky 1993a; Puttfarcken et al. 1988; Sharma et al. 1975b). To

test the effect of chronic morphine exposure on the intracellular levels of

cAMP in CHO cells expressing the mu opioid receptor, cAMP levels were

assayed by activation of the mu receptor with DAMGO before and after

morphine treatment. Before chronic morphine treatment, the forskolinstimulated cAMP level in morphine-naive cells was reduced to 70 percent

relative to controls upon treatment with 1 micromlar (μM) DAMGO.

After the cells were exposed to morphine for 24 hours, DAMGO-induced

inhibition of cAMP levels was enhanced, resulting in a decrease of cAMP

levels to 40percent relative to controls. This result suggests that

morphine treatment may enhance the effectiveness of the mu receptormediated inhibition of adenylyl cyclase activity. When the forskolinstimulated cAMP levels in naive cells were measured in the presence of
110

different concentrations of DAMGO, the intracellular cAMP levels were

reduced by DAMGO in a dose-dependent fashion, with a 50percent

effective concentration (EC50) value of about 30 nM. The effect of

DAMGO was clearly the result of the mu receptor activation, since

treatment of the cells with 1 μM DAMGO in the presence of 10 μM

naloxone completely abrogated the inhibitory effect of DAMGO on

cAMP accumulation. In contrast, DAMGO had no effect on the

intracellular cAMP levels in non-transfected parental cells. Chronic

treatment with morphine for 24hours enhanced the extent of the

DAMGO-induced inhibition of adenylyl cyclase activity, but did not

change the EC50 value of the DAMGO-induced inhibition. To test

whether the number of receptors was changed upon chronic morphine

treatment, CHO cells expressing the cloned mu opioid receptors were

chronically treated with 1 μM of morphine for 24 hours. Using

[3H]DAMGO and whole-cell binding to determine the number of the

total surface receptors, it was observed that there was no appreciable

change in the number of surface receptors, nor was there any noticeable

change in the affinity for DAMGO after chronic morphine treatment.

These results suggest that, unlike the changes in adenylyl cyclase

activities upon morphine treatment, receptor downregulation does not

occur after chronic morphine treatment in these CHO cells.

The EC50 value calculated from the dose-response curve observed with

DAMGO after chronic morphine treatment was about 30 nM, compared

to the 2 nM 50 percent inhibitory concentration (IC50) value derived

from the competition binding studies. The difference between the

functional potency and the binding affinity values may be due to the

ratio of mu receptors on the cell and the G-protein or effector molecules

to which they couple, but other possibilities may also exist. It should be

noted that the efficacy for adenylyl cyclase inhibition by the mu opioid

receptor in the CHO cells is similar to that in SK-N-SH and 7315c cells

(Frey and Kebabian 1984; Yu et al. 1990). The relative efficacy of mu

opioid receptors to inhibit adenylyl cyclase activity seems to be lower

than that of other G-protein-coupled receptors that are linked to the

inhibition of adenylyl cyclase, such as the a2-adrenergic receptor,

serotonin 1A receptor, and dopamine receptors (Fraser et al. 1989;

Raymond et al. 1992; Sokoloff et al. 1992). The differences in the

ability of each receptor type to inhibit adenylyl cyclase suggests that

different G-proteins may be involved in coupling distinct receptors to a

common effector molecule. Since more than one adenylyl cyclase has

been identified in the cell (Iyengar 1993), an alternative possibility may

be that different subtypes of adenylyl cyclases couple differently to the

various membrane receptors.
111

The protocol the authors used for studying the effect of chronic

morphine treatment is similar to that used by many investigators

(Puttfarcken and Cox 1989). First, cells are chronically exposed to a

moderate concen-tration of agonist (morphine). The cells are washed to

remove agonist, and then are acutely stimulated with various

concentrations of agonist. Because the presence of the agonist during

the chronic exposure presents a continued inhibitory input, agonist

washout before acute treatment is equivalent to disinhibition, and has

been compared to the withdrawal paradigm following the establishment

of opioid tolerance (Sharma et al. 1975b). In the authors’ cell line, both

the basal and forskolin-stimulated cAMP levels were elevated after

chronic morphine treatment and withdrawal. This suggests that a

compensatory mechanism involving increased adenylyl cyclase activity

may be responsible for the higher levels of cAMP that have been

observed upon removal of mu receptor-mediated inhibition (Puttfarcken

and Cox 1989; Sharma et al. 1975b). It is interesting to note that

chronic morphine exposure displays a differen-tial effect on cAMP

levels. Basal cAMP levels were about 60percent greater after morphine

exposure than that observed in naive cells, while the forskolin-stimulated

cAMP levels were more than doubled with morphine treatment. It is

tantalizing to speculate that this difference between basal and forskolinstimulated cAMP levels during chronic morphine exposure reflects a

differential sensitivity to acute mu receptor activation of two populations

of adenylyl cyclase molecules—those present before morphine exposure

and those synthesized during morphine pretreatment as the

compensatory mechanism becomes engaged. It is conceivable that these

two populations of adenylyl cyclases may have different compositions

with regard to cyclase subtypes, since molecular cloning has shown that

multiple adenylyl cyclases exist (Iyengar 1993).

Another phenomenon of morphine treatment is that the DAMGOinduced cAMP inhibition in the morphine-treated cells is more robust,

which resulted in doubling the percent inhibition of cAMP from

approximately 25percent in naive cells to almost 60 percent in

morphine-treated cells. These results suggest that morphine exposure

"sensitized" the system, such that acute mu receptor activation was more

effective in inhibiting the adenylyl cyclase activity. This is in contrast to

the studies with cell lines expressing endogenous mu receptors, in which

chronic morphine treatment caused a decrease in the ability of the

receptor to inhibit adenylyl cyclase (Puttfarcken and Cox 1989; Werling

et al. 1989). The lack of receptor desensitization in CHO cells suggests

that receptor modulation of adenylyl cyclase in various cell types occurs

differently. This may reflect the uniqueness in the composition of

endogenous G-proteins and/or adenylyl cyclases in CHO cells versus
112

other cell types. Also of interest is that chronic morphine treatment of

the transfected CHO cells did not cause downregulation of surface

receptors. In morphine-treated cells, neither the number of cell surface

receptors nor the Kd value for DAMGO was significantly different from

those observed with naive cells. This is in contrast to agonist-induced

downregulation in ß2-adrenergic receptors (Collins et al. 1991). The

increase in cAMP levels observed with chronic morphine treatment and

the sensitization of the signal transduction pathways involving adenylyl

cyclase suggest that this cell line could serve as a cellular model, allowing

the study of both the molecular mechanisms that link the mu opioid

receptor to its effector systems and the changes associated with morphine

tolerance and dependence.

Activation of opioid receptors has been known to affect membrane

permeability to potassium (North 1993). Stimulation of the mu opioid

receptor hyperpolarizes cellular membranes by increasing the K+

conduc-tance through an inwardly rectifying channel (North et al. 1987;

Wimpey and Chavkin 1991). The recent cloning of an inwardly

rectifying K+ channel (Dascal et al. 1993; Kubo et al. 1993) was shown

to be expressed in the brain, and the authors were interested in testing

whether the mu opioid receptor coupled to this G-protein-activated K+

channel (Chen and Yu 1994; Mestek et al. 1995). Messenger ribonucleic

acids (mRNAs) encoding both proteins were generated by in vitro

transcription and injected into Xenopus oocytes. Coupling of the

receptor to the K+ channel was assessed by a two-electrode voltage

clamp. In the oocytes injected with either the mu receptor mRNA or the

K+ channel mRNA alone, no membrane current was observed with the

mu-receptor agonist DAMGO. This indicated that there were no

endogenous currents in oocytes activated by DAMGO, and that either the

mu receptor or the K+ channel alone is not sufficient to generate

DAMGO-induced currents. However, coexpression of both proteins gave

rise to membrane currents upon DAMGO stimulation (Chen and Yu

1994; Mestek et al. 1995). Exposure of the oocytes to 1 μM of DAMGO

produced an inward membrane current that was completely blocked by

the opioid receptor antagonist naloxone. This current was carried

through the inwardly rectifying K+ channel, since the current varied in

amplitude with the concentration of K+ in the extracellular solution and

was blocked by 100μM Ba2+, a known K+ channel blocker. The

current-voltage relationship of this K+ channel was characteristic of an

inward rectifier. With progressive hyperpolarization, the magnitude of

current increases. However, as the membrane is depolarized, current flow

decreases until there is little to none at a membrane potential of 0

millivolts (mV). Thus, the mu opioid receptor is capable of coupling to

the G-protein-activated K+ channel.
113

Prolonged exposure to opioids is known to produce tolerance in neurons

(Di Chiara and North 1992). At the cellular level, tolerance development

is observed as a diminished response to opioids (Johnson and Fleming

1989). Since opioid narcotics with abuse potential such as morphine,

methadone, and fentanyl interact with the mu opioid receptor with high

affinities, the authors were interested in examining whether the

intracellular signaling by the mu opioid receptor displays tolerance.

Using the receptor-K+ channel coupling as an assay, oocytes were

subjected to repeated agonist stimu-lation to determine whether

functional desensitization of the mu receptor-K+ channel coupling

occurred. For this purpose, a protocol of repeated agonist application

was used, and "desensitization" was operationally defined as a reduction

in the second response compared to the first response. The oocyte was

superfused with high-potassium (HK) solution while the DAMGOinduced responses were measured. The superfusate was then switched to

frog Ringer's solution and the cell was subjected to the various

treatments. After treatment, DAMGO-induced responsiveness was

measured again in HK solution. Comparison between the maximal

response before and after the treatment thus indicates how much desensitization has occurred following the first stimulation with DAMGO.

Repeated stimulation of the mu receptor resulted in a moderate and

consistent desensitization as observed by a reduction in the second

response. The second response was reduced to 80 percent relative to the

first response. These results suggest that when expressed in oocytes, the

coupling between the mu opioid receptor and the G-protein-activated K+

channel undergoes a process of desensitization.

Previous studies had shown that activation of PKC was capable of

attenuating opioid receptor activity in neuroblastoma cells (Louie et al.

1990) as well as affecting ion conductances using acutely dissociated

neurons in culture (Doerner et al. 1988). The authors were interested in

observing whether stimulation of PKC affects the coupling of the mu

opioid receptor to the K+ channel. Using the protocol described above,

oocytes were superfused for 10 to 15 minutes after the initial stimulation

with phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol

ester. Oocytes were again stimulated with DAMGO. Comparison

between the peak current response to DAMGO before and after PMA

treatment indicated that PMA reduced the second response by 70percent

relative to the first. Thus, activation of PKC augmented the

desensitization of the mu opioid receptor-activated K+ current. To

control for possible non-specific effects caused by application of a

phorbol ester to the cellular membrane, oocytes were treated with 4aphorbol, a phorbol ester that does not activate PKC (Blumberg et al.
114

1984). Recordings of peak current amplitude show that the 4a-phorbol

does not augment desensitization beyond that observed with no

treatment, as membrane currents before and after 4a-phorbol treatment

exhibited moderate desensitization similar to that with no treatment. This

demonstrated that the inactive form of the phorbol ester does not

augment desensitization. Therefore, these results indicate that

augmentation of the desensitization by PMA reflects the activation of

PKC.

Physiologically, PKC is activated by diacylglycerol (DAG), a second

messenger of the phosphatidylinositol pathway. This signaling pathway

also generates another second messenger, inositol 1,4,5-trisphosphate

(IP3), which triggers an increase in intracellular calcium and results in the

activation of the multifunctional Ca2+/calmodulin-dependent protein

kinase (type II Ca2+/calmodulin-dependent protein kinase, CaM kinase

II) (Schulman and Hanson 1993). The authors were interested in testing

whether CaM kinase II could modulate the mu opioid receptor-mediated

K+ current. Using the protocol described above, oocytes were injected

with the activated form of CaM kinase II between the first and second

stimulations with DAMGO, and the effect on the receptor-induced K+

current was evaluated. CaM kinase II clearly enhanced the desensitization, causing a twofold decrease in the second response relative to

uninjected oocytes. As a negative control, the same CaM kinase II

solution was placed in a boiling water bath for 5 minutes and chilled on

ice before injection into the oocytes. Boiled CaM kinase II did not

augment desensitization beyond that observed with uninjected controls.

The authors concluded that activated CaM kinase II can therefore

augment the desensitization of the mu opioid receptor-activated K+-

current.

Since the mu opioid receptor can modulate the activity of adenylyl

cyclase, there was also interest in determining whether cAMP, the product

of adenylyl cyclase, had any effect on the mu receptor-K+ channel

coupling (Chen and Yu 1994). Treatment of the oocyte with 8-

chlorophenylthio-cAMP (8-CPT-cAMP), a membrane-permeable cAMP

analog that can diffuse into the cell and stimulate cAMP-dependent

protein kinase (PKA), completely abolished the desensitization observed

in untreated oocytes, resulting in the K+ current being similar in

amplitude for both the first and the second stimulation with DAMGO.

To determine whether the 8-CPT-cAMP effect on preventing desensitization was mediated by PKA, the catalytic subunit of PKA was injected

into the oocytes after the first DAMGO stimulation. This resulted in the

same effect as 8-CPT-cAMP incubation, indicating that the blockade of

desensitization was indeed mediated through PKA.
115

DISCUSSION

A Molecular Mechanism for Mu Opioid Receptor Function and Its Acute

Desensitization

Results from the authors’ studies indicate that cloned mu opioid receptor

cDNAs can be used to express functional mu opioid receptors that

display the expected pharmacological profile. The cellular functions

affected by the activation of the mu opioid receptor include the

inhibition of adenylyl cyclase activity and the activation of a K+ channel.

Various protein kinases have also been shown to modulate mu receptormediated cellular processes. Based on these results, the authors propose

the following model for mu receptor function and its acute

desensitization. This model, shown schematically in figure 2, is discussed

below.

A major effect of the mu opioid receptor in brain is the decrease of

neuronal membrane excitability. One of the mechanisms for this effect

is an increase in K+ conductance, accomplished by the opening of an

inward rectifier, resulting in outward K+ currents and hyperpolarization

of the cell membrane (Chavkin 1988; North 1986, 1993). With the

cloning of an inwardly rectifying K+ channel that can be activated by a

number of neurotransmitter receptors (Dascal et al. 1993; Kubo et al.

1993), it became possible to examine whether the mu opioid receptor

could also activate this channel. It has been shown that the mu opioid

receptor from both rat (Chen and Yu 1994) and human (Mestek et al.

1995) can activate this channel, causing an increase in K+ conductance.

The receptor-channel coupling is clearly mediated through

heterotrimeric GTP-binding proteins (G-proteins), since a

nonhydrolyzable GTP analog, GTP-gS, can enhance the mu receptoractivated K+ current and pertussis toxin treat-ment can decrease it (Chen

and Yu 1994). Thus, the authors’ data
116
117

suggest that the mu receptor-channel coupling may be the basis for

the mu receptor-mediated increase in K+ conductance.

Receptor-mediated signaling processes often display desensitization,

operationally defined as a decrease in the cellular response to further

agonist stimulation upon continuous or repeated exposure to agonist

(Benovic et al. 1988). This may serve as a physiological mechanism

to prevent overstimulation of the neuron. The mu opioid receptor is

the physiological target of morphine and fentanyl, analgesics used in

the clinical management of pain. Prolonged use of morphine and

related opioids can lead to the development of tolerance, necessitating

dosage increases to achieve the same degree of their initial

physiological effect. At the cellular level, tolerance manifests itself as

a desensitized responsiveness to repeated opioid applications, and it

has been hypothesized that several of the intermediates in the mu

receptor signaling pathway are involved (Nestler et al. 1993). In the

neurons of the rat locus coeruleus, desensiti-zation was observed as a

reduction in membrane hyperpolarization upon continued application

of Met5-enkephalin. A decrease in K+ conductance was shown to be

responsible for the observed effect (Harris and Williams 1991). In

oocytes expressing the mu opioid receptor and the inwardly rectifying

K+ channel, a protocol was used to evaluate desensitization by

measuring the K+ currents evoked by sequential activation of the

receptor with a mu-selective agonist. Comparison of the maximum

K+ currents thus indicates the extent of desensitization between the

first stimulus and second stimulus caused by activation of the

receptor. Using this paradigm, desensitization occurred consistently,

as observed by the reduction of the K+ current to 80 percent of the

initial response evoked by receptor activation. Thus, desensitization

of receptor-channel coupling appears to be a normal process when

studied in oocytes, suggesting that such a phenomenon may exist as

an adaptive process in neurons to modulate the responsiveness of the

mu receptor-mediated increase in K+ conductance.

Desensitization of receptor-channel coupling may involve several

mechanisms at the cellular level. For acute desensitization such as that

studied here with a timescale of fewer than 30 minutes, de novo

protein synthesis or receptor turnover are unlikely to account for the

majority of the observed effects. Covalent modification through

kinase-mediated phosphorylation, on the other hand, appears to play

an essential role. The authors demonstrated that both PKC and CaM

kinase II augment desensitization. Activation of PKC by the phorbol

ester PMA and injection of the type II CaM kinase activated by

autophosphorylation both resulted in augmentation of desensitization.
118

In contrast, treatment with the inactive 4a-phorbol ester or injection of

boiled CaM kinase II did not augment desensitization beyond that

observed in oocytes that had been untreated, suggesting that the

augmentation effect is specific to the active form of these kinases.

When activated individually, PKC and CaM kinase (Akasu and

Tokimasa 1992; Shearman et al. 1989) have been shown to decrease

K+ conductance in neurons, which supports the authors’ observations

in Xenopus oocytes. Furthermore, in rat locus coeruleus neurons

containing mu opioid receptors, treatment with staurosporine, a

nonselective kinase inhibitor that can inhibit both PKC (Tamaoki

1991) and CaM kinase II (Yanagihara et al. 1991), reduced

desensitization of mu receptor-mediated hyperpolarization (Harris

and Williams 1991). Processes that elevate the activity of these

cellular kinases, therefore, may play an important role in regulating

the extent of the mu receptor-K+ channel coupling.

Both PKC and CaM kinase II are cellular effectors of a G-proteinactivated phosphodiesterase, phospholipase C (PLC). The activation

of PLC causes the production of DAG and IP3, two intracellular

second messenger molecules that represent a bifurcation in the signal

transduction pathway (Berridge 1987). Whereas DAG activates PKC,

IP3triggers Ca2+ release from intracellular stores. Since CaM kinase

II is activated by physiological elevations in cytosolic Ca2+ levels

(MacNicol and Schulman 1992a; Schulman and Hanson 1993),

stimulation of receptors linked to PLC may cause activation of both

PKC and CaM kinase (figure 2). Other neuro-transmitter receptors

that belong to the family of G-protein-coupled receptors influence the

steady-state levels of cAMP by either stimulating or inhibiting the

activity of adenylyl cyclase, respectively, as is the case for the ßadrenergic and opioid receptors. The widespread distribution of

many G-protein-coupled receptors suggests that some may be found

within similar structures of the brain. In fact, in situ hybridization has

shown that messages encoding receptors that use either similar (Lester

et al. 1993) or different (Weiner et al. 1990) signaling pathways do

coexist within the same cell. The authors’ data suggest that PLCcoupled receptors may augment the process of desensitization, which

is observed as a reduction in receptor-channel coupling upon repeated

stimulation of the mu opioid receptor (figure 2). Thus, it is plausible

that signal transduction mechanisms may affect one another when the

receptors to which they couple are present on the same neuron.

Since opioid receptor activation has been shown to affect cAMP levels,

another important kinase in the regulation of opioid receptor activity

is PKA. Opioid receptor activation inhibits adenylyl cyclase, resulting
119

in decreased levels of cAMP and a reduction in basal PKA activity.

The authors previously demonstrated that an increased PKA activity,

either by injection of the catalytic subunit of PKA into the cell or by

exposing the cell to a membrane-permeable cAMP analog, eliminated

desensitization of the mu receptor-K+ channel coupling (Chen and Yu

1994). Thus, agents that activate PKA would function antagonistically

in relation to the mu opioid receptor-mediated channel activity. This

presents an interesting control mechanism whereby the activity of the

channel is subject to negative feedback inhibition modulated by the

mu opioid receptor via a decrease in PKA activity (figure 2).

