National Institute on Drug Abuse

RESEARCH

MONOGRAPH SERIES

Molecular

Approaches to

Drug Abuse

Research

Volume II

U.S. Department of Health and Human Services • Public Health Service • National Institutes of Health

126
Molecular Approaches to

Drug Abuse Research

Volume II: Structure,

Function, and Expression

Editor:

Theresa N.H. Lee, Ph.D.

NIDA Research Monograph 126

1992

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, MD 20857
ACKNOWLEDGMENT

This monograph is based on the papers and discussions from a technical

review on “Molecular Approaches to Drug Abuse Research” held on July 30-31,

1991, in Bethesda, MD. The technical review was sponsored by the National

Institute on Drug Abuse (NIDA).

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 passages from copyrighted sources is in the public domain 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.

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.”

DHHS publication number (ADM)92-1945

Printed 1992

i i
Contents

Page

Introduction 1

Theresa N.H. Lee

Neuronal Nicotinic Acetylcholine Receptor Diversity 4

James W. Patrick

Neurotransmitter and Drug Receptor Genes 14

George R. Uhl

The Diversity of the Dopamine Receptors 23

Olivier Civelli, James R. Bunzow, Qun-Yong Zhou, and

David K. Grandy

Cloned Dopamine Receptors: Targets in Therapy of

Drug Abuse 34

Philip Seeman

Cannabinoid Receptors: Which Cells, Where, How, and Why? 48

Lisa A. Matsuda, Tom I. Bonner, and Stephen J. Lolait

Studies on μ-Opioid-Binding Sites With Peptide Antibodies 57

Eric J. Simon, Theresa L. Gioannini, Yi-He Yao, and

Jacob M. Hiller

Molecular Cloning and Characterization of Neurotransmitter

Transporters 66

Randy D. Blakely

iii
Regulation of Gene Expression by Dopamine: Implications

in Drug Addiction 84

Orla M. Conneely, Ronan F. Power, and Bert W. O’Malley

Regulation of Opioid Gene Expression: A Model To Understand Neural

Plasticity 98

Michael J. Comb, Linda Kobierski, Hung Ming Chu, Yi Tan,

David Borsook, Karl Herrup, and Steven E. Hyman

Cellular and Molecular Analysis of Opioid Peptide Gene

Expression 113

Cynthia T. McMurray, Karen M. Pollock,

and James Douglass

The Prohormone and Proprotein Processing Enzymes PC1

and PC2: Structure, Selective Cleavage of Mouse POMC

and Human Renin at Pairs of Basic Residues, Cellular

Expression, Tissue Distribution, and mRNA Regulation 132

Nabil G. Seidah, Robert Day, Suzanne Benjannet, Normand

Rondeau, Alain Boudreault, Timothy Reudelhuber, Martin K.-H.

Schafer, Stanley J. Watson, and Michel Chrétien

List of NIDA Research Monographs 151

iv
Introduction

Theresa N.H. Lee

At the first National Institute on Drug Abuse (NIDA) technical review meeting

on “Molecular Approaches to Drug Abuse Research” held in August 1989,

one member of each of the three families of receptors and channels was

chosen to illustrate various strategies of gene cloning due to the limited number

of drug receptor genes cloned. These are the dopamine D2 receptor of the

G protein-coupled receptor family, the nicotinic acetylcholine receptor of the

ligand-gated channel family, and the potassium channel of the voltage-gated

channel family. Because of the extensive and innovative studies on the

nicotinic acetylcholine receptor, it was selected as an example to cover

postcloning research endeavors such as regulation of expression using

molecular genetics and transgenic mice as well as studies of structure and

function relationship using site-directed mutagenesis and other techniques.

The second NIDA technical review meeting on “Molecular Approaches to

Drug Abuse Research” was held on July 30 and 31, 1991, at the National

Institutes of Health. This technical review encompassed almost all the pivotal

research developments of the past 2 years in the field. What extraordinary

progress scientists in this field have made since the 1989 meeting! The

proceedings of this conference are presented in the following chapters of

this monograph.

As a logical extension of the agenda of the 1989 conference, the 1991

technical review began with Dr. James W. Patrick’s update on the diversity of

ligand/receptor interactions of neuronal nicotinic acetylcholine receptors based

on studies of functional oligomeric receptors. Presentations followed on the

successful cloning of genes of two other members of the ligand-gated channel

family—the glutamate (AMPA/Kainate) receptor and the -aminobutyric acid

(GABA) p-1 receptor by Dr. Stephen Heinemann and Dr. George R. Uhl,

respectively.