Phosphorylation by kinases is one of the most important mechanisms

for the functional regulation of many cellular proteins including

neuro-transmitter receptors and ion channels, with PKA and PKC

being two of the most widely studied kinases (Krebs 1989; Shearman

et al. 1989). Phosphorylation of ß2-adrenergic receptor by either

PKA or PKC leads to its uncoupling from G-proteins, resulting in

desensitization to further agonist stimulation (Lefkowitz and Caron

1988; Sibley and Lefkowitz 1985). In the case of voltage-dependent

Ca2+ channels such as the endogenous oocyte Ca2+ channel, PKAand PKC-mediated phosphory-lation is able to potentiate channel

activity (Bourinet et al. 1992; Chen et al. 1993c). Cystic fibrosis

transmembrane conductance regulator (CFTR), a Cl- channel

associated with cystic fibrosis, is also regulated by cAMP through the

PKA pathway (Anderson et al. 1991; Bear et al. 1991). The authors

have attempted to investigate the role of each kinase in the

mechanisms involved in acute desensitization. Figure2 depicts the

individual effects of these kinases on opioid receptor-mediated K+

channel activity. Although precedents exist for their acting

independently, these kinases are also subject to "cross-talk," which is

the ability of one signal transduction mechanism to affect another

(MacNicol and Schulman 1992b; Yamakawa et al. 1992). Taken

together, the data suggest that an intricate network of modulation

among receptors, G-protein effectors, and protein kinases may exist.

It should be noted that although each element in the model has been

demonstrated in oocytes and/or neurons, the complete scheme has not

yet been confirmed with respect to the cross-modulation that may

occur among multiple receptor types. With the cloning of the mu

opioid receptors and the other proteins involved in cellular signaling,

future efforts will undoubtedly further researchers’ general

understanding of the molecular mechanisms that underlie the

regulation of receptor-mediated neuronal activity, with particular

focus on the modulatory network involved with mu opioid receptor

function.
120

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AUTHORS

Anton Mestek, Ph.D.

Postdoctoral Research Associate

Yan Chen, Ph.D.

Postdoctoral Research Associate

Lei Yu, Ph.D.

Professor

Department of Medical and Molecular Genetics

Indiana University School of Medicine

Medical Research and Library Building

975 West Walnut Street, IB 151

Indianapolis, IN 46202
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Cloning and Characterization of

Multiple Opioid Receptors

Huda Akil, Fan Meng, Alfred Mansour, Robert Thompson,

Guo-Xi Xie, and Stanley Watson

THE CLONING OF THE THREE MAJOR OPIOID RECEPTOR

TYPES

Until recently, the study of opioid receptors, while greatly profiting

from the rich array of synthetic peptides and alkaloids with high

affinity and specificity, has been hampered by the absence of opioid

receptor clones. Matters were changed, however, in December 1992,

when two groups, Evans' and Kieffer's, independently cloned the

mouse delta receptor from the NG-108 cell line (Evans et al. 1992;

Kieffer et al. 1992). This work opened the door for rapid advances in

this area. Several groups, including the authors, subsequently cloned

the remaining two major types of opioid receptors from various

rodent species (Chen et al. 1993; Fukuda et al. 1993; Kong et al.

1993; Meng et al. 1993; Minami et al. 1993; Thompson et al. 1993;

Wang et al. 1993; Xie et al. 1994; Yasuda et al. 1993). The authors

have independently cloned the three prototypical mu, delta, and kappa

opioid receptors from rat brain, as well as the kappa receptor from

guinea pig brain. The authors also have partial delta and mu receptor

clones from guinea pig brain, which are currently being fully cloned

and characterized (Fickel et al. 1994). In addition, in searching for

other opioid receptor subtypes from rat and guinea pig brain, the

authors have isolated several clones, also obtained by other

laboratories (Mollereau et al. 1994), which appear to encode a protein

closely related to the opioid family, but fails to bind a number of

opiate alkaloids or opioid peptides. Thus, the question of the

existence of opioid receptor subtypes at the molecular level remains to

be fully elucidated.

SELECTIVITY OF THE CLONED RECEPTORS TOWARD THE

ENDOGENOUS OPIOID LIGANDS

Since the endogenous opioid system is particularly rich in

endogenous ligands, comprising three separate genes each giving rise

to multiple active peptides, the issue of the exact relationship between
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these endogenous ligands and the opioid receptors is of great

importance. Previous studies in tissue homogenates led the authors to

suspect that there is no one-to-one correspondence between any given

precursor and any given receptor. Now that these receptors can be

expressed indi-vidually, it is possible to carefully ascertain the

selectivity profile of each of the endogenous ligands toward the

individual receptors. The question of whether each precursor has at

least one ligand that "sees" each of the receptors, or whether there is

any exclusivity, can be addressed. For example, researchers already

know that the mu receptor can interact with members of each of the

families, but is kappa truly a "dynorphin" receptor, or can it be

accessed by proenkephalin (proEnk) products? By comparing all

three receptors and all the ligands side by side, a much more complete

picture can be obtained than was had before. An equally important

issue is that of efficacy at the receptors. While it may be assumed that

all endogenous ligands are agonists, there is evidence that naturally

occurring opioids, such as beta-endorphin1-27, can be antagonists in

behavioral studies (Bals-Kubik et al. 1988; Tseng and Li 1986). This

needs to be confirmed at the cellular level, and leads to the question of

whether any of the other endogenous ligands can serve as antagonists

or as partial agonists at one or more of the receptors.

A set of studies that is currently being performed in the authors’

laboratory involves the evaluation of the selectivities of the

endogenous opioid peptides for the cloned mu, delta, and kappa

receptors. A series of proEnk, prodynorphin, and pro-opiomelanocortin peptides has been evaluated for affinity to the mu, delta,

and kappa receptors that have been transiently transfected into COS-1

cells. Thus far, the data suggest that prodynorphin peptides have a

high affinity for all three opioid receptor types, with approximately a

tenfold selectivity for the kappa receptors. A surprising finding that is

being further explored is that extended proEnk peptides such as

Methione-Enkephalin-Arginine-Phenylalanine or MethioneEnkephalin-Arginine-Glycine-Leucine also have high affinity for

kappa receptors in addition to mu and delta. Interestingly, Leuenkephalin emerges as the most selective endogenous ligand,

exhibiting a more than hundredfold preference for delta over kappa

binding. On the other hand, the kappa receptor appears to have the

widest range of selectivity vis-a-vis endogenous ligands, whereas mu

and delta show relatively less discrimination (within one order of

magnitude).
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STRUCTURAL PROPERTIES OF THE OPIOID RECEPTORS—

BINDING TO PEPTIDES VERSUS SMALL MOLECULES

The three opioid receptors cloned to date are all members of the

guanosine triphosphate binding protein (G-protein)-coupled family

of receptors containing seven transmembrane alpha helixes. While

they exhibit significant homology to the somatostatin receptor family,

they are particularly similar among themselves, exhibiting 61 percent

level of identity at the amino acid level. The fact that opioid receptors

belong to the seven transmembrane family of G-protein-coupled

receptors tells a fair amount about their general topography based on

analogy to bacterial rhodopsin, which has been visualized by electron

microscopy (Findlay and Pappin 1986; Henderson et al. 1990). The

seven alpha-helical transmembrane segments are thought to be

arranged in a circular manner allowing the macromolecule to form a

ligand binding cavity, and exposing three intracellular loops and the

carboxy terminus to the cytoplasmic milieu and three extracellular

loops and the amino terminus to the outside environment (Humblet

and Mizadegen 1992). The intra-cellular loops, particularly the

second and third loop, along with the carboxy terminal domain, are

thought to be the site of interaction with G-proteins. The N-terminal

domain and extracellular loops are in a position to play a role in

receptor selectivity. It has been shown for several small

neurotransmitter receptors that actual ligand binding takes place via

specific interactions within the pocket formed by the trans-membrane

domains. A great deal of this information derives from mutants and

chimeras that have been constructed for many members of this

superfamily, particularly the beta-adrenergic receptor (Dixon et al.

1987; Dohlman et al. 1987). The structure/function relationship of

the dopamine receptors by site-directed mutagenesis has been studied

in the authors’ laboratory (Mansour et al. 1992), as well as by

constructing chimeras between dopamine type 1 (D1) and dopamine

type 2 (D2) (Meng et al. 1992).

In spite of the general model presented above, a great deal remains to

be learned about the family of G-protein-coupled receptors in terms

of actual three-dimensional arrangement, means of achieving ligand

selectivity, molecular basis of drug efficacy and receptor-effector

coupling, require-ments for selective G-protein interactions, and

mechanisms of receptor regulation such as desensitization. For

example, the homology of the mammalian receptors to rhodopsin,

even in the transmembrane helixes, appears imperfect when examined

in greater detail (Pardo et al. 1992). In addition, while they are

discussed as a superfamily, recent studies have pointed to differences
130

as well as similarities between these receptors, especially between some

inhibitory G-protein (Gi)-coupled receptors and the prototypical

stimulatory G-protein (Gs)-coupled ß-adrenergic receptor (Dixon et

al. 1987; Dohlman et al. 1987). Most importantly, while researchers’

understanding of the interactions of small ligands with G-protein

receptors is reasonable, understanding how peptide ligands bind and

activate these structures is more limited.

Studying the opioid receptors at this structural level is of interest for

several reasons. Such an analysis might help scientists understand the

cellular basis of tolerance by describing mechanisms of receptor

desensitization (e.g., phosphorylation), internalization, and downregulation. Equally important, however, this level of analysis will

allow researchers to understand, at the molecular level, the issue of

receptor heterogeneity and of ligand selectivity in the context of the

opioid system, which is unusually rich in endogenous ligands and in

pharmacological probes. It is particularly advantageous that, in the

opioid system, multiple peptides and multiple alkaloids interact with

one family of receptors. A first-order question is, do they interact at

the same sites (i.e., with the same critical residues) in the receptor? A

related question is, do these receptors achieve selectivity toward

peptide ligands in the same way that they achieve selectivity toward

alkaloid ligands? The authors’ working hypothesis is as follows:

Both peptide and alkaloid agonists are likely to interact with the same

region of the "binding pocket" of the receptor (though not necessarily

in exactly the same way) to trigger a chain of allosteric events, which

leads to a change in the interaction with G-proteins, thereby initiating

the signal transduction cascade. However, it is possible that the

selectivity between opioid peptides and opioid receptors is achieved

through mechanisms quite distinct from those at play in alkaloid

selectivity.

All known mammalian opioid peptides begin with the sequence TyrGly-Gly-Phe- (YGGF), followed by Leu or Met. Clearly, this

sequence, sometimes termed the "message," is critical to ensure

interaction with the binding pocket of all opioid receptors, but the

selectivity rests in the remaining sequence, the carboxy terminal

extension beyond the penta-peptide (ranging from 2 to 26 residues in

length), which provides the "address" (Schwyzer 1986). This carboxy

terminal domain, at least when it is long, is likely to interact with the

N-terminal domain or extracellular loops in a distinctive manner that

may contribute to selectivity and that cannot be achieved by the much

smaller alkaloids. The authors have proposed this model for kappa

selectivity (Robinson and Berridge 1993) as the unique presence of
131

negative charges were noted on the second extra-cellular loop of the

receptor, which the authors proposed to be interacting with the

positive charge of the Arg-Arg residues in the kappa-selective

prodynorphin products. The authors have already obtained some

evidence in support of this model (see below). Interestingly, a similar

model has been subsequently suggested for the thrombin receptor

(Gerszten et al. 1994). This model for opioid selectivity does not

exclude the possibility that opioid peptides, short or long, may also

achieve discrimination by other means (e.g., by interacting with

residues or sequences within the binding cavity).

The authors have embarked on a series of studies using the

construction of chimeric receptors and the use of site-directed

mutagenesis in order to examine the structural basis of receptor

selectivity and specificity. In particular, the authors are very interested

in the question of how this set of receptors achieves selectivity toward

the family of endogenous ligands. A related question is whether

peptides and opiate alkaloids bind to the receptor in the same way, or

whether the complex structure of a peptide endows it with unique

ways of interacting with the receptor protein.

Structure Function Analysis of the Receptors: The Chimera Approach

The authors have identified specific locations in each of the three

opioid receptors that are good candidates for introducing mutations in

order to make cassettes for the construction of chimeras. The

mutations have been engineered into all three receptors, and the

authors have constructed over 20 of the possible chimeras. Each

receptor can now be divided into seven distinct domains, labeled "a"

through "g" moving from the N- to the C-terminal domains. One type

of study carried out to date has revolved around delta/kappa chimeric

receptors constructed by using native restriction sites in these two

receptors. Because both the rat kappa and delta receptors contain an

Afl3 restriction site in the middle of transmembrane 3 (TM3) and a

Bgl2 site at the beginning of TM5, the authors were able to construct

six chimeras directly from the wild-type receptors. Two of the

chimeric receptors did not show any binding to [3H]ethylketo. The

arrangement of their DNA fragments appears to be correct as judged

by several restriction enzyme digests. Their inability to bind is

probably due to a disruption of some subtype-specific interactions

among the different domains in a receptor. At this stage, it was not

possible to localize these interactions because the domains involved

were relatively large; this issue is being addressed with single-segmentexchanged chimeric receptors. However, the authors were able to
132

obtain complete binding profiles for the four remaining chimeras

along with the wild-type kappa and delta receptors when they are

labeled by 1 nano-molar (nM) [3H]ethylketocyclazocine (EKC).

Multiple scatchard plot analyses were carried out using several classes

of ligands, including nonselective ligands, highly selective kappa and

delta ligands, and endogenous ligands, particularly members of the

prodynorphin family. The following conclusions can be derived

from these data:

1. The authors had hypothesized that high-affinity binding of the

prodynorphin peptides to the kappa receptor was related to the

presence of the highly negatively charged N-terminal and extracellular loop 2 in that receptor (Meng et al. 1993). The present

results seem consistent with this hypothesis. A domain that includes

extracellular loop 2 (negatively charged) appears to be particularly

critical. When replaced by the delta sequence, the resulting chimeric

receptor shows very low affinity for prodynorphin products while

retaining excellent affinity for EKC, naloxone, or naltrexone. On the

other hand, when these kappa domains are preserved but the regions

C-terminal to them are replaced with delta fragments, the resulting

receptor exhibits excellent kappa affinity. Thus, extracellular regions,

particularly extracellular loop 2, may be critical for both the high

affinity and high selectivity of DynA for the kappa receptor.

2. There is a high-affinity Tyr-Gly-Gly-Phe binding pocket in the

delta receptor. This pocket is likely localized in the TM5-TM7 region

in the delta receptor. This can be seen by comparing the binding of

delta-selective ligands (peptides or alkaloids) to a chimeric receptor

that contains domains TM5-TM7 of delta versus one that does not

(i.e., delta receptor with kappa TM5-TM7). Whenever the C-terminal

domain of delta is preserved, high delta affinity is maintained, but

replacing it with its kappa equivalent abolishes this high-affinity delta

binding.

3. Taken together, these results suggest that, in general, the delta

recep-tor binds its selective ligands differently from the kappa

receptor. Thus, the N-terminal half appears more critical for kappa

binding and the C-terminal part for delta binding. This is revealed by

the fact that a receptor with a kappa N-terminus and a delta Cterminus binds almost all ligands tested with an affinity comparable to

wild type, whereas its mirror image (delta-N-terminus/kappa-Cterminus) binds all the specific ligands with low affinity but still binds

the nonspecific ligands with good affinity, showing that the protein is

being expressed and a generic opiate binding pocket is formed.
133

4. Several other observations can be made regarding the binding of

alkaloids versus peptides, and of highly selective versus nonselective

ligands. There are also some interesting exceptions to the general

rules that the authors have tried to derive, suggesting that certain

ligands with unusual structures have unique modes of interfacing with

the receptor proteins.

Several mutants have been constructed. In the case of kappa, the

authors examined the effect of simultaneously mutating the three

negative charges on extracellular domain 2 to test the possible role of

this region in interacting with the positive charges found in

dynorphin. These mutations resulted in a small decrease in the

affinity of Dyn A, Dyn B, and alpha neoendorphin by less than one

order of magnitude. This finding suggests that while the negative

charges may play a small role in the binding of prodynorphin

products, other features of the extracellular loop may be even more

critical for kappa selectivity. Binding of EKC and norbinaltorphimine (nor-BNI) was not significantly altered by any

manipulations of this region, supporting the notion that the

extracellular loop may be particularly important in interaction with

the peptides.

Structure Function Analysis of the Receptors: The Modeling

Approach

The authors have used as a starting point the fact that all three cloned

opioid receptors interact with the Tyr-Gly-Gly-Phe sequence. The

most likely sites of interactions within this tetrapeptide are the NH3 +

of the N-terminal Tyr, the OH group on the phenyl ring of this same

Tyr1, possibly the NH group of Gly3, and the potential aromatic

interactions with the phenyl ring of Tyr1 and Phe4. These sites are

likely to bind to the receptors through hydrogen bonds, charge

interactions, or hydro-phobic interactions. Therefore, it is presumed

that there are comple-mentary sites on opioid receptors that interact

with these active groups. As a working hypothesis, it can be assumed

that these are among the amino acid residues that are conserved across

the three opioid receptors. Some of these residues should be unique

to the opioid receptor family, and distinctive from sites found on

other members of the G-protein super-family. However, this latter

criterion should not be used to exclude important residues such as the

Asp in TM3, since this negatively charged residue is used by other

receptors for ionic interactions and may also be recruited in opioid

receptors to perform a similar function. Thus, the authors sought to
134

identify residues within the TM domains that are common to delta,

mu, and kappa receptors and which, by their charge, their hydrogen

bonding potential, or their potential for interaction with aromatic

nuclei, may be involved in binding the opioid core sequence.

With these notions in mind, two models of the binding pocket of

opioid ligands have been developed, one within the authors’ group

and the other in collaboration with Dr. Henry Mosberg and

colleagues. The details of these models and their similarities and

differences are beyond the scope of this paper. Suffice it to say that

although independently derived, they share some common amino

acids as key in the binding pocket, but they differ in their orientations

of the ligands within the pocket. The advantage of having two

working models is to force researchers to consider alternatives in

interpreting empirical results. Mutation studies will first examine the

residues chosen by both models as being important in the binding.

Once so-called critical residues have been ascertained, focus will then

shift toward testing orientation of ligand within the pocket. Testing

has already begun on the proposed critical sites in the mu receptor.

Preliminary results with several mutants created based on modeling

are encouraging and show the usefulness of this modeling approach,

which has led to specific residues of the receptors in domains not

previously seen as important anchor points in the monoaminergic

receptors.

Taken together, structural studies of the opioid receptors carried out

to date, along with the findings of others in the field, strongly

reinforce the view that these receptors are much more complex than

previously anticipated, that multiple domains are used for multiple

types of inter-actions, and that small molecules versus large peptides

may interact very differently with these receptors. It is anticipated that

continued efforts in this arena will lead to a better understanding not

only of the opioid receptors but also of peptide receptors in general.

ANATOMICAL STUDIES

Regardless of their relative preferences at the pharmacological level,

opioid signaling in a particular region or circuit depends primarily on

the local opioid anatomy. For example, even if Leu-Enk exhibits a

preference for the delta receptor, if no delta sites are found in the

vicinity, but mu sites are, Leu-Enk may act as a mu agonist. When

both mu and delta sites are present, then the difference in selectivity

becomes a way in which to code the presence and concentration of
135

ligand in a more com-plex way than would be possible with one site.

If this logic is extended to multiple endogenous ligands deriving from

one or more precursor, and is interacting with multiple opioid

receptors, it can be seen how this system changes a binary signal

(receptor bound or unbound) to an analog communication

mechanism. Thus, ideally, to describe a system under-lying a given

behavior or function, it would be necessary to delineate the local

complement of receptors and endogenous ligands with their range of

affinities, selectivities, and efficacies.

The location of opioid receptors vis-a-vis their endogenous ligands

has been the subject of much interest and discussion—reports of

"mismatch" between the two (Herkenham and McLean 1986) have

described several types of lack of concordance between the

distribution of receptors and ligands. While this can be seen as the

basis of nonsynaptic communi-cation, with diffusion of the ligands to

distant sites, there are a number of alternative interpretations,

including the fact that there is not a one-to-one correspondence

between a given receptor and the selectivities of products deriving

from a single opioid precursor (not even for kappa and proDyn, as

shown above). Thus, ligand-receptor matching needs to include the

relation of all opioid receptors to all endogenous ligands.

Furthermore, the level of resolution possible with receptor

autoradiography does not allow detailed anatomical studies possible

with in situ hybridization (ISH) and immunocytochemistry (ICC) with

specific antibodies directed at the individual receptors. The use of

these approaches has only recently become possible, and these tools

can now be used to readdress the ques-tion of the anatomical

relationship between opioid peptides and receptors.

Anatomical procedures for identifying the receptor messenger

ribonucleic acids (mRNAs) that encode the three classical opioid

receptors have been developed. ISH studies demonstrate that cells

expressing the mu, delta, and kappa receptor mRNAs are differentially

distributed in the central nervous system (CNS) and spinal cord and

correspond well to known receptor binding distributions defined by

receptor autoradiography. Three separate studies were carried out

comparing the individual receptor mRNAs to their respective binding

sites using combined ISH and receptor autoradiographic techniques.

In addition, a study was completed revealing the overall distribution of

kappa receptors, in comparison to the expression of pro-dynorphin,

using adjacent sections. This body of work has been published

detailing the anatomy of the cells that express the opioid receptor

genes in relation to various known anatomical and functional
136

characteristics of the endogenous opioid system in the brain (Mansour

etal. 1993, 1994a, 1994b).

To the authors’ anatomical armamentarium have recently been added

antibodies that have been raised to nonhomologous regions of the mu

and kappa receptors. The mu antibody is the best characterized with

fairly complete immunohistochemical maps in both colchicine and

non-colchicine-treated animals. The distribution of mu receptor

protein corresponds well to mu receptor binding and mRNA

expression with high levels of expression in such regions as the striatal

patches, medial habenula, interpeduncular nucleus, and the dorsal

horn of spinal cord. The importance of developing this and other

opioid receptor antibodies lies in the higher cellular resolution that

can be achieved and in the visualization of fibers and terminals, which

is imperative in under-standing the anatomy of these receptors. Both

immunofluoresence and diaminobenzidine (DAB)/nickel chloride

visualization procedures have been developed, allowing for the direct

co-localization with mRNA probes and antibodies to other molecules

of interest.

The kappa antibody is in the latter stages of development. Specific

immunohistochemical staining is observed in such regions as the

nucleus accumbens, paraventricular hypothalamus, median eminence,

substantia nigra (pars reticulata), and periaqueductal grey. The ideal

immuno-histochemical conditions have presently, however, not been

achieved, and further studies are in progress. The delta antibodies are

in the early stages of development, and peptides are being produced

in order to inoculate rabbits. These antibodies when fully

characterized will be invaluable in studying the opioid receptor

proteins anatomically and in regulatory studies.

SUMMARY

Over the course of 1 to 2 years, the field has moved swiftly to

investigate the functional and structural properties of the newly cloned

opioid receptors. Achieving a better understanding of these

macromolecules is likely to have profound implications for drug

design aimed at the production of better analgesic drugs, for a more

fundamental under-standing of mechanisms of action of drugs of

abuse, and for a more comprehensive knowledge base regarding the

biology of opioid peptides in particular, and neuroactive peptides in

general.
137

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AUTHORS

Huda Akil, Ph.D.

Gardner C. Quarton Professor of Neuroscience

Research Scientist and Director of Neurosciences

Fan Meng, Ph.D.

Research Fellow

Alfred Mansour, Ph.D.

Research Investigator

Robert Thompson, Ph.D.

Postdoctoral Fellow

Mental Health Research Institute

University of Michigan

205 Zina Pitcher Place

Ann Arbor, MI 48109-0720
140

Guo-Xi Xie, Ph.D.

Research Scientist

Spectra Biomedical, Inc.

2nd Floor

4040 Campbell Avenue

Menlo Park, CA 94025

Stanley Watson, M.D., Ph.D.

Associate Director and Research Scientist

Associate Chair for Research

and

Theophile Raphael Professor of Neurosciences

Department of Psychiatry

Mental Health Research Institute

University of Michigan

205 Zina Pitcher Place

Ann Arbor, MI 48109-0720
141

Regulation of Acute and Chronic

Opioid Receptor Functions by

OBCAM, a Cell Adhesion-Like

Molecule

Horace H. Loh and Andrew P. Smith

INTRODUCTION

Opioid receptors were one of the first class of cell surface receptors to

be identified using in vitro binding assays with brain tissue, but for

many years efforts to purify and clone them were unsuccessful

because of their sensitivity to detergents, their heterogeneity, and the

lack of a simple biochemical assay for their function (Loh and Smith

1990). Several laboratories, including the authors’ own, reported

purification of opioid-binding proteins (Cho et al. 1986; Gioannini et

al. 1985; Maneckjee et al. 1985; Newman and Barnard 1984), but

were unable to demonstrate unequivocally that they possessed all the

pharmacological properties of in vivo receptors.

This situation has been dramatically altered by the recent successful

cloning of the delta opioid receptor from NG108-15 cells, as reported

at the end of 1992 by two independent laboratories (Evans et al.

1992; Kieffer et al. 1992). The two laboratories used a similar

approach (expression cloning), preparing a cDNA library from

NG108-15 cells, transfecting pools of this cDNA into mammalian

cells, and then assaying the cells for binding of a radioactive opioid

ligand. The cloned, expressed receptor showed typical binding

properties expected of an opioid receptor, including high affinity,

stereoselectivity, and preference for a particular class of opioids,

namely delta; in addition, Evans and colleagues (1992) demonstrated

that binding to the expressed receptor inhibited adenylyl cyclase, as is

the case with native delta-opioid receptors on NG108-15 cells.

In NG108-15 cells (Koski and Klee 1981), as well as in at least some

areas of the mammalian central nervous system (CNS) (Attali and

Vogel 1989; Blume et al. 1979; Childers and Snyder 1978; Law et al.

1981), opioid receptors are coupled to guanosine triphosphate

binding proteins (G-proteins), so it was no surprise that the predicted

amino acid sequence of the cloned opioid receptor cDNA was

homologous to other members of the G-protein-coupled receptor
142

superfamily (Dohlman et al. 1987); in particular, the sequence

displayed seven characteristic hydrophobic regions that are presumed

to span the cell membrane. The cloned delta opioid receptor

(hereafter referred to as DOR) cDNA possesses several other features

in common with other G-protein-coupled receptors, including

aspartate residues in characteristic positions in transmembrane regions

2 and 3, which are thought to be involved in ligand binding and

coupling to adenylyl cyclase; cysteine residues in extracellular loops 2

and 3, which may form a disulfide bridge between these regions;

several putative glycosylation sites in the N-terminal region; and

several putative phos-phorylation sites in the C-terminal region, which

in other G-protein-coupled receptors are thought to be involved in

regulation of the receptor during chronic agonist treatment (e.g.,

desensitization and downregulation). Among the other members of

this superfamily, the somatostatin receptors are most closely related to

the delta opioid receptor, exhibiting 35 to 40percent sequence

homology, and this is consistent with the report that somatostatin

ligands can bind to opioid receptors.

Whenever the cDNA for a receptor becomes available, it becomes of

interest to use it as a probe to screen libraries in search of structurally

similar sequences. In the case of the delta opioid receptor, this search

has taken on particular importance, for it seemed possible that the

other types of opioid receptors that have been defined

pharmacologically, in parti-cular the mu receptor (the major mediator

of opioid antinociception) and the kappa receptor, might have similar

nucleotide sequences. This has proved to be the case. Chen and

associates (1993) isolated from rat brain a cDNA that expressed

receptor selective for mu ligands such as [D-Ala2-MePhe4, Gly-ol5]-

enkephalin (DAMGO), and which was negatively coupled to adenylyl

cyclase. Yasuda and colleagues (1993) also isolated a clone from rat

brain that appears to express a receptor selective for kappa opioids.

Both of these receptors exhibit about 60 percent amino acid

homology with the delta opioid receptor as well as with each other.

The greatest homology, as expected, is in the seven putative

transmem-brane regions, while the least is in the N-terminal and Cterminal sequences, and in the second and third extracellular loops

formed by amino acids between transmembrane regions 4 and 5 and

6 and 7.

Of particular surprise and interest, however, is the very strong

sequence homology—about 90 percent—of the third cytoplasmic

loop, between transmembrane regions 5 and 6, in all three of these

opioid receptor sequences. In other members of the G-protein-
143

coupled superfamily, this region is thought to be critical in coupling

to G-proteins, and consistent with the wide variety of G-proteins

available for coupling to different receptors, the homology in this area

is relatively low even between highly related receptors. For example,

the somatostatin 1 and 2 receptors have about 40 percent sequence

identity in this region, as do the ß1- and ß2-adrenergic receptors. The

unexpectedly high homology in the case of opioid receptors suggests

that these three receptors may couple to the same G-proteins. This

raises the question of how these receptors are regulated so that they

mediate distinct physiological responses.

Studies in the authors’ laboratory have begun to address the question

of opioid receptor regulation. To determine which G-proteins the

receptors couple to, a technique involving labeling G-proteins with a

stable guanosine triphosphate (GTP) analog has been employed.

After stable transfection of mu or delta opioid receptor cDNA into

Chinese hamster ovary (CHO) cells, the authors determined the ability

of various opioid ligands to induce covalent labeling of the alpha

subunit of G-proteins by the radioactive GTP analog [32P]-alphaazidoanilido GTP. In CHO cells, four different Gas could be

separated by SDS/urea gel electrophoresis and identified by

appropriate antibodies: Go2a, Gi2a, Gi3a, and another, unidentified

Goa. All four of these Gas were labeled by selective opioid ligands

(mu or delta) in CHO cells transfected with mu or delta opioid

receptor cDNA, though there was some preference demonstrated. In

most cases, there was no correlation between the potency of a ligand

to label a G-protein and its affinity for the expressed receptor or its

potency to inhibit adenylyl cyclase.

The authors have also begun to identify the genetic elements that may

be responsible for regulating the synthesis of opioid receptors. The

sequence of the mu opioid receptor gene has been determined, and a

number of sequences have been found that fit the consensus for

certain regulatory elements. These include AP-1 and AP-2, which are

regulated by cyclic adenosine monophosphate (AMP), and NF-GMb

and NF-IL6, which are involved in the regulation of cytokine

receptors. The existence of potential cyclic AMP (cAMP) regulatory

elements is of interest, as opioid receptors in many systems inhibit

cAMP synthesis. On the other hand, the discovery of potential

immune regulatory sites is intriguing in light of accumulating

evidence of links between opioids and the immune system.

Another possibility, however, is that opioid receptors may be regulated

by some other molecule. For the past several years, the authors’
144

laboratory has characterized a protein, opioid binding cell adhesion

molecule (OBCAM), that appears to play a regulatory role in opioid

receptor function. OBCAM has homology to neural cell adhesion

molecule, amalgam, and other cell adhesion molecules, possessing

three immuno-globulin domains that are presumed to be oriented on

the extracellular surface of the cell membrane. Since it has no

putative cytoplasmic domain, it is difficult to see how it could behave

as a complete signal transducing unit, yet it might still regulate opioid

receptor function in some way. In this chapter, some of the evidence

that OBCAM plays such a role will be discussed.

ROLE OF OBCAM IN OPIOID RECEPTOR BINDING AND ACUTE

ACTION OF OPIOIDS

OBCAM was originally isolated on the basis of its ability to bind

opioids, though it required the additional presence of acidic lipids

(Hasegawa et al. 1987). As discussed above, both the lack of a

putative cytoplasmic domain in OBCAM, as well as the demonstration

that G-protein-coupled receptors bind opioids, make it unlikely that

OBCAM directly binds opioids. Nevertheless, several studies in the

authors’ laboratory indicate that OBCAM may regulate opioid

binding.

First, antibodies have been raised both to purified OBCAM and to peptides

corresponding to portions of its predicted amino acid sequence, and have

demonstrated that these antibodies inhibit opioid binding to the purified

protein as well as to brain membranes (Roy et al. 1988a, 1988b). In

addition, antibodies to OBCAM also block opioid antinociception when

injected into the brain. These antibody data do not establish that OBCAM

itself binds opioid ligands in situ, but they strongly suggest some kind of

close association between OBCAM and the opioid receptor.

A second line of evidence indicating that OBCAM regulates opioid binding

has come from studies using antisense cDNA. This technique has gained

widespread use in recent years as a relatively easy and highly specific way

of testing the role of a particular gene product in some function by

selectively inhibiting expression of that gene. The authors have applied

this technique to NG108-15 neuroblastoma x glioma hybrid cells, which

contain a homogeneous population of delta opioid receptors coupled to

adenylyl cyclase (Chang and Cuatrecasas 1979; Sharma et al. 1975a). By

transfecting neuroblastoma x glioma NG108-15 cells with antisense cDNA

to OBCAM, a stable cell line has been created (ST7-3) in which opioid

binding is greatly reduced relative to that of cells transfected with OBCAM
145

sense (ST8-4), as well as nontransfected cells (Ann et al. 1992). The

selectivity of this effect is suggested by the observation that binding of

ligands to other cell surface receptors in ST7-3 cells was unaffected.

Scatchard analysis of the binding indicated that most of the reduction was

due to a decrease in receptor number, not affinity. Moreover, the

remaining receptors could be further downregulated by chronic opioid

agonist treatment of the cells. Thus, it appears that the OBCAM antisense

has greatly reduced the number of opioid receptors on the cell without

affecting their intrinsic response to acute or chronic opioid agonist

treatment. This result again is consistent with an association between

OBCAM and another molecule functioning as the receptor.

The antisense OBCAM cDNA has also been used to create a line of

transgenic mice. The antisense was injected into mouse oocytes, which

were implanted into pseudopregnant females; upon birth and maturation,

the mice were selectively bred to create a stable line of transgenics.

Founder as well as first- and second-generation transgenic mice showed a

reduced response to the antinociceptive effect of morphine, as determined

by the tailflick test, and also reduced sensitivity to acute tolerance to

morphine. In the latter test, the animals were pretreated with a fixed dose

of morphine, followed by determination of the morphine median analgesic

dose (AD50) in the presence of a fixed dose of the antagonist naloxone.

Normal mice show an increased AD50 in the presence of naloxone, and the

higher the pretreatment dose, the higher the AD50. This increase was

greatly reduced in the transgenic mice.

These studies all suggest that OBCAM plays a role in opioid receptor

function, but do not really address the question of how it could do so. The

authors have recently shown that coupling to G-proteins is altered in ST7-3

(OBCAM antisense-transfected) cells (Gavitrapong et al. 1993). To

demonstrate this, the authors made use of the fact that cholera toxin (CTX)

induces adenosine diphosphate (ADP)-ribosylation of G-proteins only in

the presence of ligand, which promotes coupling between receptor and Gprotein. Thus ribosylation can be used as a convenient assay of the degree

of coupling induced by ligand for a specific type of receptor. In

untransfected cells or cells transfected with OBCAM sense (ST8-4), CTX

was shown to induce, in the presence of opioid agonist, ADP-ribosylation

of Gi and Go on the basis of the reactivity of SDS gel bands with

antibodies for these G-proteins. In the antisense (ST7-3) cells, in contrast,

this labeling was greatly inhibited; as shown in table 1, the median effective

dose (ED50) for DADLE to induce labeling was increased thirtyfold to

fiftyfold.
146

TABLE 1. DADLE-induced, CTX-catalyzed ADP-ribosylation.

Membrane ED50 (nM)

NG108-15 20

ST8-4 (sense) 30

ST7-3 (antisense) -1,000

The reduced coupling between receptor and G-protein in ST7-3 cells

was also manifested in reduced ability of opioid ligand to inhibit

either basal or forskolin-stimulated adenylyl cyclase. The basal data

are shown in figure 1a. While [2-D-Ala-5-D-Leu-] enkephalin

(DADLE) at
147

concentrations of 1 to 10 nanomalors (nM) inhibited cyclase approximately 50percent in untransfected or sense-transfected cells, maximal

inhibition was less than 20 percent in antisense-transfected cells, and even

then required much higher DADLE concentrations. Furthermore, the

antisense effect appeared to be specific to opioid receptors, as inhibition

of adenylyl cyclase by adrenergic and muscarinic ligands was the same

in ST7-3 cells as in ST8-4 or untransfected cells (figures 1b and 1c).

Finally, the ability of DADLE to stimulate GTPase, a normal concomitant

of coupling, was also inhibited in ST7-3 cells.

In summary, the presence of antisense OBCAM cDNA in NG108-15 cells

has somehow interfered with coupling of opioid receptors to G-proteins,

reducing the ability of the receptors to inhibit adenylyl
148

cyclase. It is not clear how OBCAM, an extracellular molecule, could

couple a seven transmembrane receptor to G-proteins, which are

located on the cytoplasmic face of the cell membrane. One possibility

is that OBCAM interaction with the receptor alters its conformation, so

that those portions of the receptor that are located in the cell, and

couple directly to G-proteins, are altered. Alternatively, the presence

of OBCAM may be necessary to promote a lipid environment in

which receptor and G-protein may couple.
149

ROLE OF OBCAM IN CHRONIC OPIOID EFFECTS

One of the signal features of opioid drugs is the ability to induce

tolerance and dependence upon chronic administration to humans or

animals. Tolerance may be defined as a state in which the dose of

drug required to achieve a given effect is larger than normal.

Dependence is a state in which regular doses of drug are required to

prevent withdrawal symptoms.

The molecular basis of opioid tolerance and dependence is unknown,

but a useful model system is provided by NG108-15 cells. When

treated chronically with an appropriate opioid agonist such as

DADLE, these cells become less responsive to opioids in a tolerantlike manner (Sharma et al. 1975b). Studies of the molecular basis of

these effects have shown that several processes underlie this reduced

sensitivity, including uncoupling of the opioid receptors from

adenylyl cyclase, an effector for opioids in this system, and

downregulation of the receptors, a process by which they are lost from

the cell surface (Law et al. 1984).

Studies in the authors’ laboratory have suggested that OBCAM plays

a role in these processes also. The fate of OBCAM during chronic

opioid treatment of NG108-15 cells has been studied by the use of

fluorescent antibody labeling in conjunction with confocal

microscopy. The antibody used was raised against a 12-amino-acid

peptide (MN-3) corresponding to a portion of OBCAM in the third

immunoglobulin domain. Initial studies demonstrated heavy

fluorescence on the surface of NG108-15 cells treated with this

antibody, which was blocked by pretreatment of the antibody with the

peptide. In addition, cells transfected with OBCAM antisense cDNA

(see above) showed much less immunofluorescence.

When NG108-15 cells were chronically treated with a delta opioid

agonist such as DADLE, there was a significant reduction in the

amount of antibody staining. The dose dependence and timecourse

of this reduction closely paralleled the downregulation of opioid

receptors; furthermore, the ability of a number of different agonists to

induce downregulation of OBCAM-like immunoreactivity also

paralleled their ability to downregulate opioid receptors. Thus delta

agonists were the most effective, while mu and kappa opioids were

much less so; the addition of an antagonist such as naloxone blocked

the downregulation of OBCAM-like immunofluorescence. The

specificity of this effect for opioid receptors was demonstrated by

control experiments in which OBCAM-like immunoreactivity was
150

unchanged following chronic treatment of the cells with ligands for

other types of receptors, such as muscarinic and adrenergic.

Furthermore, chronic treatment with DADLE had no effect on the

surface levels of neuronal cell adhesion molecule (N-CAM).

The recent cloning of the delta opioid receptor has made it possible to

study the relationship of this receptor with OBCAM more directly, by

transfection of cDNA into mammalian cells. With the collaboration of

Dr.Ping Law, the authors have obtained CHO cells stably transfected

with DOR cDNA. OBCAM expression, as measured by the

appearance of M2 antibody fluorescence, is greatly increased in these

cells, though interes-tingly, there is no correlation with increased

opioid binding; that is, the level of immunofluorescence is about the

same in the transfected CHO cells as in untransfected NG108-15 cells,

though the level of opioid recep-tor binding is several times higher in

the former (Ko and Loh, unpublished data). In addition, the authors

have stably transfected neuro 2A cells with DOR, and OBCAM

immunofluorescence does not correlate with opioid binding levels in

different stable transfectants. Nevertheless, this work, together with the

downregulation studies, indicates that manipu-lations that raise or

lower the level of opioid receptors on the surface of NG108-15 cells

result in a corresponding increase or decrease in OBCAM levels.

CONCLUSION

The cloning of mu, delta, and kappa opioid receptors has ended a

two- decade search in pharmacology, but raised new questions

concerning the mechanisms by which these receptors are regulated.

The authors’ studies with OBCAM suggest that this cell adhesion-like

molecule may play a role in both the acute and chronic actions of

opioids. Thus OBCAM seems to be necessary for normal coupling

between opioid receptors and G-proteins, as well as undergoing

downregulation in parallel to that of opioid receptors upon chronic

opioid treatment. Further studies should clarify the role of OBCAM

in opioid receptor function.

REFERENCES

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H.H. Specific reduction of delta-opioid receptor binding in

transfected NG108-15 cells. J Biol Chem 267:7921-7926, 1992.
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Attali, B., and Vogel, Z. Long-term opiate exposure leads to

reduction of the alphai-1 subunit of GTP-binding proteins. J

Neurochem 53:1636-1639, 1989.

Blume, A.J.; Lichtchstein, D.; and Boone, G. Coupling of opiate

receptors to adenylate cyclase: Requirements for Na+ and GTP. Proc

Natl Acad Sci U S A 76:5626-5630, 1979.