Perhaps as a reflection of the intense interest in the molecular cloning of the

opioid receptor genes, there have been an unprecedented number of genes

cloned in the G protein-coupled receptor family in these 2 years. These

scientists have made international news recently with their breakthroughs in

1
receptor gene cloning and subsequent remarkable studies. Some of the

examples presented in the 1991 conference were Dr. Olivier Civelli and Dr.

Philip Seeman on the molecular biology and pharmacology of dopamine

receptors D1, D2, D3, D4, and D5 and Dr. Lisa A. Matsuda on the cannabinoid

receptor. Dr. Eric J. Simon gave an update on the μ-opioid receptor. Even

though the cloning of the -opioid receptor gene was not conclusive at the

time of the conference, the recent cloning and expression of the long-awaited

prohormone and proprotein convertases PC1 and PC2 genes opened up

numerous avenues in the field (Dr. Nabil G. Seidah).

This technical review also coincided with another breakthrough in the field,

namely, the successful cloning of the gene for the dopamine transporter with

12 transmembrane domains. This work will undoubtedly help elucidate the

molecular mechanism of cocaine action. The leader of one of the groups that

accomplished this important task, Dr. Susan Amara, has also joined the NIDA

scientific community recently as a grantee. Although Dr. Amara was unable to

attend the conference, Dr. Randy D. Blakely spoke instead on the molecular

cloning and characterization of GABA and norepinephrine transporter genes.

This technical review would not have been complete without coverage of

the enormous contributions of NIDA scientists to our understanding of the

regulation of expression of opioid peptides. Dr. Michael J. Comb and Dr.

James Douglass provided two superb examples with their presentations on

this subject. From a different perspective, Dr. Orla M. Conneely discussed

the recent evidence from their laboratory that members of the steroid/thyroid

receptor family of transcription factors could be activated by dopamine and

dopamine D1 receptor agonists.

The 1991 technical review also commemorated the establishment of the

Molecular Biology and Genetics Program initiated 3 years earlier in the Division

of Preclinical Research of NIDA. This program now encompasses nearly every

important area employing molecular approaches to drug abuse research; this is

clearly illustrated in the technical review and this monograph. I am extremely

proud to point out that, of the 11 people contributing to this monograph, 8 are

members of the NIDA scientific community, and another 2 are in the process

of applying for support.

Drug abuse researchers interested in molecular approaches will find this

coming decade challenging and fruitful, with ground-breaking results expediting

understanding of the underlying basis of addiction to generate better strategies

for effective treatment, education, and prevention.

2
AUTHOR

Theresa N.H. Lee, Ph.D.

Program Director

Molecular Biology and Genetics Program

Biomedical Branch

Division of Preclinical Research

National Institute on Drug Abuse

Parklawn Building, Room 10A-19

5600 Fishers Lane

Rockville, MD 20857

3
Neuronal Nicotinic Acetylcholine

Receptor Diversity

James W. Patrick

INTRODUCTION

Society must deal with the dangerous dichotomy of drugs, a dichotomy that

arises from the fact that the drugs that provide the best treatment for many

illnesses also present tremendous opportunity for abuse. Furthermore, both

naturally occurring and synthetic drugs provide extremely powerful tools for

the study of biological systems. The use of these drugs as research tools has

resulted in a refinement of understanding of both the drugs and their biological

sites of action. This has, in turn, led to the development of ever more specific

and powerful drugs with subsequent new opportunities for abuse. This trend is

expected to continue because recent studies have revealed an unexpected

and, for the moment, bewildering diversity of drug receptors. This diversity

suggests that eventually one will be able to engineer exquisitely specific drugs

targeted to specific areas of the brain with minimal side effects. On the one

hand, one expects that this work will reveal new sites and mechanisms of

action of drugs and, unfortunately, will offer more opportunity for abuse through

the development of new classes of drugs. On the other hand, one hopes that

the insights gained in this work will result in the production of drugs with minimal

abuse potential or perhaps generate therapies to deal with abuse. There is

every reason to believe that new insights into receptors, new drugs, and rapidly

expanding access to the nervous system will lead to a new level of appreciation

and understanding of both the function of the brain and the uses and abuses of

drugs.

The rationale that underlies much of the research in this field is based on

the recent observation that there is a large family of genes that encode

neurotransmitter receptors and that almost all the ligand-gated ion channels

are members of this superfamily. Because these receptors are all members

of the same family they share structural and functional features. However,

they differ in many important ways, and these differences provide powerful tools

with which to probe their important common features. The family of genes that

compose the nicotinic receptors is studied to understand the structure, function,

4
and regulation of the nicotinic receptors. The information thus obtained has

provided the foundation for all other studies of ligand-gated ion channels and

will very likely continue to be important for understanding other receptors such

as the members of the glutamate, GABA, glycine, or serotonin receptor gene

families. Therefore, insights provided into the structure and function of the

neuronal nicotinic receptors will help one’s understanding of the ligand-gated

ion channels in general.