Chang, K.J., and Cuatrecasas, P. Multiple opiate receptors:

Enkephalins and morphine bind to receptors of different specificity. J

Biol Chem 254:2610-2618, 1979.

Chen, Y.; Mestek, A.; Liu, J.; Hurley, J.A.; and Yu, L. Molecular

cloning and functional expression of a mu-opioid receptor from rat

brain. Mol Pharmacol 44:8-12, 1993.

Childers, S.R., and Snyder, S.H. Guanine nucleotides differentiate

agonist and antagonist interactions with opiate receptors. Life Sci

33:759-762, 1978.

Cho, T.M.; Hasegawa, J.; Ge, B.L.; and Loh, H.H. Purification to

apparent homogeneity of a mu-specific opioid receptor from rat

brain. Proc Natl Acad Sci U S A 83:4138-4142, 1986.

Dohlman, H.G.; Caron, M.G.; and Lefkowitz, R.J. A family of

receptors coupled to guanine nucleotide regulatory proteins.

Biochemistry 26:2657-2664, 1987.

Evans, C.J.; Keith, D.E., Jr.; Morrison, H.; Magendzo, K.; and

Edwards,R.H. Cloning of a delta opioid receptor by functional

expression. Science 258:1952-1955, 1992.

Gavitrapong, P.; Zhang, X.; Loh, H.H.; and Lee, N.M. Transfection

of NG108-15 cells with antisense opioid-binding cell adhesion

molecule cDNA alters opioid receptor-G-protein interaction. J Biol

Chem 268, 1993.

Gioannini, T.L.; Howard, A.D.; Hiller, J.M.; and Simon, E.J.

Purification of an active opioid-binding protein from bovine striatum.

J Biol Chem 260:15117-15121, 1985.

Hasegawa, J.-I.; Loh, H.H.; and Lee, N.M. Lipid requirement for

mu opioid receptor binding. J Neurochem 49:1007-1012, 1987.

Kieffer, B.L.; Befort, K.; Gaveriaux-Ruff, C.; and Hirth, C.G. The

delta-opioid receptor: Isolation of a cDNA by expression cloning and

pharmacological characterization. Proc Natl Acad Sci U S A

89:12048-12052, 1992.

Koski, G., and Klee, W.A. Opiates inhibit adenylate cyclase by

stimulating GTP hydrolysis. Proc Natl Acad Sci U S A 78:4185-4189,

1981.
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Law, P.Y.; Griffin, M.T.; and Loh, H.H. Mechanisms of multiple

cellular adaptation processes in clonal cell lines during chronic opiate

treatment. In: Sharp, C.W., ed. Mechanisms of Tolerance and

Dependence. National Institute on Drug Abuse Research Monograph

54. Washington, DC: Supt. of Docs., U.S. Govt. Print. Off., 1984.

Law, P.Y.; Wu, J.; Koehler, J.E.; and Loh, H.H. Demonstration and

characterization of opiate inhibition of the striatal adenylate cyclase.

JNeurochem 36:1834-1846, 1981.

Loh, H.H., and Smith, A.P. Molecular characterization of opioid

receptors. Ann Rev Pharmacol Toxicol 30:123-147, 1990.

Maneckjee, R.; Zukin, R.S.; Archer, S.; Michael, J.; and OseiGyimah, P. Purification and characterization of the mu opioid

receptor from rat brain using affinity chromatography. Proc Natl

Acad Sci U S A 82:594-598, 1985.

Newman, P.L., and Barnard, E.A. Identification of an opioid

receptor subunit carrying the mu binding site. Biochemistry 23:5385-

5389, 1984.

Roy, S.; Zhu, Y.X.; Lee, N.M.; and Loh, H.H. Different molecular

weight forms of opioid receptors revealed by polyclonal antibodies.

Biochem Biophys Res Commun 150:237-244, 1988a.

Roy, S.; Zhu, Y.X.; Loh, H.H.; and Lee, N.M. A monoclonal

antibody that inhibits opioid binding to rat brain membranes.

Biochem Biophys Res Commun 154:688-693, 1988b.

Sharma, S.; Nirenberg, M.; and Klee, W. Morphine receptors are

regulators of adenylate cyclase activity. Proc Natl Acad Sci U S A

72:590-594, 1975a.

Sharma, S.K.; Klee, W.A.; and Nirenberg, M. Dual regulation of

adenylate cyclase accounts for narcotic dependence and tolerance.

Proc Natl Acad Sci U S A 72:3092-3096, 1975b.

Yasuda, K.; Raynor, K.; Kong, H.; Breder, C.D.; Takeda, J.; Reisine,

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ACKNOWLEDGMENT

This work is supported by NIH grant nos. DA70554, DA00564,

DA01583, and DA05695.
153

AUTHORS

Horace H. Loh, Ph.D.

Frederick Stark Professor and Head

Andrew P. Smith, Ph.D.

Research Associate

Department of Pharmacology

University of Minnesota Medical School

3-249 Millard Hall

435 Delaware Street SE.

Minneapolis, MN 55455-0347
154

Discrete Structural Domains and

Cell-Specific Expression Determine

Functional Selectivity of the

Dopamine and Norepinephrine

Transporters

Kari J. Buck, Dominique Lorang, and Susan G. Amara

INTRODUCTION

The dopamine (DA) transporter (DAT) and norepinephrine (NE)

transporter (NET) terminate catecholaminergic neurotransmission at

synapses by high-affinity reuptake into presynaptic terminals and are

the initial sites of action for a variety of drugs of abuse and

therapeutic antidepressants. Recent cDNA cloning studies have

demonstrated that NET and DAT are members of a family of sodium

and chloride ion-dependent carriers, and that single cDNAs for NET

and DAT effectively reconstitute many properties of the native

transporters, including appro-priate pharmacology and ion

dependence. Although catecholamine transporters have a high degree

of sequence similarity, they are distin-guished by their monoamine

substrate selectivities and their differential sensitivities to a wide

spectrum of transport antagonists. DAT mediates uptake of DA, but it

is an inefficient carrier of NE and other biogenic amines (Giros et al.

1991, 1992; Kilty et al. 1991; Shimada et al. 1991; Usdin et al. 1991),

whereas NET transports both DA and NE (Pacholczyk et al. 1991).

1-methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is also a

substrate of DAT. This compound causes a selective and irrever-sible

loss of nigrostriatal DA neurons and provides the basis for an

experimental model of Parkinson's disease (Boyson 1991; Snyder and

D'Amato 1986).

Reuptake systems for monoamines are also sensitive to a wide range

of uptake inhibitors (e.g., therapeutic drugs including antidepressants

and drugs of abuse such as cocaine). NET exhibits marked sensitivity

to many therapeutic antidepressants, including desipramine

(Pacholczyk et al. 1991). Clinical and behavioral studies of tricyclic

antidepressant drugs suggest that inhibition of NE reuptake correlates

with antidepressant activity (Baldessarini 1985). In contrast, drugs
155

such as cocaine, amphetamine, and methylphenidate, which

nonselectively inhibit DAT and NET, are poor antidepressants, despite

the fact that they have stimulant and euphoric effects in some

individuals (Baldessarini 1985).

The structural determinants responsible for the functional properties

of NET and DAT, such as selectivity for substrates and

antidepressants, shared recognition of cocaine and amphetamine, and

conserved mechanistic features (e.g., sodium and chloride ion

coupling and substrate translocation) remain to be elucidated. NET

and DAT cDNAs predict protein sequences of 617 and 618 amino

acids, and hydropathy analyses of the sequences indicate 12

hydrophobic regions proposed to represent membrane-spanning

domains (Amara and Kuhar 1993). The two transporters are most

similar in the putative transmembrane domains (TM), and least

conserved in the NH2 and COOH termini thought to be oriented on

the cytoplasmic face of the plasma membrane. A large extracellular

domain between TM3 and TM4 shows a similar lack of conservation.

In order to assign the kinetic and pharmacologic properties of NET

and DAT to general structural domains, a series of recombinant

chimeric transporter genes was generated and expressed in

mammalian cells (Buck and Amara 1994). This approach has proven

successful in the structural analysis of guanosine triphosphate binding

protein (G-protein)-coupled receptors (Frielle et al. 1988; Kobilka et

al. 1988), G-proteins (Masters et al. 1988), and ligand-gated ion

channels (Elsele et al. 1993; Li et al. 1992). Unlike other

conventional methods of mapping functional domains, such as

analyses of site-directed or deletion mutants in which the function of

interest is frequently destroyed, chimeras can provide an assayable

phenotype that allows positive inferences to be drawn from functions

associated with specific protein domains. Analyses of the functional

properties of NET/DAT chimeric transporters have delineated

structural domains that determine apparent transport affinity and

translocation selectivity of catecholamines and MPP+ (Buck and

Amara 1994), and sensitivity to desipramine and GBR12935 (1-[2-

(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine).

Pharmacologic and Kinetic Selectivity of Wild-Type Catecholamine

Transporters

NET and DAT have strikingly similar sequences, with 64 percent

amino acid identity and nearly 85 percent sequence similarity

considering conservative amino acid differences. Despite their

structural similarities, these two transporters demonstrate
156

physiologically and clinically important differences in selectivity for

substrates and for a variety of uptake inhibitors. The functional

properties of NET and DAT were examined by expression in HeLa

cells using a vaccinia virus-based transient expression system. These

studies show that NET has high apparent affinity for both NE and DA

transport kinetic constant (KT)=0.4±.1 micromolars (μM) and

0.2±0.1 μM, respectively), whereas DAT expresses lower apparent

affinity for NE and DA uptake (KT=5.7±2.7 μM and 3.2±0.4 μM,

respectively) (figure 1, see also table1). In these kinetic analyses,

differences in substrate affinity for the kinetically relevant site(s) are

reflected in the Michaelis constant (KT). However, this constant is

also influenced by rate constants for a number of events that occur

subsequent to substrate recognition (e.g., trans-location and

dissociation). In addition to the apparent affinity constant for

substrate transport (KT), a second kinetic parameter, the velocity of

transport at steadystate (Vmax), can be used to assess the capacity of a

carrier to catalyze translocation. Vmax reflects the turnover number

of the carrier, which varies with different substrates. However, Vmax

is also influenced by expression levels, and therefore cannot be

compared between cells with different numbers of carriers at the

surface. Thus, for each transporter, translocation efficacy is reported

as a rank order Vmax in cells transfected and assayed in parallel for

dopamine and norepinephrine transport (table 1). Differences in the

rank order Vmax between trans-porters reflect the relative efficiency

with which different substrates are translocated. For example, cells

transfected with DAT efficiently translocate DA as compared to NE

(rank Vmax 1:9 for NE and DA). In contrast, NET accumulates

dopamine nearly as efficiently as NE (rank Vmax 2:1 for NE and

DA). These data clearly show that KT and rank Vmax can vary

independently (figure 1).

Chimeric Catecholamine Transporters

To determine which structural domains confer distinct functional

properties of NET and DAT, a series of chimeric recombinant

transporters from NET and DAT cDNAs was constructed using a novel in

vivo method that generates chimeras which junction in regions of

sequence similarity (Buck and Amara 1994) (figure2). Briefly,

DAT/NET and NET/DAT chimeras were engineered by subcloning the

coding regions of NET and DAT in tandem into a pB SKII- plasmid

vector. The DAT/NET and NET/DAT constructs were linearized and

used to transform bacteria. The chimeric constructs are formed from the
157
158

TABLE 1. Substrate selectivity of wild-type and chimera transporters.

KT (μM) (apparent affinity constant) Vmax (uptake efficacy)

Transporte

r

NE DA Relative

(NE:DA)

Normalize

d (DA)

NET 0.4±0.1 0.2±0.1 2:1 1.6

ND 11 0.4±0.1 0.3±0.1 1:1 1.3

ND 10 0.9±0.4 0.6±0.3 1:1 1.1

ND 4 1.6±0.1 1.5±0.5 1:1 1.3

ND 3 1.7±0.5 0.8±0.2 1:1 0.6

ND 1 2.6±0.8 2.6±0.7 1:4 1.0

DAT 5.7±2.7 3.2±0.4 1:9 1.)

DN 10 2.4±0.8 2.2±0.8 1:20 1.8

DN 9 2.3±0.6 2.1±0.5 1:17 1.7

DN 3 2.0±0.6 1.1±0.4 1:5 0.8

DN2 2.1±0.3 1.8±0.2 4:5 0.9

DN1 0.4±0.1 0.2±0.1 1:2 0.7

NOTE: Kinetic analysis of substrate transport in HeLa cells

expressing chimeric transporters or wild-type NET or DAT. Chimeras

are identified as ND or DN, and numbered to reflect the

transmembrane domain near which they junction. The kinetic

constants, KT, and Vmax for each of the substrates were obtained by

nonlinear least-squares fits of substrate/velocity profiles using data

analysis program. The apparent affinity constant (KT) for transport

of the substrates NE and DA is reported as the mean KT ± SEM as

determined from three to six independent experiments performed in

quadruplicate. For each transporter, translocation efficacy is reported

as a rank order Vmax for DA and NE transport in cells transfected

and assayed in parallel.
159
160

linear DNA within the bacterial host in a process that probably

involves endogenous exonuclease digestion of linear DNA, base

pairing of homologous regions, and subsequent bacterial repair of the

plasmid. The approach used favors generation of chimeras which

junction at single sites in regions of conserved nucleic acid sequence,

and typically resulted in functional chimeric transporters. Expression

of the chimeric trans-porters in HeLa cells using a vaccinia virus/T7

polymerase-based expression system (Blakely et al. 1991) allowed an

assessment of their apparent affinity constants (KT) and translocation

efficacy (rank Vmax) for each catecholamine, and sensitivity to

uptake antagonists such as DMI and GBR 12935. To provide an

indication of the robust activity of these chimeras, the Vmax of each

chimera was compared to the wild-type DAT activity observed in a

parallel experiment (table1, normalized Vmax). Specific functional

properties of NET and DAT were found to be correlated with

particular protein sequence elements of the chimeric transporters,

allowing differential properties of the two transporters to be assigned

to specific domains. The structures of this series of chimeras, their

apparent substrate affinity constants for uptake of NE and DA, and

their selectivity for inhibition by desipramine are compared relative to

wild-type NET and DAT in figure 3.

Transport is attenuated in all chimeras that junction in a region

spanning TM5 through TM8 (including DN5, ND7, DN8, and ND8,

figure 3c). Chimera DN5 demonstrated a dramatic reduction in

transport (i.e., Vmax <5percent for NE uptake by NET) with no

decrease in apparent substrate affinity relative to DN3 and DN9.

However, surface [125I]RTI-55 binding to intact cells expressing

these chimeric transporters was approximately 40 to 50 percent of

binding to intact cells transfected with NET or 25 to 35percent of

binding to cells expressing DAT. RTI-55 is a structural analog of

cocaine with high affinity for DAT (Boja et al. 1991), which also

recognizes NET although with lower affinity. These results suggest

that this region spanning TM5 through TM8 may contain important

elements of a catalytic domain involved in substrate translocation.

However, in general the low transport capacity of chimeras

junctioning between TM5 and TM8 limited detailed analysis of their

transport kinetics, and may have restricted the ability to localize

additional determinants influencing translocation efficacy and

substrate KT which may also exist between TM5 and TM8. However,

apparent substrate affinity and translocation efficacy were readily

compared in chimeric transporters that junction in or before TM4

(e.g.,DN1, DN2, DN3, ND1, ND3, ND4) or after TM9 (e.g., DN9,

DN10, ND10, and ND11) (figure 3b).
161

Discrete Domains Influence Catecholamine Substrate Selectivity

A goal of these studies was to identify structural domains that

influence apparent affinity (KT) and catalytic efficacy (rank Vmax)

of NET and DAT. The kinetics of catecholamine uptake by wild-type

and chimeric trans-porters are summarized in table 1. The apparent

substrate affinities of DN1 are consistent with those of NET, indicating

that substitution of NET NH2-terminal sequences with that of DAT

does not reduce apparent affinity relative to NET (table 1 and figure

3). DN3 has a fivefold higher KT for DA, and fivefold higher KT for

NE than DN1 or NET (table1). These two chimeras define a domain

that has a pronounced effect on substrate KT within a region spanning

TM1 and TM3. The comple-mentary chimeras ND1 and ND3 further

underscore the importance of this region in differentiating substrate

KT values of NET and DAT. Furthermore, because ND1 has a

modest two- to threefold lower apparent affinity than ND3 for both

substrates, additional determinant(s) may reside near the aminoterminus of TM1. A region spanning TM10 and TM11 also

contributes to differences in KT between NET and DAT for

catecholamines.

DAT substrate selectivity for DA is largely due to the rate (Vmax)

with which it transports DA relative to NE (see figure1). Chimeras

DN3 and ND1 translocate DA more efficiently than NE (rank Vmax >

4:1 for DA and NE), indicating that amino-terminal determinants also

influence relative Vmax. Thus, TM1 through TM3 play an important

role in determining substrate KT, and TM2 through TM3 influence

rank Vmax. DN9 and DN10 even more closely resemble DAT in

their capacity to translocate DA relative to NE. These results suggest

that some deter-minants that enhance the efficacy with which DA is

transported relative to NE may fall within the central domain (TM5

through TM8) identified above as involved in catalyzing substrate

translocation (see rank order Vmax in table 1).

Desipramine Specificity Involves Two Structural Domains

The NET and serotonin transporters are important initial targets for a

number of tricyclic and other antidepressants used in the treatment of

human depression. An important goal of the authors’ studies was to

identify the structural domain(s) involved in differential sensitivity of

NET and DAT for antidepressants. Desipramine is one of the most

potent tricyclic antidepressants in blocking NET, but is more than a
162
163
164

thousandfold less potent as an inhibitor of DAT (figure 3a).

Antagonism of DA uptake by the chimeric transporters identifies two

domains of primary and secondary importance that are required for

the effective blockade of catecholamine transport by desipramine

(figure 3b).

The most influential determinants of desiramine selectivity for NET

lie within a region spanning TM5 through TM7, delineated in part by

ND7 and DN5, which differ seventeenfold in their sensitivity to

desipramine. These two chimeras (as well as ND8, ND10, ND11, DN1,

DN2, and DN3), each with nanomlar (nM) affinity for desipramine,

share a region of sequence from NET extending from the aminoterminus of TM5 through TM7. In contrast, chimeric transporters

that pharmacologically resemble DAT (e.g., despramine inhibition

constant (Ki) = 9.1 to 10.0 μM for ND1, DN8, DN9, DN10) all

possess sequence from the analogous region of DAT (figure 3). ND4

has lower sensitivity to desipramine than either DAT or NET,

suggesting that this chimera may reflect a disruption in desiramine

recognition rather than a shift toward DAT-like sensitivity. Several

chimeric transporters (DN2, DN3, DN5, and ND3) displayed

intermediate sensitivity for uptake antagonism by desipramine, and

delineate a secondary domain spanning TM1 through TM3 that also

influences desipramine recognition. Chimeras with NET sequence in

the primary domain and DAT sequence in the secondary domain

demonstrate intermediate selectivity for desipramine, which more

closely resembles NET (e.g., Ki = 352 nM, 178 nM, and 189 nM for

DN2, DN3, DN5) (figure 3). In contrast, a chimera with DAT

sequence in the primary domain and NET sequence in the secondary

domain displays an intermediate antidepressant sensitivity much more

similar to DAT (e.g.,ND3 with Ki= 2.7 μM). Chimera DN1 displays

specificity for desipramine consistent with NET, indicating the

cytoplasmic NH2-terminus does not contribute to this secondary

domain. Thus, secondary deter-minants for desipramine potency lie

within a region spanning TM1 through TM3, and may overlap with

the amino-terminal domain described above, which influences both

KT and rank Vmax for the catecholamines DA and NE. In addition,

the domain that has the greatest impact on desipramine Ki is within a

region spanning TM5 through TM7, and may overlap the domain

thought to be involved in catalyzing substrate translocation.

GBR 12935 Inhibition of Dopamine Uptake

GBR 12935, a potent uptake antagonist, is moderately selective for

DAT relative to NET (Anderson 1987). Chimeric transporters (e.g.,
165

ND4 and ND7 with Ki values of 3±1 nM and 18±5 nM, respectively)

delineate a domain spanning TM4 through TM7, which confers

modest selectivity for GBR 12935 (figure 3). This domain may

overlap with a region extending from TM5 through TM8, which

contributes to selective translocation of DA and MPP+ by DAT. This

domain may also overlap in part with a homologous region of NET,

which is largely responsible for desipramine selectivity. Interestingly,

antagonism of DA uptake by GBR 12935 is not impaired in ND4

relative to other chimeric trans-porters, as was inhibition by

desipramine. These results suggest that ND4 may dissociate the

inhibitory actions of GBR 12935 and desipramine.

Distribution of the Dopamine and Norepinephrine Transporters in the

Central Nervous System

The distribution of the transporters in different neuronal populations

in large part determines which substrates they are likely to encounter

and which pathways they are most likely to influence. The two

carriers show a distinct and nonoverlapping distribution. NEcontaining cell bodies in the locus coeruleus and lateral tegmentum of

the brainstem express NET messenger ribonucleic acid (mRNA),

whereas DAT mRNA is expressed in dopaminergic cell groups of the

mesencephalon, and the A12 and A13 dopaminergic cell groups of

the diencephalon (Lorang et al. 1994). The cell type-specific

expression of catecholamine transporters suggests that DAT and NET

gene expression may be closely linked to cellular mechanisms that

specify transmitter phenotype. Thus, the role of the transporters in

DAT and NET influence catecholaminergic neurotrans-mission and

appear to be determined by their distribution in the brain, as well as

differences in their pharmacologic and kinetic properties.

Studies combining in situ hybridization of DAT and NET with

immuno-histochemical detection of enzymes for catecholamine

synthesis indicate that these two transporters are expressed only in

catecholaminergic neurons. NET mRNA labeling is found in regions

of the medulla oblon-gata and pons known to contain noradrenergic

cell bodies (corresponding to the A4, A5, A6, and A7 cell groups) and

neurons in the lateral teg-mentum and nucleus of the solitary tract that

correspond to the locations of A1 and A2 catecholaminergic neurons.

The locus coeruleus A6 cell group contains the greatest density of

heavily labeled NET mRNA-containing neurons. No evidence for

NET mRNA in tyrosine hydroxylase (TH)-positive dopaminergic

neurons was observed even though NET has a higher affinity for DA

than NE (Buck and Amara 1994; Pacholcyzk et al. 1991). Despite
166

the possibility that NET could serve as the transporter for epinephrine,

no NET mRNA was detected in phenylethanolamine-N-methyl

transferase (PNMT)-immunoreactive neurons.

Combined immunohistochemical/in situ hybridization analysis (see

figure4) of NET mRNA in catecholaminergic neurons revealed that

an overwhelming majority of dopamine ß-hydroxylase (DBH)-

immuno-reactive cells in the A1-A7 cell groups also contain NET

mRNA. Noradrenergic cells do not appear to express DAT mRNA,

which is restricted to dopaminergic cells of the diencephalon and

mesencephalon of the central nervous system (CNS). Northern blots

show a single DAT
167

transcript in neurons that comprise both the mesostriatal and

mesocorti-colimbic dopaminergic pathways. Thus, neurons that send

projections to the ventral and dorsal striatum appear to express the

same DAT mRNA as do neurons that project to limbic regions,

isocortex, and the spinal cord. The cells showing the most intense in

situ hybridization to DAT mRNA are found in the substantia nigra

and the ventral tegmental area, which corres-pond to the A8, A9, and

A10 mesencephalic dopaminergic cell groups. Immunohistochemical

analysis confirms that greater than 90percent of the THimmunoreactive cells of the zona compacta and zona lateralis of the

substantia nigra, and throughout the ventral tegmental area, are also

intensely labeled for DAT mRNA. In the diencephalon, intense

hybridi-zation with DAT mRNA is found in the zona incerta in cells

presumably belonging to the A13 cell group, and moderate levels of

labeling are found over neurons in the A12 cell group in the arcuate

nucleus. Interestingly, TH-positive dopaminergic neurons in the

preoptic area, periventricular nuclei, and the posterior hypothalamus

do not appear to contain DAT mRNA, suggesting that DA release

from terminals does not undergo rapid inactivation by reuptake into

presynaptic cells. Thus, despite marked similarities in their primary

structures and shared pharmacological properties, the nonoverlapping

and differential expression patterns of DAT and NET suggest that

these transporters function independently in distinct neuronal

pathways. Moreover, the possibility that the expression or activity of

these transporters is differentially regulated in these pathways provides

a possible basis for understanding how pharmacological agents that

act on both transporters exert such diverse effects on

catecholaminergic neurotransmission.

DISCUSSION

In order to establish a potentially general approach to study the

superfamily of sodium and chloride ion-dependent carriers, the authors

constructed and expressed a series of functional recombinant chimeric

transporters in which similar sequence domains and distinct functional

properties of catecholamine transporters are exchanged (Buck and Amara

1994). These studies examine the structural domains responsible for

differential selectivity of NET and DAT for a variety of substrates (e.g.,-

DA, NE, MPP+) and uptake inhibitors (e.g.,GBR 12935 and desipramine),

and are the first step in identifying the specific structural or regulatory

determinants responsible for the distinct functional properties of NET and

DAT. Antagonist recognition and substrate translocation are likely to

involve some shared determinants of NET and DAT. However, studies
168

employing chimeric catecholamine transporters focus on structural

features unique to NET and DAT that contribute to differences in their

apparent substrate affinities, translocation efficacies, and antagonist

selectivities. These data are summarized in the model shown in figure 5.
169

Structural Determinants of Antidepressant and Catecholamine

Selectivity

Structure activity studies indicate that a protonated amine group is a

critical feature of both transported substrates and a number of

therapeutic antidepressants recognized by monoamine transporters

(De Paulis et al. 1978; Koe 1976; Maxwell and White 1978; Maxwell

et al. 1969, 1976). The terminal ammonium of monoamines and

antidepressants may associate with a negatively charged residue of the

transporters, whereas planar aromatic moieties of these compounds

may associate with an analogous surface by hydrophobic and/or van

der Waals bonding. Current studies delineate determinants within a

region spanning TM1 through TM3 which have a pronounced effect

on apparent substrate affinity. Additional determinants within a

region spanning TM2 and TM3 may be involved in selective

translocation of DA relative to NE by DAT. A homologous region of

NET may influence selective antagonism by desipramine. Within

these domains, negatively charged aspartate (D) and glutamate (E)

residues at positions NETD64/DATD68, NETD75/DATD79, and

NETE113/DATE117 are conserved in monoamine carriers and may

recognize the terminal ammonium of substrate catecholamines and

tricyclic antidepressants. NETD75 and DATD79 are of particular

interest because they are uniquely conserved in monoamine carriers.

Mutation of aspartate residue DATD79 to glutamate or to neutral

alanine or glycine residues has been reported to dramatically impair

DA uptake (Vmax values were < 7 percent to 24 percent of wild-type

values), and results in a threefold to sixfold decrease in the apparent

affinity for DA uptake (Kitayama et al. 1992). Results obtained from

studies of catecholamine transporter chimeras indicate that amino

acids in close proximity to these conserved acidic residues are

important in determining both the different apparent affinity and

differential translocation efficacy of DAT and NET and differential

sensitivity to antidepressants (figure 5). Within TM1, a single amino

acid difference (valine for isoleucine) between NET and DAT is

observed. In TM2, three conservative differences and a

nonconservation substitution (NETT99 versus DATL103) are found,

while the sequence between TM2 and TM3 is somewhat less

conserved. These results underscore the possible importance of

conservative amino acid substitutions, which have been shown to be

critical for agonist pharmacology in other membrane proteins

(Schmieden et al. 1992).

Studies of catecholamine binding to mutant adrenergic receptors

indicate that serine and aspartate residues within the hydrophic
170

regions are also important in catecholamine recognition (Chung et al.

1988; Fraser et al. 1988; Strader et al. 1988, 1989; Wang et al. 1991).

Through hydrogen bonds, serine residues may possibly interact with

meta- and parahydroxyl moieties of the phenyl ring of the substrate

catecholamines. In DAT, replacement of the serine residues at

positions DATS356 and DATS359 in TM7 by alanine or glycine has

recently been reported to cause reductions in DA and MPP+ uptake

(Kitayama et al. 1992). These serine residues are conserved in NET,

suggesting that these amino acids are not directly responsible for

differences in the transport properties of the two carriers. However,

DATS358 is not conserved in NET (which contains alanine). These

serine residues fall within a region extending from TM5 to TM7,

which in the current studies appear to be involved in translocation

selectivity, and may also play a more basic role in the mechanism of

substrate translocation. DATS358 or one of several other

nonconserved residues within this domain may therefore be important

in determining greater translocation efficacy for DA than NE by

DAT. This domain does not appear to influence apparent substrate

affinity, suggesting that substrate recognition is not altered. Thus, it

appears that this central domain is primarily responsible for events

occurring subsequent to substrate recognition. Interestingly, it has

been suggested that a substrate dissociation or recycling processes

following substrate recognition may be rate limiting in transport

kinetics (Friedrich and Bonisch 1986; Schomig and Bonisch 1988;

Zimanyi et al. 1989).

Overlapping Domains for Antagonist Affinity and Substrate

Translocation

Structure-function analysis of the chimeric transporters indicates that

overlapping domains may influence transporter affinity for

desipramine (a potent NET antagonist) and GBR 12935 (a selective

DAT inhibitor), and translocation efficacy (Vmax) (summarized in

figure 5). Chimeras junctioning between TM5 and TM8 also

demonstrate attenuated transport (i.e., the capacity of these chimeras

to transport DA is < 5 percent that of NET), suggesting that they may

define a structural domain involved in substrate translocation. Despite

the low transport efficacy of these chimeras, the potency of various

inhibitors was readily assessed. Primary determinants of desipramine

affinity are found within a region spanning TM5 through TM7

delineated in part by DN5 and ND7. Similarly, determinants of

substrate translocation selectivity lie within a region extending from

TM3 through TM9.
171

An overlapping region spanning TM5 through TM8 may also define

a domain critical for substrate translocation. These results imply that

this domain may influence transport by positioning determinants

involved in substrate translocation and antagonist recognition. To

date, no infor-mation is available on the positioning of

transmembrane domains relative to each other, or if the transporter is

multimeric. In spite of these potential limitations, all of the chimeras

reported here are functional to a significant degree, indicating that the

secondary and tertiary structures of NET and DAT are largely

maintained. Thus, structure-function studies of chimeric transporters

can provide evidence that some domains are more important for

certain functions than for others, although they may not distinguish

between direct effects on residues interacting with the substrates and

indirect effects modifying transporter conformation. As more

information becomes available on the higher order structure of

members of the family of sodium-dependent transporters, precise

inter-actions between residues and domains identified in these studies

and their role in transport will become apparent.

SUMMARY

The successful generation and functional expression of a series of

recombinant chimeric transporters, in which distinct functional

properties of NET and DAT are exchanged, have allowed the

assignment of a number of important functional properties of MPP+

and antidepressant-sensitive catecholamine transporters to specific

domains within their primary structure. These studies are the first

comprehensive structure-function analysis of members of the rapidly

growing superfamily of Na+/Cl- carriers using chimeric transporters.

This represents the first step in identifying the specific structural or

regulatory determinants that differentiate NET and DAT. An

appreciation of the potentially distinct sites for substrate recognition,

translocation, and transport inhibition of NET and DAT may facilitate

the development of more selective drugs for the treatment of stimulant

addiction, human depression, and other affective disorders.

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ACKNOWLEDGMENTS

This chapter was prepared with support from the National Institute on

Drug Abuse grants DA07595 (SGA) and DA05542 (DL), National

Institute on Alcoholism and Alcohol Abuse grant AA07468 (KJB),

and a Hitchings Award from the Burroughs Wellcome Fund (SGA).

AUTHORS

Kari J. Buck, Ph.D.

Vollum Institute for Advanced Biomedical Research

Department of Medical Psychology

Dominique Lorang, Ph.D.

Vollum Institute

Susan Amara, Ph.D.

Vollum Institute

Howard Hughes Medical Institute

Oregon Health Sciences University

2181 Southwest Sam Jackson Park Road

Portland, OR 97201-3098
176

Drug Interactions with Vesicular

Amine Transport

Doris Peter, Juan Jimenez, Yongjian Liu, Andrew

Merickel, David Krantz, and Robert H. Edwards

INTRODUCTION

A large body of evidence has implicated monoamine

neurotransmitters in a range of behavioral phenomena including

mood. Reserpine depletes monoamines and induces a syndrome

resembling depression (Frize 1954), giving rise to the monoamine

hypothesis of affective disorders. In addition, many drugs used to

treat depression act by inhibiting the reuptake of norepinephrine and

serotonin from the synapse (Axelrod et al. 1961; Iversen 1976). The

reinforcing properties of cocaine derive from its action to inhibit the

reuptake of dopamine. Antipsychotic drugs also interfere with

signaling by dopamine by interacting with dopamine receptors. Thus,

extensive pharmacologic observations have implicated monoamines in

psychiatric disease and drug abuse. In each case, the mechanism of

drug action has revealed important features of normal signaling by

monoamines, such as the role of specific receptors and transport

proteins. In the case of psychostimulants, however, the specific

mechanism of action remains unclear. In contrast to cocaine, which

blocks the reuptake of dopamine from the synapse, amphetamines

induce the release of monoamine stored within the presynaptic cell,

apparently through a mechanism fundamentally different from the

quantal release of classic synaptic transmission.

Classical synaptic transmission involves the regulated release of

neurotransmitter in response to neural activity. Although regulated

release can result from the reversal of electrogenic plasma membrane

neurotransmitter transporters by depolarization (Attwell et al. 1993;

Schwartz 1987), the vast majority of regulated release in the nervous

system results from the regulated fusion of vesicles with the plasma

membrane. Thus, synaptic transmission usually requires the storage

of neurotransmitters in specialized secretory vesicles.

Neural peptides and classical transmitters differ in the mode by which

they enter secretory vesicles specialized for regulated release. In the

case of neural peptides and hormones, protein precursors translocate
177

into the lumen of the endoplasmic reticulum during translation. The

proteins then sort into large, dense core vesicles (or secretory granules

in endocrine cells) and undergo proteolytic processing and other

modifications before regulated release. In the case of classical

transmitters, however, packaging of both newly synthesized transmitter

and transmitter accumulated by plasma membrane reuptake occurs in

the cytoplasm. Thus, storage in secretory vesicles depends on specific

transport from the cytoplasm into the vesicle.

Classical studies have identified four distinct vesicular transport

activities for monoamines: acetylcholine, glutamate, and the inhibitory

transmitters gamma-aminobutyric acid (GABA) and glycine. The

availability of bovine adrenal chromaffin granules with easily

detectable transport activity for monoamines has enabled

characterization of the bioenergetic basis for this class of

neurotransmitter transport. In contrast to the plasma mem-brane

transporters that use the Na gradient across the plasma membrane to

remove transmitter from the synapse and so terminate its action, the

vesicular transporter expressed by chromaffin granules uses the

proton electrochemical gradient produced by a vacuolar H+-ATPase

to drive transport (Johnson 1988; Kanner and Schuldiner 1987).

Specifically, the transporter exchanges two protons in the lumen of

the vesicle for one monoamine in the cytoplasm (Knoth et al. 1981).

Since the H+-ATPase usually generates a pH of approximately two

(log units) in secretory granules, this active transport system can

produce concentration gradients of up to 104 inside the vesicle

relative to outside. Other vesicular neuro-transmitter transporters

appear to use a similar mechanism. Like the vesicular amine

transporter, the transporter for acetylcholine also uses predominantly

the pH component of the proton electrochemical gradient

(Anderson et al. 1982), whereas the vesicular glutamate transporter

uses mainly the electrical component of the gradient ( y ) (Carlson et

al. 1989; Maycox et al. 1988) and the GABA transporter uses both

pH and y (Hell et al. 1990). Interestingly, the bioenergetics of

vesicular amine transport appear to have an important role in the

action of psychostimulants such as amphetamines.

Amphetamines appear to induce the release of stored monoamines by

interfering with plasma membrane transport and vesicular amine

storage rather than by inducing vesicle fusion with the plasma

membrane. Inhibitors of plasma membrane amine transport block the

effects of amphetamines, suggesting that reversal of the transporter

mediates the release of monoamines from the cytoplasm into the

synapse (Rudnick and Wall 1992a, 1992b, 1992c; Sulzer et al. 1993),
178

possibly by exchange of extracellular amphetamine for cytoplasmic

monoamine. The lipophilic properties of amphetamines may enable

them to diffuse rapidly back out of the cell after uptake, providing for

virtually unlimited exchange and net efflux of the transmitter.

Alternatively, protracted uptake of the amphetamine due to rapid

diffusion back across the plasma membrane may eventually run down

the Na+ gradient that drives transport, allowing monoamines to

equilibrate across the plasma membrane in accord with their

concentration and potentially accounting for the associated neural

toxicity. In the case of either model, however, emptying the

cytoplasmic pool of monoamine into the synapse would have little

physiological effect unless the normally low cytoplasmic

concentrations are increased by efflux from the storage vesicles.

The mechanism by which amphetamines induce the release of stored

monoamines into the cytoplasm remains uncertain. Amphetamines

may exchange for monoamines in the lumen of the vesicle. Recent

obser-vations, however, suggest that amphetamines act as weak bases

to disrupt the pH gradient across the vesicle membrane (Schuldiner et

al. 1993b; Sulzer and Rayport 1990). Previous work has shown that

in the presence of pH, chromaffin granules retain loaded

monoamines for up to an hour (Maron et al. 1983). In the absence of

pH, previously loaded mono-amines rapidly leak from the vesicle,

suggesting that pH prevents reversal of the transporter. Even without

weak bases such as the amphetamines, efflux could occur under

conditions of energy failure that deplete the ATP in nerve terminals

and allow pH to dissipate. The studies describing efflux did not,

however, clearly indicate a role for the transporter in efflux. To

understand these and other questions about the role of vesicular

neurotransmitter transport in synaptic transmission, neuropsychiatric

disease, and drug abuse, the authors have isolated cDNA clones for

several vesicular neurotrans-mitter transporters and developed

biochemical assays to characterize their functional properties,

including their interaction with psychostimulants.

VESICULAR AMINE TRANSPORT CONFERS RESISTANCE TO

MPP+

The potent neurotoxin N-methyl-4-phenyltetrahydropyridine (MPTP)

produces a syndrome with remarkable clinical and pathological

similarity to idiopathic Parkinson's disease (PD) (Langston et al.

1983). As with PD, MPTP produces clinical bradykinesia and

rigidity. Pathologically, MPTP also results in the relatively selective
179

degeneration of dopaminergic neurons in the substantia nigra. In

addition, the MPTP syndrome responds to L-dopa, with the eventual

development of typical disabling dyskinesias.

The strong resemblance between MPTP toxicity and idiopathic PD has

suggested that the study of MPTP toxicity will reveal mechanisms that

also participate in the pathogenesis of PD. As a neutral lipophilic

compound, MPTP easily penetrates the blood-brain barrier.

Monoamine oxidase B, presumably expressed by glia, then converts

MPTP to the active metabolite N-methyl-4-phenylpyridinium (MPP+)

(Heikkila et al. 1984; Langston et al. 1984; Markey et al. 1984).

Plasma membrane amine transporters recognize and accumulate

MPP+ within monoamine cell groups, accounting for the selectivity of

degeneration (Javitch et al. 1985). Inside the cell, MPP+ enters

mitochondria and inhibits respiration, apparently at the level of

complex I in the respiratory chain (Krueger et al. 1990).

A growing body of evidence supports the relevance of MPTP toxicity

for idiopathic PD. The drug selegiline hydrochloride prevents the

MPTP syndrome by inhibiting monoamine oxidase B and also

appears to slow the rate of progression in PD (Parkinson Study Group

1989). Further, defects in complex I of the respiratory chain appear

in PD as well as MPTP toxicity (Mizuno et al. 1989; Ozawa et al.

1990; Parker et al. 1989; Shoffner et al. 1991). However, particular

features of the MPTP syndrome are still not understood, and these

features may play an important role in idiopathic PD.

Adrenal chromaffin cells express a plasma membrane norepinephrine

transporter and accumulate large amounts of MPP+ after systemic

injection of MPTP, but, in contrast to dopaminergic neurons in the

substantia nigra, these cells do not degenerate (Johannessen et al.

1985; Reinhard et al. 1987). Similarly, rat pheochromocytoma PC12

cells (derived from the adrenal medulla) accumulate MPP+ through a

plasma membrane amine transporter but show toxicity only to

extremely high concentrations (Denton and Howard 1987; Snyder et

al. 1986). Although inhibition of plasma membrane amine transport

blocks MPP+ toxicity entirely in PC12 cells, the Chinese hamster

ovary (CHO) fibroblast cell line lacks plasma membrane amine

transport activity and shows more sensitivity to MPP+ than do PC12

cells. Thus, this potent neurotoxin appears to have more toxicity for a

fibroblast cell line than for a neural cell line.

To understand the basis of resistance to MPP+, the authors transferred

DNA sequences from the relatively MPP+-resistant PC12 cells into the
180

relatively MPP+-sensitive CHO fibroblasts and selected the

transformants in MPP+. After selection for several weeks, one colony

of resistant cells appeared. To dissect the mechanism of their

resistance to MPP+, the authors first determined whether the toxin

affected respiration (Liu et al. 1992a). In contrast to wild-type CHO

cells, which show the rapid inhibition of respiration by MPP+, the

resistant cells showed no inhibition, indicating that resistance did not

derive from a compensatory mechanism but rather from a primary

failure of toxin action. The authors also determined that the resistant

cells showed wild-type sensitivity to the other complex I inhibitor

rotenone, indicating specificity of the resistance mechanism for

MPP+. The authors then found that reserpine completely abolished

resistance to the toxin and did not affect the sensitivity of wild-type

cells. This suggested that sequences encoding vesicular amine

transport had transferred from PC12 cells to CHO fibroblasts. It was

presumed that the transporter protects against the toxin by

sequestering it in vesicles, away from its primary site of action in

mitochondria. To confirm this mechanism of resistance, CHO cells

were loaded with large amounts of exogenous dopamine and the

intrinsic fluorescence of this transmitter was used to determine its

intracellular localization. Whereas wild-type CHO cells showed

diffuse staining, MPP+-resistant cells showed a striking, particulate

pattern that reverted to wild type in the presence of reserpine, strongly

supporting the hypothesis that vesicular amine transport protected the

resistant cells by sequestering the toxin in vesicles.

To isolate the sequences responsible for resistance to MPP+, the

authors used plasmid rescue (Liu et al. 1992b). Retransfection of the

rescued plasmids led to the eventual isolation of a single clone that

conferred resistance to MPP+. This clone also conferred vesicular

amine transport as determined by dopamine-loaded fluorescence,

even in transfected cells not selected in MPP+. Also developed was a

quantitative assay for vesicular amine transport using membrane

vesicles from the transfected cells. Briefly, the cells are disrupted at

narrow clearance (10 micromolars (μm)), the debris sedimented by

centrifugation at low speed, and the supernatant incubated in the

presence of tritiated monoamine for varying intervals, then rapidly

diluted, filtered, and the bound radioactivity measured. As expected,

transport activity depends on the presence of pH generated by the

vacuolar H+-ATPase; it also shows an affinity for monoamine

substrates in the low micromolar range and inhibition by low

nanomolar concentrations of reserpine. Surprisingly, tetrabenazine

inhibited transport only at high concentrations, but cocaine and

tricyclic antidepressant did not inhibit the activity at all. Thus, the
181

cDNA conferred virtually all of the physiological and

pharmacological characteristics expected for the vesicular amine

transporter.

The sequence of the cDNA conferring MPP+ resistance and vesicular

amine transport predicted a novel protein with 12 transmembrane

domains (Liu et al. 1992b). Although many transport proteins are

predicted to have 12 transmembrane domains, the primary amino acid

sequence of the vesicular amine transporter showed no similarity to the

plasma membrane neurotransmitter transporters, or other mammalian

transporters and thus appeared to define a novel mammalian gene family

that is now known to include the vesicular transporters for other

neurotransmitters such as acetylcholine (Erickson et al. 1994; Roghani et

al. 1994; Varoqui et al. 1994) (figure 1). However, the first six

transmembrane domains of the vesicular amine transporter show weak but

definite homology to a class of bacterial antibiotic resistance proteins (Liu

et al. 1992b). Interestingly, these proteins transport antibiotics out of

bacteria, a phenomenon topo-logically equivalent to the transport of

MPP+ into vesicles. Further, the bacterial transporters also act by proton

exchange (Kaneko et al. 1985). In the case of the bacterial multidrug

resistance transporter, reserpine inhibits its activity (Neyfakh et al. 1991).

Thus, the vesicular amine transporter shows functional as well as structural

similarity to these bacterial proteins. The relationship suggests that

vesicular neurotransmitter transport evolved from these ancient

detoxifying systems. Together with cloning of the vesicular amine

transporter by selection in MPP+, the relationship raises the possibility that

vesicular transport plays two roles in the nervous system: one in

packaging transmitter for regulated release and the other in neural

protection. However, MPTP or another exogenous toxin has not been

identified in idiopathic PD (Tanner and Langston 1990).

Vesicular transport may protect against the normal transmitter dopamine

itself. Monoamines and dopamine in particular oxidize very easily,

producing free radicals that injure neural cells by a mechanism that does

not involve interaction with a specific receptor (Cohen 1990; Michel and

Hefti 1990; Rosenberg 1988). Vesicular amine transport would clearly

protect against this form of endogenous toxicity as well as against the

toxicity of MPP+. Thus, a defect in vesicular amine transport could

contribute to the pathogenesis of idiopathic PD. The toxicity associated

with amphetamines may also result from the efflux of monoamine stores

into the cytoplasm. Indeed, recent imaging studies of primary dopaminergic neuronal cultures show localization of free-radical injury induced

by amphetamines to the sites of vesicle accumulation (Cubells et al.

1994).
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183
184

TWO DISTINCT GENES ENCODE VESICULAR AMINE

TRANSPORT

Classical studies have shown that monoamine cell populations in the

central nervous system (CNS) express vesicular amine transport

activity that is inhibited by reserpine and tetrabenazine. However,

Northern blot analysis of polyA+ ribonucleic acid (RNA) from

different tissues including the brain showed expression of sequences

encoding the chromaffin granule amine transporter (CGAT or

VMAT-1) in only the adrenal gland. Although low levels of

expression or expression by a small proportion of cells could account

for the failure to detect a signal, the authors pursued the alternative

possibility that the brain expresses a distinct vesicular amine

transporter. Screening a brainstem cDNA library with the CGAT

cDNA as probe under moderately reduced stringency revealed a

second distinct but closely related transporter, originally termed the

synaptic vesicle amine transporter (SVAT or VMAT-2) (Liu et al.

1992b) (figure 2). Northern analysis showed expression of this

sequence in the brainstem but not the adrenal gland. In situ

hybridization also showed expression by the expected dopaminergic

cell groups in the substantia nigra and ventral tegmental area, the

noradrenergic neurons of the locus coeruleus, and serotonergic

neurons of the dorsal raphe, consistent with the previously

demonstrated recognition of multiple monoamine transmitters with

similar affinity by the transporter from bovine chromaffin granules.

Thus, chromaffin cells and central neurons express distinct but highly

related vesicular monoamine transporters.

Surprisingly, an amine transporter purified from bovine chromaffin

granules corresponds more closely to the central rat transporter than to

the adrenal transporter (Howell et al. 1994). Purification of proteins

labeled by 3H-reserpine had in fact yielded two proteins that differ in

isoelectric point (Stern-Bach et al. 1990), with the sequence of one

corresponding to VMAT-2 (Howell et al. 1994). Additional study has

further shown that the bovine and rat adrenal glands contain both

transporters, although the rat adrenal contains an overwhelming

preponderance of the expected VMAT-1 transporter (Peter, et al., in

press). Further, the purified bovine transporter showed high sensitivity to

inhibition by tetrabenazine (Howell et al. 1994), whereas rat VMAT-1 did

not (Liu et al. 1992b). To under-stand the basis for these differences in

pharmacology as well as to identify other differences that may play a role

in synaptic transmission and possibly protect against neural degeneration,

the authors have characterized the functional properties of cloned VMAT1 and VMAT-2 (Peter et al. 1994).
185

Using expression in a heterologous system, it has been determined that

VMAT-1 and VMAT-2 differ in their physiological properties as well as

in their pharmacology (Peter et al. 1994). Both VMAT-1 and VMAT-2

protect against MPP+ toxicity after expression in CHO cells, and these

stably transformed cell lines have been used to determine their functional

characteristics. VMAT-2 has an approximately twofold to threefold

higher affinity for most monoamine substrates than VMAT-1 (table 1).

Both transporters have the highest affinity for serotonin, followed by

dopamine, then norepinephrine and epinephrine. However, the transporters differ dramatically in their affinity for histamine, with low micromolar concentrations inhibiting transport of 3H-serotonin by VMAT-2

but two orders of magnitude more required to inhibit VMAT-1.

To further compare the physiologic and pharmacologic properties of

VMAT-1 and VMAT-2, the authors have investigated the interaction of

the transporters with the antihypertensive drug reserpine. Using membranes prepared from stably transfected CHO cell lines, it has been found

that reserpine inhibits transport of amines by both VMAT-1 and VMAT-2

with high potency and binds to VMAT-1 with two distinct affinities (Peter

et al. 1994; Schuldiner et al. 1993a). Previous work had shown that

monoamines inhibit reserpine binding with an affinity similar to their

apparent affinity as substrates for transport, indicating that reserpine binds

at or near the site of substrate recognition. In addition, the imposition of

pH accelerates the rate of reserpine binding, suggesting that reserpine

can detect conformational changes in the protein that occur during the

transport cycle (Rudnick et al. 1990; Scherman and Henry 1984; Weaver

and Deupree 1982). Both of these observations have been confirmed for

VMAT-1 expressed in CHO cells (Schuldiner et al. 1993a). Binding to

reserpine also enables quantitation of the transporter and hence

calculation of the turnover number for VMAT-1 (~10/min) and VMAT-2 

400/min at saturating substrate concentrations and 29°C (Peter et al.

1994). Thus, the transporter expressed by dopaminergic cells susceptible

to MPTP (VMAT-2) has a higher apparent affinity for substrates and a

faster turnover than the transporter expressed by more resistant cells in the

adrenal medulla (VMAT-1) and so cannot account for the differential

vulnerability seen in MPTP toxicity and PD. Rather, the results suggest

that vesicular amine transport acts to protect against endogenous or

exogenous toxins in both cell types but does not suffice to protect

neurons in the substantia nigra.

In contrast to the apparent similarity of their interaction with reserpine,

VMAT-1 and VMAT-2 differ in their interaction with another inhibitor of

transport, tetrabenazine. As noted above, high concentrations of tetra-
186
187

TABLE 1. Apparent affinity of VMAT-1 and VMAT-2 for

monoamine substrates and MPP+. Standard transport assay (Liu et al.

1992b) was performed at 29oC for 2 minutes using 3H-serotonin.

The values indicate the Km for serotonin and the Kis for other

compounds.

Substrate VMAT-1 VMAT-2

μM μM

Serotonin 0.85 ± 0.23 0.19 ± 0.04

Dopamine 1.56 ± 0.35 0.32 ± 0.04

Epinephrine 1.86 ± 0.11 0.47 ± 0.05

Norepinephrine 2.5 ± 0.4 0.33 ± 0.06

Histamine 436 ± 36 3.06 ± 1.0

MPP+ 2.8 ± 0.6 1.6 ± 0.45

SOURCE: Reproduced from Peter et al. 1994 with the permission of

the American Society for Biochemistry and Molecular Biology.

benazine are required to inhibit transport by VMAT-1, whereas the

purified bovine chromaffin granule transporter shows sensitivity to

low nanomolar amounts. Consistent with the sequence similarity to

the purified bovine transporter, VMAT-2 also shows sensitivity to

nanomolar concentrations of tetrabenazine (figure 3) (Peter et al.

1994). The differential inhibition of VMAT-1 and VMAT-2 by

tetrabenazine, although not anticipated, does account for several

classic pharmacological observations. In contrast to reserpine, which

depletes both peripheral and central monoamine stores, tetrabenazine

depletes predominantly central stores and so causes less hypotension

(Carlsson 1965). Differences in the turnover of monoamine stores in

the adrenal gland and the brain had been invoked to explain this

differential effect (Scherman and Boschi 1988). However, differential

inhibition of VMAT-1 and VMAT-2 by tetrabenazine now seems far

more likely to account for the observations.

In further contrast to reserpine, classical studies have shown that only

large amounts of monoamine transmitters displace 3Hdihydrotetrabenazine from the bovine transporter (Scherman and

Henry 1984) and pH does not
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189

influence binding, indicating that tetrabenazine does not bind at the

site of substrate recognition and recognition of the drug does not

change during the transport cycle. However, tetrabenazine can

prevent reserpine binding, suggesting an allosteric interaction between

the two sites (Darchen et al. 1989). In the case of VMAT-1 and

VMAT-2, tetrabenazine also inhibits 3H-reserpine binding but with

distinct potencies that correspond to the differential sensitivity of

transport to tetrabenazine (Peter et al. 1994). Thus, the difference in

tetrabenazine sensitivity between VMAT-1 and VMAT-2 does not

reflect a difference in the interaction between these two sites. Rather,

additional study with 3H-dihydrotetrabenazine has shown binding to

VMAT-2 but not VMAT-1, indicating a simple difference in drug

recognition (Peter et al. 1994). The vesicular amine transporters also

differ in their interaction with psychostimulants.

VESICULAR AMINE TRANSPORT AND PSYCHOSTIMULANTS

The authors have found that the two vesicular amine transporters

differ in their interaction with amphetamines. Methamphetamine

inhibits the transport of 3H-serotonin by VMAT-2 much more

potently than transport by VMAT-1 (figure 4) (Peter et al. 1994).

Although amphetamines can inhibit transport by the dissipation of

pH (Sulzer and Rayport 1990), the differential inhibition of VMAT1 and VMAT-2 and the stereospecificity of the inhibition make a

direct interaction far more likely. Indeed, meth-amphetamine inhibits

reserpine binding to VMAT-2 with greater potency than to VMAT-1,

indicating interaction with the site of substrate recog-nition (Peter et

al. 1994). Nonetheless, the significance of this interaction for the

mechanism by which psychostimulants induce monoamine efflux

remains uncertain. In particular, the lipophilic nature of

amphetamines may not require specific transport into vesicles to

dissipate pH. Alternatively, recognition by the transporter may

promote efflux through an exchange mechanism. However,

dissection of the role that the transporter plays in efflux induced by

dissipation of pH and efflux induced by amphetamines requires the

development of an appropriate heterologous expression system.

Since amphetamines act by inducing the release of stored monoamines,

the site of storage also appears critical to their action as toxins. Indeed,

amphetamines produce free radical injury localized to the sites where

vesicles accumulate (Cubells et al. 1994). The site of monoamine storage

appears to differ from other classical transmitters. Small (40 nm), clear

synaptic vesicles contain such classical transmitters as acetylcholine,
190
191

GABA, glycine, and glutamate; they mediate typical fast synaptic

trans-mission and cluster over the nerve terminal (De Camilli and Jahn

1990; Sudhof and Jahn 1991; Trimble et al. 1991). However,

monoamines appear to be stored with neural peptides in larger, dense

core vesicles in at least some tissues such as the adrenal gland (and

PC12 cells). In contrast to synaptic vesicles, dense core vesicles (or

secretory granules in endocrine cells) mediate the relatively slow

release of neuromodulators and occur in the cell body and dendrites

as well as the nerve terminal. Thus, the site of monoamine storage has

profound consequences for its role in signaling. Interestingly,

smaller, occasionally dense cored vesicles that cluster over the synapse

appear to store monoamines in the CNS.

Since localization of the vesicular amine transporters determines the site

of monoamine storage, the authors have examined the distribution of

endoge-nous VMAT-1 in the neuroendocrine PC12 cell line using a

polyclonal antibody generated against a peptide derived from the Cterminus of the protein. By both immunofluorescence and density

gradient centrifugation through several different media, VMAT-1 sorts to

dense core vesicles (figure 5), accounting for the pattern of monoamine

storage (Liu et al. 1994). Only small amounts of immunoreactive

material appear in lighter synaptic-like microvesicles. Thus, in contrast to

the numerous peptides that sort to the regulated secretory pathway,

VMAT-1 is the first integral mem-brane protein identified that is

preferentially expressed on dense core vesicles rather than synaptic

vesicles. Since neural peptides apparently sort to this pathway by

aggregation (Burgess and Kelly 1987; Chanat and Huttner 1991), the

availability of a membrane protein may enable identi-fication of a specific

sorting signal. The identification of this signal may then help to explain

how aggregated lumenal proteins such as neural peptides sort to the

pathway. In addition, VMAT-1 contains signals for endocytosis,

accounting for the detection of transport activity using membranes from

CHO cells (Liu et al. 1992b). However, the storage of central

monoamines in smaller vesicles requires explanation. It may derive from

the expression of a transporter (VMAT-2) with distinct sorting sequences

or from expression within a different cell type. As a result of sorting to

different types of vesicle, monoamines may play distinct roles in synaptic

transmission and influence the activity of amphetamines.

CONCLUSIONS

The authors have used selection in the neurotoxin MPP+ to isolate a

cDNA clone encoding vesicular amine transport. The protein protects
192

against MPP+ by sequestering it in vesicles, away from its primary site of

action in mitochondria. Interestingly, the sequence of the cDNA predicts

a protein with 12 transmembrane domains but no strong relationship to

previously reported sequences other than several bacterial antibioticresistant proteins, suggesting evolution from ancient detoxification

systems. Molecular cloning has further demonstrated that two distinct
193
194

proteins mediate vesicular amine transport in the adrenal gland and

central nervous system (VMAT-1 and VMAT-2, respectively). Using

membrane vesicles from cells transfected with the two cDNAs, it has

been found that they differ in physiological and pharmacological

properties, including the interaction with amphetamines. In

conjunction with the development of appropriate expression systems

and efflux assays, the availability of the cDNA clones will enable

scientists to dissect the mechanism of amphetamine action. In

particular, does amphetamine-induced efflux involve reversal of the

vesicular transporter? Do amphetamines simply dissipate pH or do

they interact directly with the transport protein? The storage of

monoamines within dense core vesicles and synaptic vesicles will also

influence the site of monoamine release and hence the

psychostimulant and neurotoxic action of amphetamines. It has been

found that VMAT-1 sorts preferentially to dense core vesicles in PC12

cells. To understand the psychostimulant and neurotoxic actions of

amphetamines, researchers must now examine the subcellular

localization of VMAT-2 in the CNS. In particular, how does the

intracellular trafficking differ from VMAT-1 in the adrenal gland and

PC12 cells? What is the mechanism of sorting?

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AUTHORS

Doris Peter, B.S.

Graduate Student

Microbiology and Immunology

Juan Jimenez, B.S.

Medical Student

Yongjian Liu, M.D.

Postdoctoral Fellow

Andrew Merickel, B.A.

Gradute Student

Neuroscience

David Krantz, M.D., Ph.D.

Psychiatry resident
200

Departments of Neurology and Biological Chemistry

Molecular Biology Institute

UCLA School of Medicine

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Los Angeles, CA 90024

Robert H. Edwards, M.D.

UCSF Neurology

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Strategies for Identifying Genes

Underlying Drug Abuse

Susceptibility

John C. Crabbe, Kari J. Buck, Pamela Metten, and

John K. Belknap

INTRODUCTION

It is becoming widely accepted that most, if not all, drug responses are

subject to influence by genetic factors. The magnitude, duration, and

even the quality or direction of response can differ among individuals

with differing genotypes. Most drug responses are not simple genetic

traits; rather, multiple genes can be demonstrated to influence a given

response. In addition, any gene is likely to affect multiple traits, a

condition called pleiotropism. To the extent that two traits are pleiotropically influenced by some of the same genes, they are said to be

genetically correlated. Use of genetic animal models has achieved

significant progress in the analysis of drug response traits.

The goal of this chapter is to discuss two related methods for tracing

the pathways from genes to drug-related behaviors. An example of

one method, Quantitative Trait Locus (QTL) gene mapping, will

demonstrate that a gene has been identified that appears to affect the

severity of withdrawal from multiple drugs. The utility of this method

for extrapolating animal model findings to humans will be discussed.

STUDIES WITH PANELS OF INBRED STRAINS

One of the classic methods of pharmacogenetics depends upon the

analysis of inbred strains. In a seminal report, McClearn and Rodgers

(1959) reported that inbred mouse strains differed markedly in their

consumption of alcohol solutions. Because an inbred strain is created

by mating close genetic relatives for many generations, all mice of the

same sex within an inbred strain are virtually identical genetically.

There are more than 100 inbred strains of mice commercially

available. If individuals from several inbred strains are tested for a

response in carefully controlled environ-mental conditions,

differences among strain mean values are due to genetic differences,

while pooled individual differences within strains are by definition
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nongenetic or environmental (see figure 1). Therefore, partitioning

variance in responses leads to a direct estimate of the proportion of

trait variability due to genetic influences, technically known as the

heritability (Falconer 1989).

Comparisons of multiple strains on multiple traits can then be used to

estimate genetic correlations. Statistically, this is accomplished by

partitioning trait covariance into among-strain and within-strain components (Falconer 1989). An experiment testing two strains for two

traits is uninformative: multiple strains are required before

correlations can be estimated with any degree of confidence. How

many strains should be examined depends on the genetic structure of

the traits examined, which is usually not known. One estimate of the

number suggested in practice as sufficient to detect genetic

correlations of modest degree with modest statistical power is 7 to 10

strains (Crabbe et al. 1990).

In one example of this approach, mice from 15 inbred strains were

tested for the severity of acute withdrawal following intraperitoneal

(IP) injection of 4 grams per kilogram (g/kg) ethanol by assessing the

magnitude of their handling-induced convulsions over several hours.

The same mice were tested 1 to 2 weeks later for withdrawal severity

after 60milligrams per kg (mg/kg) pentobarbital. Strains differed

significantly in withdrawal from both drugs. Figure 2 shows the

genetic correlation resulting from the analysis of mean strain

responses on the two traits. The significant corre-lation seen suggests

that about half the genetic variability in each response is shared, and is

presumably due to the pleiotropic influence of some genes on both

responses (Metten and Crabbe 1994). Significant genetic correlations between ethanol and nitrous oxide withdrawal severity have

also been reported in a similar analysis of a set of recombinant inbred

strains (Belknap et al. 1993a). Studies with mice selectively bred to

express severe (withdrawal seizure prone, WSP) or mild (withdrawal

seizure resistant, WSR) ethanol withdrawal handling-induced

convulsions have revealed that they differ in severity of withdrawal

from diazepam, abecarnil, pentobarbital, phenobarbital, nitrous oxide,

several other alcohols, and acetaldehyde. WSP mice have more severe

acute or chronic withdrawal than WSR mice from all the above agents

(Belknap et al. 1987, 1988, 1989; Crabbe et al. 1991). Together,

these results suggest that some genes affect withdrawal from several

different central nervous system depressant agents.
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A large literature documenting strain differences in response to

virtually all abused drugs has accumulated over the years. A major

advantage of inbred strains is their genetic stability, so studies

performed 10 years ago can be directly compared with those

employing the same strains performed last week. Strain differences in

drug responses have been reviewed (Broadhurst 1978; Crabbe and

Harris 1991).

A systematic attempt to determine patterns of genetic commonality has

recently been undertaken in Portland. Over a 4-year period, 15 inbred

strains of mice were studied after administration of saline or 1 of 4 doses

of morphine (Belknap et al., submitted), ethanol (Crabbe et al. 1994a),

diazepam (Gallaher et al., submitted), or pentobarbital (Crabbe et al., in

process). To the extent possible, the same battery of tests was employed.

All drugs were tested for their effects on open-field activity and body

temperature; in addition, preference drinking of different concentrations

of the drugs in tap water, and subsequently in saccharine solutions, was
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determined (Belknap et al. 1993b, 1993c; Belknap et al., in process).

Other drug-specific responses were also examined (e.g.,loss of righting

reflex after ethanol and pentobarbital).

An example of results from these studies is given in figure 3. It can be

seen that strains differed markedly in the sensitivity to the locomotor

stimulant effects of IP morphine. Not only were there quantitative
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differences in response magnitude, but qualitative differences could

be seen: the A/HeJ, DBA/1J, and DBA/2J strains responded with

reduced activity at all morphine doses. Similar qualitative strain

differences in morphine-stimulated activity have been known for

many years (Shuster et al. 1975). Strain differences of the magnitude

shown in figure 3 were detected for virtually all responses to all drugs.

Two traits were tested for all four of the drugs examined. To identify

common patterns of genetic influence on these traits, a

multidimensional scaling (MDS) analysis was performed (Kruskal

1964; Kruskal and Wish 1978). Mean locomotor activity, monitored

for 15 minutes, and the mean change in body temperature 30 minutes

after injection were calculated for each strain. For each variable, strain

mean values for the saline groups were subtracted from the drugdosage group means. A (genetic) correlation matrix among strain

means was calculated and subjected to least squares MDS. The results

of this analysis are shown in figure 4. The resulting plot shows the

position of each drug dose response relative to all others, mapped by

its weighting on the two principal dimensions identified by the MDS

analysis. Linear distance represents similarity among the plotted

variables in the pattern of inbred strain (genetic) differences. Points

close together share a great deal of common genetic influence (i.e.,

are genetically correlated), while points far apart do not. The

numbers near each point identify the dose in milligrams per kilogram

or grams per kilogram of EtOH. The two primary dimensions (linear

components) mapped accounted for 85 percent of the total genetic

variance in this data set.

Several results of this analysis can be identified. Different doses of

the same drug on a given response showed a substantial degree of

common genetic influence; this was to be expected. It also appeared

that both activity and temperature responses to morphine were

genetically related at all four doses. This cluster of responses was

reasonably distinct from other clusters of variables/doses/drugs. In

addition, most doses of diaze-pam and pentobarbital (for both activity

and temperature responses), and the activating effects of moderate

doses of ethanol, were grouped in a common cluster. All three of

these drugs influence gamma aminobutyric acid A (GABAA)

receptor-gated chloride ion conductance (Harris and Allan 1989;

Macdonald and Olsen 1994). Therefore, modulation of this receptor

complex is a possible common mechanism for the mediation of the

genetic similarity of this response cluster. It is of interest that ethanol

effects on body temperature were genetically distinct from ethanolinduced activity responses. It seems reasonable to suggest that ethanol
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hypothermia is not likely to be influenced to a major degree by the

genetically determined mechanisms underlying pentobarbital and

diaze-pam hypothermia. Finally, the effects of the lowest doses of

ethanol and pentobarbital on activity appeared to be genetically

distinct from the higher doses, and are similar to the effects of the

lowest dose of pentobarbital on body temperature.

This is only one example of the application of multivariate methods to

the analysis of genetic similarity among drugs and responses. There

is an accumulating wealth of knowledge about drug responses in a few

specified genotypes. It would be of interest to know, for example,

whether the several drug responses hypothesized to index drug

reward, intravenous (IV) self-administration, preference drinking,

place-conditioned and taste-conditioned responses, and forward

locomotion and its sensitization with repeated administration of drug

shared a common genetic influence. Extension of multiple-strain

studies to include neurochemical and neuro-pharmacological

responses would be of utility in identifying common mechanisms of

drug action.

QTL GENE MAPPING

Traditional analyses of drug sensitivity using genetic animal models

such as those described above are able to estimate degree of genetic

influence, and to identify response clusters of genetic

codetermination. Many studies not reviewed above have also

employed genetic animal models in cases where identified candidate

genes are implicated in a given drug effect. (See Crabbe and Harris

1991 and Crabbe et al. 1994b for reviews.) For most drug responses,

no clear candidate gene is indicated, and several questions remain to

be addressed. It would be useful to determine how many genes

influence the trait, what their function is, and where they are located in

the genome. Traits affected by multiple genes usually display a

continuous degree of response (rather than all-or-none responses),

and the genes responsible are referred to as QTLs. Although each

QTL may have only a modest effect on the drug response,

collectively, the QTLs can determine a major proportion of drug

responsiveness.

The recently developed technique of QTL gene mapping (Paterson et

al. 1988) can be employed to identify the chromosomal location of

QTLs. QTL mapping depends upon linkage, or the tendency of

genes physically close to one another, to be inherited as a unit.
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Unlinked genes, in con-trast, tend to be inherited independently

according to the principles of segregation and independent

assortment. Discussions of this method have recently been published

(CrabbeandBelknap 1992; Lander and Botstein 1989; Plomin and

McClearn 1993; Tanksley 1993).

Most pharmacogenetic research with gene mapping has employed

recombinant inbred (RI) strains derived from the cross of C57BL/6J

(B6) and DBA/2J (D2) inbred mouse strains. This panel of strains was

developed by inbreeding from the F2 population derived from

B6XD2 hybrid mice. More than 20 BXD RI strains exist; each

represents the more or less random shuffling of the genetic deck for

all genes where B6 and D2 mice possess different alleles. Each RI

strain is like any other inbred strain in that all its members are

homozygous for each gene and are identical to each other.

Furthermore, each RI strain can only have inherited either the B6 or

the D2 allele (for any gene where the B6 and D2 progenitor strains

had the same allele, all BXD RI strains also have only that allele).

The use of RI strains for gene mapping was first recognized by Taylor

(1978) and Bailey (1981). The BXD RI panel of strains is powerful

for gene mapping because it comprises a substantial number of strains

that have been genotyped for more than 1,500 marker loci that are

poly-morphic in the B6 and D2 progenitors. Once the utility of

restriction fragment length polymorphisms (RFLPs) for gene

mapping was clearly recognized (Lander and Botstein 1989), many

RFLPs were identified in the BXD RI strains. More recently, markers

based on polymerase chain reaction (PCR) amplification of

deoxyribonucleic acid (DNA) micro-satellite simple sequence repeats

(SSRs) have been typed in great numbers in the BXD and other RI

strains (Deitrich et al. 1992). Plomin and colleagues (1991) first

applied QTL analysis to behavioral responses in BXD RI mice.

One method for identifying and mapping QTLs is to adopt a twostage procedure (Belknap et al. 1993a). An example of a QTL

analysis for alcohol withdrawal severity will be given to illustrate the

procedure. For the first stage, a database was established comprising

the allelic status of each RI strain at each mapped marker locus. For a

given marker, any RI strain possessing B6 alleles was given a value of

0, and any RI strain possessing D2 alleles a score of 1. The RI strains

were then tested for acute ethanol withdrawal and each strain's mean

response determined. Ethanol withdrawal severity (the quantitative

trait) was then correlated with allelic status for each marker gene in the

database.
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Six groups of markers on four chromosomes were significantly

associated with ethanol withdrawal severity (Belknap et al. 1993a). A

similar analysis was performed for nitrous oxide withdrawal severity.

The locations of the putative QTLs identified in these analyses are

given in figure 5. Each such associated chromosomal region suggests

the presence of a QTL affecting withdrawal severity. In this first stage

of the analysis, it is undesirable to reject any potential QTL as

unassociated; therefore, a relatively weak criterion of significance (p <

0.05) was adopted. However, because of the large number of

correlations calculated, some QTLs detected during this initial stage

will represent false-positive (chance) associations (Belknap 1992; see

also other articles in the same issue for methodological considerations

and alternative strategies). Therefore, it was necessary to verify the

putative QTLs detected with further analyses.

In the second stage of testing, a QTL associated with both ethanol and

nitrous oxide withdrawal severity was selected for further study. This

apparent QTL appeared to account for as much as 48percent of the

genetic variability in ethanol withdrawal severity; the markers most

highly associated (r = 0.69, p < 0.001) between allelic status and

withdrawal severity for 19 BXD RI strains were D2Mit9 and Scn2a,

which mapped to a position 37 centiMorgans (cM) from the

centromere of chromosome 2 (Silver et al. 1993). If a QTL in this

region were actually associated with withdrawal severity, then allelic

status of individual mice at markers in this region should also predict

withdrawal severity. To verify this, approxi-mately 150 F2 mice

derived from crosses of C57BL/6J and DBA/2J mice were tested for

ethanol withdrawal severity. They were then genotyped for six nearby

SSR markers using PCR.

Figure 6 shows how individual F2 mice display segregation and

independent assortment at a polymorphic marker, and the codominant

inheritance of heterozygotes. For informative markers, about one-half of

the F2 mice were homozygous for the B6 (or D2) allele and one-half were

heterozygotes. Ethanol withdrawal severity was significantly associated

with allelic status only at the marker D2Mit9, but not at other more distant

markers (Buck et al., in process). Figure 7 shows that the pattern of the

withdrawal response in F2 mice that were B6B6 homozygotes at D2Mit9

was significantly less pronounced and of shorter duration than that in F2

mice that were D2D2 homozygotes (Buck et al., in process).
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This region of mouse chromosome 2 is syntenic with human 2q24-q37,

which suggests that there is likely to be an analogous gene in humans.

The great degree of homology between mouse and human chromosome

maps (Copeland et al. 1993) is one of the most attractive features of QTL
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mapping in mice: any map site that can be verified in mice can likely

be extrapolated to a human map site. In addition, there are plausible

candidate genes near D2Mit9. Gad-1 codes for glutamic acid

decarboxy-lase, an enzyme catalyzing synthesis of GABA, and maps

to very nearly the same location as D2Mit9. A cluster of genes

coding for a-subunits of brain voltage-dependent sodium channels

(Scn1a, Scn2a, and Scn3a), which influence the action potential in
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excitable cells including neurons, also map nearby. Experiments are

underway to determine whether functional differences in these

candidate genes influence drug withdrawal severity.

FUTURE DIRECTIONS

Several interesting approaches are made possible by the QTL

mapping approach. QTL maps for one drug response trait can

obviously be compared with those for another (see figure 5). A

recent example was able to identify several QTLs influencing both

acute ethanol hypothermia and the development of tolerance to this

effect (Crabbe et al. 1994c). In addition, a growing number of

potential candidate genes are now mapped in mouse (e.g.,

neurotransmitter receptors and transporters, synthetic and metabolic

enzymes, ion channels). A composite map for multiple drug traits can

reveal genomic regions where presumed QTLs affecting multiple

responses are located. Combining QTL maps for drug response traits

and potential candidate genes can reveal associations across biological

levels of analysis (e.g., behavior with candidate gene). A recent

preliminary attempt to construct such a composite map compared

QTLs from more than 20 responses to ethanol and multiple responses

to methamphetamine, morphine, nitrous oxide, and other drugs

(Crabbe et al. 1994b). In that analysis, several responses to alcohol,

morphine, methamphetamine, and haloperidol all were significantly

associated with a region spanning 27 to 31 cM on chromosome 9.

This outcome is interesting because the gene encoding the dopamine

D2 receptor gene (Drd2) has been mapped to mouse chromosome 9,

27 cM from the telomere. Another region of interest was on

chromosome 6, where a putative QTL influencing six

methamphetamine responses was identified.

At this Technical Review, the existence of a transgenic mouse absent

5-hydroxytryptamine 1B (5-HT1B) receptors was demonstrated (Hen,

this volume). The gene encoding this receptor (Htr1b) has been

mapped to chromosome 9 at 40cM, near the dilute coat color locus at

42 cM. The composite QTL map mentioned above detected

associations of two methamphetamine, three morphine, and three

ethanol responses with markers in this region. One of these traits was

methamphetamine-induced hypophagia (Angeli-Gade, unpublished

data). Hypophagia is known to be induced by stimulation of 5-HT1B

receptors (Kitchener and Dourish 1994). It would be of interest to see

whether the 5-HT1B knockout mice differed in the drug responses

suggested by the QTL associations. It should be noted that nearly all
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the QTL analyses collected on the composite map described are

preliminary and represent only first-stage (BXD RI) associations; each

will need to be verified in a segregating genetic population.

Nonetheless, the rapid advances in gene mapping in the area of

pharmacogenetics make the prospect of such studies exciting.

Other avenues are also opened by the QTL approach. Using the

infor-mation derived from PCR genotyping of individual mice for

markers flanking a QTL of interest, it will be possible to breed mice

selectively for the genotype of interest (Plomin and McClearn 1993).

This approach in distinct from the usual practice of selective breeding

based on phenotypes, where the underlying responsible genes remain

anonymous. With selection based on genotype, individuals possessing

several risk-promoting QTLs could be generated and studied for their

drug responses. A similar approach would be to produce congenic

lines by backcrossing individuals possessing selected genotypes to an

appropriate background strain (Bailey 1981). For example, F1 mice

possessing DBA/2J alleles promoting high pentobarbital withdrawal

could be repeatedly backcrossed with low-risk C57BL/6J mice. After

many generations of selection and backcrossing, a congenic strain will

be produced, which possesses C57BL/6J genotype at all genes except

for a very small genomic region surrounding the QTL of interest.

This approach is already proving feasible using phenotypic markers

for selection of alcohol drinking (Dudek and Underwood 1993).

The potential advantage of congenic selected lines is in their utility for

subsequent applications with molecular biological methods. Even

once a QTL of interest has been identified, it remains a daunting task

to find the (relatively) nearby candidate gene that is actually

influencing the trait. It has recently been demonstrated that the

technique of representational difference analysis (RDA) can be used

to identify polymorphisms that can be amplified by PCR, through

comparison of two closely related DNA samples (Lisitsyn et al. 1994).

Genetically directed RDA has revealed numerous previously

unidentified polymorphisms closely targeted to the region of interest.

If such a region were defined by the difference between a normal

C57BL/6J and a selected congenic C57BL/6J strain possessing only a

small segment of DNA containing DBA/2J alleles leading to high

pentobarbital withdrawal, genetically directed RDA might eventually

increase the QTL map precision to about 200 kilobases (0.2 cM), or

about 0.05 percent of the mouse genome. This might make

positional cloning of the nearby gene feasible.
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CONCLUSIONS

Pharmacogenetic analyses of drug sensitivity have been fruitful in

identifying the important influence of often anonymous genes on

virtually all drug response traits. Genetic animal models are now

being employed using procedures designed to detect the influence of

QTLs on drug responses and to identify their chromosomal location.

The rapid identification of such QTLs, and the emerging pattern of

influence of drug-related QTLs, show promise as a strategy for

linking mouse and human genetic determinants of susceptibility to

abuse drugs.

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ACKNOWLEDGMENTS

Thanks to Dr. Benjamin Taylor for the RI marker database. Animal

studies were approved by the Animal Care and Use Committee and

conducted in accordance with NIH and USDA guidelines. Supported

by DA05228, NIDA contract no. 271-90-7405, AA08621, AA06243,

and grants from the Research Service of the Department of Veterans

Affairs. Dr. Buck was supported by Training Grant AA07468.

AUTHORS

John C. Crabbe, Ph.D.

Professor

Departments of Medical Psychology and Pharmacology

Oregon Health Sciences University (OSHU)

and

Research Career Scientist

Department of Veterans Affairs Medical Center

Kari J. Buck, Ph.D.

Assistant Professor

Department of Medical Psychology
219

and

Vollum Institute for Advanced Biomedical Research

OSHU

Pamela Metten, M.S.

Graduate Student

Department of Medical Psychology

John K. Belknap, Ph.D.

Professor

Department of Medical Psychology

OHSU

and

Research Career Scientist

Department of Veterans Affairs Medical Center

3710 Southwest U.S. Veterans Hospital Road

Portland, OR 97201
220

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221

26 THE BEHAVIORAL ASPECTS OF SMOKING.

Norman A. Krasnegor, Ph.D., ed. (Reprint from 1979 Surgeon General's Report

on Smoking and Health.)

NCADI#M26 NTIS PB #80-118755/AS (A09) $27.00

42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL FLUIDS.

Richard L. Hawks, Ph.D., ed.

NCADI #M42 NTIS PB #83-136044/AS (A07) $27.00

50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF

ABUSE. John Grabowski, Ph.D., ed.

NCADI #M50 NTIS PB #85-150381/AS (A07) $27.00

52 TESTING DRUGS FOR PHYSICAL DEPENDENCE POTENTIAL AND

ABUSE LIABILITY. Joseph V. Brady, Ph.D., and Scott E. Lukas, Ph.D., eds.

NCADI #M52 NTIS PB #85-150373/AS (A08) $27.00

53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION. John

Grabowski, Ph.D., and Sharon M. Hall, Ph.D., eds.

NCADI #M53 NTIS PB #89-123186/AS (A07) $27.00

54 MECHANISMS OF TOLERANCE AND DEPENDENCE.

Charles Wm. Sharp, Ph.D., ed.

NCADI #M54 NTIS PB #89-103279/AS (A19) $52.00

56 ETIOLOGY OF DRUG ABUSE: IMPLICATIONS FOR PREVENTION.

Coryl LaRue Jones, Ph.D., and

Robert J. Battjes, D.S.W., eds.

NCADI #M56 NTIS PB #89-123160/AS (A13) $36.50

61 COCAINE USE IN AMERICA: EPIDEMIOLOGIC AND CLINICAL

PERSPECTIVES. Nicholas J. Kozel, M.S., and

Edgar H. Adams, M.S., eds.

NCADI #M61 NTIS PB #89-131866/AS (A11) $36.50

62 NEUROSCIENCE METHODS IN DRUG ABUSE RESEARCH.

Roger M. Brown, Ph.D., and David P. Friedman, Ph.D., eds.

NCADI #M62 NTIS PB #89-130660/AS (A08) $27.00

63 PREVENTION RESEARCH: DETERRING DRUG ABUSE AMONG

CHILDREN AND ADOLESCENTS. Catherine S. Bell, M.S., and Robert J.

Battjes, D.S.W., eds.

NCADI #M63 NTIS PB #89-103287/AS (A11) $36.50

64 PHENCYCLIDINE: AN UPDATE. Doris H. Clouet, Ph.D., ed.

NCADI #M64 NTIS PB #89-131858/AS (A12) $36.50

65 WOMEN AND DRUGS: A NEW ERA FOR RESEARCH.

Barbara A. Ray, Ph.D., and Monique C. Braude, Ph.D., eds.

NCADI #M65 NTIS PB #89-130637/AS (A06) $27.00
222

69 OPIOID PEPTIDES: MEDICINAL CHEMISTRY.

Rao S. Rapaka, Ph.D.; Gene Barnett, Ph.D.; and

Richard L. Hawks, Ph.D., eds.

NCADI #M69 NTIS PB #89-158422/AS (A17) $44.50

70 OPIOID PEPTIDES: MOLECULAR PHARMACOLOGY,

BIOSYNTHESIS, AND ANALYSIS. Rao S. Rapaka, Ph.D., and Richard L.

Hawks, Ph.D., eds.

NCADI #M70 NTIS PB #89-158430/AS (A18) $52.00

72 RELAPSE AND RECOVERY IN DRUG ABUSE.

Frank M. Tims, Ph.D., and Carl G. Leukefeld, D.S.W., eds.

NCADI #M72 NTIS PB #89-151963/AS (A09) $36.50

74 NEUROBIOLOGY OF BEHAVIORAL CONTROL IN DRUG ABUSE.

Stephen I. Szara, M.D., D.Sc., ed.

NCADI #M74 NTIS PB #89-151989/AS (A07) $27.00

78 THE ROLE OF NEUROPLASTICITY IN THE RESPONSE TO DRUGS.

David P. Friedman, Ph.D., and Doris H. Clouet, Ph.D., eds.

NCADI #M78 NTIS PB #88-245683/AS (A10) $36.50

79 STRUCTURE-ACTIVITY RELATIONSHIPS OF THE CANNABINOIDS.

Rao S. Rapaka, Ph.D., and

Alexandros Makriyannis, Ph.D., eds.

NCADI #M79 NTIS PB #89-109201/AS (A10) $36.50

80 NEEDLE SHARING AMONG INTRAVENOUS DRUG ABUSERS:

NATIONAL AND INTERNATIONAL PERSPECTIVES.

Robert J. Battjes, D.S.W., and Roy W. Pickens, Ph.D., eds.

NCADI #M80 NTIS PB #88-236138/AS (A09) $36.50

82 OPIOIDS IN THE HIPPOCAMPUS. Jacqueline F. McGinty, Ph.D., and

David P. Friedman, Ph.D., eds.

NCADI #M82 NTIS PB #88-245691/AS (A06) $27.00

83 HEALTH HAZARDS OF NITRITE INHALANTS.

Harry W. Haverkos, M.D., and John A. Dougherty, Ph.D., eds.

NCADI #M83 NTIS PB #89-125496/AS (A06) $27.00

84 LEARNING FACTORS IN SUBSTANCE ABUSE.

Barbara A. Ray, Ph.D., ed.

NCADI #M84 NTIS PB #89-125504/AS (A10) $36.50

85 EPIDEMIOLOGY OF INHALANT ABUSE: AN UPDATE.

Raquel A. Crider, Ph.D., and Beatrice A. Rouse, Ph.D., eds.

NCADI #M85 NTIS PB #89-123178/AS (A10) $36.50

87 OPIOID PEPTIDES: AN UPDATE. Rao S. Rapaka, Ph.D., and

Bhola N. Dhawan, M.D., eds.

NCADI #M87 NTIS PB #89-158430/AS (A11) $36.50
223

88 MECHANISMS OF COCAINE ABUSE AND TOXICITY.

Doris H. Clouet, Ph.D.; Khursheed Asghar, Ph.D.; and

Roger M. Brown, Ph.D., eds.

NCADI #M88 NTIS PB #89-125512/AS (A16) $44.50

89 BIOLOGICAL VULNERABILITY TO DRUG ABUSE.

Roy W. Pickens, Ph.D., and Dace S. Svikis, B.A., eds.

NCADI #M89 NTIS PB #89-125520/AS (A09) $27.00

92 TESTING FOR ABUSE LIABILITY OF DRUGS IN HUMANS.

Marian W. Fischman, Ph.D., and Nancy K. Mello, Ph.D., eds.

NCADI #M92 NTIS PB #90-148933/AS (A17) $44.50

94 PHARMACOLOGY AND TOXICOLOGY OF AMPHETAMINE AND

RELATED DESIGNER DRUGS. Khursheed Asghar, Ph.D., and Errol De Souza,

Ph.D., eds.

NCADI #M94 NTIS PB #90-148958/AS (A16) $44.50

95 PROBLEMS OF DRUG DEPENDENCE, 1989. PROCEEDINGS OF THE

51st ANNUAL SCIENTIFIC MEETING. THE COMMITTEE ON PROBLEMS

OF DRUG DEPENDENCE, INC.

Louis S. Harris, Ph.D., ed.

NCADI #M95 NTIS PB #90-237660/AS (A99) $67.00

96 DRUGS OF ABUSE: CHEMISTRY, PHARMACOLOGY, IMMUNOLOGY,

AND AIDS. Phuong Thi Kim Pham, Ph.D., and Kenner Rice, Ph.D., eds.

NCADI #M96 NTIS PB #90-237678/AS (A11) $36.50

97 NEUROBIOLOGY OF DRUG ABUSE: LEARNING AND MEMORY.

Lynda Erinoff, Ph.D., ed.

NCADI #M97 NTIS PB #90-237686/AS (A11) $36.50

98 THE COLLECTION AND INTERPRETATION OF DATA FROM

HIDDEN POPULATIONS.

Elizabeth Y. Lambert, M.Sc., ed.

NCADI #M98 NTIS PB #90-237694/AS (A08) $27.00

99 RESEARCH FINDINGS ON SMOKING OF ABUSED SUBSTANCES. C.

Nora Chiang, Ph.D., and

Richard L. Hawks, Ph.D., eds.

NCADI #M99 NTIS PB #91-141119 (A09) $27.00

100 DRUGS IN THE WORKPLACE: RESEARCH AND EVALUATION

DATA. VOL II. Steven W. Gust, Ph.D.; J.Michael Walsh, Ph.D.; Linda B.

Thomas, B.S.;

and Dennis J. Crouch, M.B.A., eds.

NCADI #M100 GPO Stock #017-024-01458-3 $8.00

101 RESIDUAL EFFECTS OF ABUSED DRUGS ON BEHAVIOR.

John W. Spencer, Ph.D., and John J. Boren, Ph.D., eds.

NCADI #M101 NTIS PB #91-172858/AS (A09) $27.00
224

102 ANABOLIC STEROID ABUSE. Geraline C. Lin, Ph.D., and

Lynda Erinoff, Ph.D., eds.

NCADI #M102 NTIS PB #91-172866/AS (A11) $36.50

106 IMPROVING DRUG ABUSE TREATMENT.

Roy W. Pickens, Ph.D.; Carl G. Leukefeld, D.S.W.; and

Charles R. Schuster, Ph.D., eds.

NCADI #M106 NTIS PB #92-105873(A18) $50.00

107 DRUG ABUSE PREVENTION INTERVENTION RESEARCH:

METHODOLOGICAL ISSUES. Carl G. Leukefeld, D.S.W., and William J.

Bukoski, Ph.D., eds.

NCADI #M107 NTIS PB #92-160985 (A13) $36.50

108 CARDIOVASCULAR TOXICITY OF COCAINE: UNDERLYING

MECHANISMS. Pushpa V. Thadani, Ph.D., ed.

NCADI #M108 NTIS PB #92-106608 (A11) $36.50

109 LONGITUDINAL STUDIES OF HIV INFECTION IN INTRAVENOUS

DRUG USERS: METHODOLOGICAL ISSUES IN NATURAL HISTORY

RESEARCH. Peter Hartsock, Dr.P.H., and Sander G. Genser, M.D., M.P.H., eds.

NCADI #M109 NTIS PB #92-106616 (A08) $27.00

111 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH:

RECEPTOR CLONING, NEUROTRANSMITTER EXPRESSION, AND

MOLECULAR GENETICS: VOLUME I. Theresa N.H. Lee, Ph.D., ed.

NCADI #M111 NTIS PB #92-135743 (A10) $36.50

112 EMERGING TECHNOLOGIES AND NEW DIRECTIONS IN DRUG

ABUSE RESEARCH. Rao S. Rapaka, Ph.D.;

Alexandros Makriyannis, Ph.D.; and Michael J. Kuhar, Ph.D., eds.

NCADI #M112 NTIS PB #92-155449 (A15) $44.50

113 ECONOMIC COSTS, COST EFFECTIVENESS, FINANCING, AND

COMMUNITY-BASED DRUG TREATMENT.

William S. Cartwright, Ph.D., and James M. Kaple, Ph.D., eds.

NCADI #M113 NTIS PB #92-155795 (A10) $36.50

114 METHODOLOGICAL ISSUES IN CONTROLLED STUDIES ON

EFFECTS OF PRENATAL EXPOSURE TO DRUG ABUSE.

M. Marlyne Kilbey, Ph.D., and Khursheed Asghar, Ph.D., eds.

NCADI #M114 NTIS PB #92-146216 (A16) $44.50

115 METHAMPHETAMINE ABUSE: EPIDEMIOLOGIC ISSUES AND

IMPLICATIONS. Marissa A. Miller, D.V.M., M.P.H., and

Nicholas J. Kozel, M.S., eds.

NCADI #M115 NTIS PB #92-146224/ll (AO7) $27.00
225

116 DRUG DISCRIMINATION: APPLICATIONS TO DRUG ABUSE

RESEARCH. R.A. Glennon, Ph.D.;

Toubjörn U.C. Järbe, Ph.D.; and J. Frankenheim, Ph.D., eds.

NCADI #M116 NTIS PB #94-169471 (A20) $52.00

117 METHODOLOGICAL ISSUES IN EPIDEMIOLOGY, PREVENTION,

AND TREATMENT RESEARCH ON DRUG-EXPOSED WOMEN AND THEIR

CHILDREN.

M. Marlyve Kilbey, Ph.D., and Kursheed Asghar, Ph.D., eds.

GPO Stock #O17-024-01472-9 $12.00

NCADI #M117 NTIS PB #93-102101/LL (A18) $52.00

118 DRUG ABUSE TREATMENT IN PRISONS AND JAILS.

Carl G. Leukefeld, D.S.W., and Frank M. Tims, Ph.D., eds.

GPO Stock #O17-024-01473-7 $16.00

NCADI #M118 NTIS PB #93-102143/LL (A14) $44.50

120 BIOAVAILABILITY OF DRUGS TO THE BRAIN AND THE BLOODBRAIN BARRIER. Jerry Frankenheim, Ph.D., and

Roger M. Brown, Ph.D., eds.

GPO Stock #017-024-01481-8 $10.00

NCADI #M120 NTIS PB #92-214956/LL (A12) $36.50

121 BUPRENORPHINE: AN ALTERNATIVE TREATMENT FOR OPIOID

DEPENDENCE. Jack D. Blaine, Ph.D., ed.

GPO Stock #017-024-01482-6 $5.00

NCADI #M121 NTIS PB #93-129781/LL (A08) $27.00

123 ACUTE COCAINE INTOXICATION: CURRENT METHODS OF

TREATMENT. Heinz Sorer, Ph.D., ed.

GPO Stock #017-024-01501-6 $6.50

NCADI #M123 NTIS PB #94-115433/LL (A09) $27.00

124 NEUROBIOLOGICAL APPROACHES TO BRAIN-BEHAVIOR

INTERACTION. Roger M. Brown, Ph.D., and

Joseph Fracella, Ph.D., eds.

GPO Stock #017-024-01492-3 $9.00

NCADI #M124 NTIS PB #93-203834/LL (A12) $36.50

125 ACTIVATION OF IMMEDIATE EARLY GENES BY DRUGS OF

ABUSE. Reinhard Grzanna, Ph.D., and

Roger M. Brown, Ph.D., eds.

GPO Stock #017-024-01503-2 $7.50

NCADI #M125 NTIS PB #94-169489 (A12) $36.50

126 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH VOLUME

II: STRUCTURE, FUNCTION, AND EXPRESSION. Theresa N.H. Lee, Ph.D.,

ed.

NCADI #M126 NTIS PB #94-169497 (A08) $27.00

127 PROGRESS AND ISSUES IN CASE MANAGEMENT.

Rebecca S. Ashery, D.S.W., ed.

NCADI #M127 NTIS PB #94-169505 (A18) $52.00
226

128 STATISTICAL ISSUES IN CLINICAL TRIALS FOR TREATMENT OF

OPIATE DEPENDENCE.

Ram B. Jain, Ph.D., ed.

NCADI #M128 NTIS PB #93-203826/LL (A09) $27.00

129 INHALANT ABUSE: A VOLATILE RESEARCH AGENDA. Charles W.

Sharp, Ph.D.; Fred Beauvais, Ph.D.; and

Richard Spence, Ph.D., eds.

GPO Stock #017-024-01496-6 $12.00

NCADI #M129 NTIS PB #93-183119/LL (A15) $44.50

130 DRUG ABUSE AMONG MINORITY YOUTH: ADVANCES IN

RESEARCH AND METHODOLOGY. Mario De La Rosa, Ph.D., and Juan-Luis

Recio Adrados, Ph.D., eds.

GPO Stock #017-024-01506-7 $14.00

NCADI #M130 NTIS PB #94-169513 (A15) $44.50

131 IMPACT OF PRESCRIPTION DRUG DIVERSION CONTROL SYSTEMS

ON MEDICAL PRACTICE AND PATIENT CARE.

James R. Cooper, Ph.D.; Dorynne J. Czechowicz, M.D.;

Stephen P. Molinari, J.D., R.Ph.; and Robert C. Peterson, Ph.D., eds.

GPO Stock #017-024-01505-9 $14.00

NCADI #M131 NTIS PB #94-169521 (A15) $44.50

132 PROBLEMS OF DRUG DEPENDENCE, 1992: PROCEEDINGS OF THE

54TH ANNUAL SCIENTIFIC MEETING OF THE COLLEGE ON PROBLEMS

OF DRUG DEPENDENCE.

Louis Harris, Ph.D., ed.

GPO Stock #017-024-01502-4 $23.00

NCADI #M132 NTIS PB #94-115508/LL (A99)

133 SIGMA, PCP, AND NMDA RECEPTORS.

Errol B. De Souza, Ph.D.; Doris Clouet, Ph.D., and

Edythe D. London, Ph.D., eds.

NCADI #M133 NTIS PB #94-169539 (A12) $36.50

134 MEDICATIONS DEVELOPMENT: DRUG DISCOVERY, DATABASES,

AND COMPUTER-AIDED DRUG DESIGN.

Rao S. Rapaka, Ph.D., and Richard L. Hawks, Ph.D., eds.

GPO Stock #017-024-01511-3 $11.00

NCADI #M134 NTIS PB #94-169547 (A14) $44.50

135 COCAINE TREATMENT: RESEARCH AND CLINICAL

PERSPECTIVES. Frank M. Tims, Ph.D., and

Carl G. Leukefeld, D.S.W., eds.

GPO Stock #017-024-01520-2 $11.00

NCADI #M135 NTIS PB #94-169554 (A13) $36.50

136 ASSESSING NEUROTOXICITY OF DRUGS OF ABUSE.

Lynda Erinoff, Ph.D., ed.

GPO Stock #017-024-01518-1 $11.00

NCADI #M136 NTIS PB #94-169562 (A13) $36.50
227

137 BEHAVIORAL TREATMENTS FOR DRUG ABUSE AND

DEPENDENCE. Lisa Simon Onken, Ph.D.; Jack D. Blaine, M.D.; and John J.

Boren, Ph.D., eds.

GPO Stock #017-024-01519-9 $13.00

NCADI #M137 NTIS PB #94-169570 (A15) $44.50

138 IMAGING TECHNIQUES IN MEDICATIONS DEVELOPMENT:

CLINICAL AND PRECLINICAL ASPECTS. Heinz Sorer, Ph.D., and Rao S.

Rapaka, Ph.D., eds.

NCADI #M138

139 SCIENTIFIC METHODS FOR PREVENTION INTERVENTION

RESEARCH. Arturo Cazares, M.D., M.P.H., and

Lula A. Beatty, Ph.D., eds.

NCADI #M139

140 PROBLEMS OF DRUG DEPENDENCE, 1993: PROCEEDINGS OF THE

55TH ANNUAL SCIENTIFIC MEETING, THE COLLEGE ON PROBLEMS OF

DRUG DEPENDENCE, INC. VOLUME I: PLENARY SESSION SYMPOSIA

AND ANNUAL REPORTS. Louis S. Harris, Ph.D., ed.

NCADI #M140

141 PROBLEMS OF DRUG DEPENDENCE, 1993: PROCEEDINGS OF THE

55TH ANNUAL SCIENTIFIC MEETING, THE COLLEGE ON PROBLEMS OF

DRUG DEPENDENCE, INC. VOLUME II: ABSTRACTS. Louis S. Harris,

Ph.D., ed.

NCADI #M141

142 ADVANCES IN DATA ANALYSIS FOR PREVENTION

INTERVENTION RESEARCH. Linda M. Collins, Ph.D., and

Larry A. Seitz, Ph.D., eds.

NCADI #M142

143 THE CONTEXT OF HIV RISK AMONG DRUG USERS AND THEIR

SEXUAL PARTNERS. Robert J. Battjes, D.S.W.;

Zili Sloboda, Sc.D.; and William C. Grace, Ph.D., eds.

NCADI #M143

144 THERAPEUTIC COMMUNITY: ADVANCES IN RESEARCH 

AND APPLICATION. Frank M. Tims, Ph.D.;

George De Leon, Ph.D.; and Nancy Jainchill, Ph.D., eds.

NCADI #M144
228

145 NEUROBIOLOGICAL MODELS FOR EVALUATING MECHANISMS

UNDERLYING COCAINE ADDICTION.

Lynda Erinoff, Ph.D., and Roger M. Brown, Ph.D., eds.

NCADI #M145

146 HALLUCINOGENS: AN UPDATE. Geraline C. Lin, Ph.D., andRichard

A. Glennon, Ph.D., eds.

NCADI #M146

147 DISCOVERY OF NOVEL OPIOID MEDICATIONS.

Rao S. Rapaka, Ph.D., and Heinz Sorer, Ph.D., eds.

NCADI #M147

148 EPIDEMIOLOGY OF INHALANT ABUSE: AN INTERNATIONAL

PERSPECTIVE.

Nicholas J. Kozel, M.S.; Zili Sloboda, Sc.D.;

and Mario R. De La Rosa, Ph.D., eds.

NCADI # M148

149 MEDICATIONS DEVELOPMENT FOR THE TREATMENT OF

PREGNANT ADDICTS AND THEIR INFANTS.

C. Nora Chiang, Ph.D., and Loretta P. Finnegan, M.D., eds.

NCADI # M149

150 INTEGRATING BEHAVIORAL THERAPIES WITH MEDICATIONS IN

THE TREATMENT OF DRUG DEPENDENCE.

Lisa Simon Onken, Ph.D.; Jack D. Blaine, M.D.; and

John J. Boren, Ph.D., eds.

NCADI # M150

151 SOCIAL NETWORKS, DRUG ABUSE, AND HIV TRANSMISSION.

Richard H. Needle, Ph.D., M.P.H.;

Susan L. Coyle, Ph.D.; Sander G. Genser, M.D., M.P.H.; and

Robert T. Trotter II, Ph.D., eds.

NCADI # M151

154 MEMBRANES AND BARRIERS: TARGETED DRUG DELIVERY. Rao

S. Rapaka, Ph.D., ed.

NCADI # M154
229

155 REVIEWING THE BEHAVIORAL SCIENCE KNOWLEDGE BASE ON

TECHNOLOGY TRANSFER.

Thomas E. Backer, Ph.D.; Susan L. David; and GeraldSoucy,Ph.D., eds.

NCADI # M155

156 ADOLESCENT DRUG ABUSE: CLINICAL ASSESSMENT AND

THERAPEUTIC INTERVENTIONS.

Elizabeth Rahdert, Ph.D.; Zili Sloboda, Ph.D.; and DorynneCzechowicz, Ph.D.,

eds.

NCADI # M156

157 QUALITATIVE METHODS IN THE PREVENTION OF DRUG ABUSE

AND HIV RESEARCH. Elizabeth Y. Lambert, M.Sc.; Rebecca S. Ashery,

D.S.W.; and

Richard H. Needle, Ph.D., M.P.H., eds.

NCADI # M157

158 BIOLOGICAL MECHANISMS AND PERINATAL EXPOSURE TO

ABUSED DRUGS. Pushpa V. Thadani, Ph.D., ed.

NCADI # M158