NICOTINIC ACETYLCHOLINE RECEPTORS

The nicotinic acetylcholine receptor has long been the prototypical ligand-gated

ion channel, and most of what is known about the structure and function of

these ion channels derives from studies of the muscle-type nicotinic receptor

isolated from the electric rays and fishes. Significant progress in this area has

come in the last 5 to 10 years following the application of the techniques of

molecular biology to the study of the muscle nicotinic receptor. These studies

led to the elucidation of the primary structure of the subunits of the receptor

(Ballivet et al. 1981; Noda et al. 1982, 1983a, 1983b; Sumikawa et al. 1982;

Claudio et al. 1983), its synthesis from RNA derived from cDNA clones (Mishina

et al. 1984), the determination of functional domains by mutagenesis (Mishina

et al. 1985), mapping of antibody binding sites (Tzartos et al. 1988, 1990),

the discovery of a new receptor subunit (Takai et al. 1985) and its role in

synaptogenesis at the neuromuscular junction (Methfessel and Sakmann

1986; Sakmann et al. 1985; Gu and Hall 1988), and the identification of

genomic sequences that control synthesis of receptor subunits (Gardner

et al. 1987). Understanding of the nicotinic receptor and the neuromuscular

junction in normal and diseased states has advanced dramatically as a result

of these studies.

The acetylcholine receptor at the neuromuscular junction was chosen for

these studies because good ligands were available and because the junction

was well studied and amenable to the types of experiments the author and his

colleagues thought were necessary. However, it seemed likely that a great

deal could be learned from the nicotinic receptors found at other nicotinic

cholinergic synapses. It also seemed likely that this information would be

important for understanding the receptor at the neuromuscular junction and

for understanding the role these receptors play in neurons. It has long been

known that cholinergic transmission at sympathetic ganglia is nicotinic, that the

nicotinic acetylcholine receptors on neurons differ from those found at the

neuromuscular junction, and that nicotinic receptors might play a very important

role in neurotransmitter release. The potential for understanding the nicotinic

receptors and ligand-gated ion channels in general, as well as the potential for

appreciating new roles for cholinergic function on neurons, led many

laboratories to the neuronal nicotinic acetylcholine receptors.

5
One approach was based on the idea that sequences encoding the muscle-type

nicotinic acetylcholine receptors would hybridize to, and thus identify, the

sequences encoding the neuronal nicotinic acetylcholine receptors. This

general approach led to the identification in the rat and chicken of a family

of genes encoding subunits of nicotinic acetylcholine receptors that are

expressed in the central nervous system (CNS) (for reviews, see Luetje et al.

1990a; Nordberg et al. 1989). Currently, the products of six different genes

(alpha2, alpha3, alpha4, alpha7, beta2, and beta4) are known to generate at

least seven different functional receptors (Nef et al. 1988; Deneris et al. 1988;

Wada et al. 1988; Boulter et al. 1986, 1987; Goldman et al. 1986; Duvoisin et

al. 1989; Couturier et al. 1990; Schoepfer et al. 1990). There are additional

members of this gene family (alpha5, alpha8, and beta3) whose gene products

have not yet been shown to be associated with a function (Schoepfer et al.

1990; Boulter et al. 1990; Deneris et al. 1989). The proteins encoded by these

nine genes have homologous extracellular, transmembrane, and cytoplasmic

domains and, in general, are classified as alpha or beta subunits. The alpha

subunits are identified by contiguous cysteines in the extracellular domain. The

beta (or nonalpha) subunits are identified by the lack of these cysteines and by

the ability of either beta2 or beta4 to substitute for the beta1 subunit in the

formation of a functional muscle-type nicotinic receptor.

The proteins derived from six of these genes associate in various combinations

to form functional receptors in the Xenopus oocyte. The alpha2, alpha3, and

alpha4 subunits each form functional receptors in combination with either beta2

or beta4. Receptors thus formed vary with respect to their single channel

properties (Papke et al. 1989) and pharmacology (Luetje and Patrick 1991;

Luetje et al. 1990b) depending on which of the three different alpha subunits

are present. Likewise, the substitution of a beta4 subunit for a alpha2 subunit

alters the response of the receptor to various agonists and antagonists

(Duvoisin et al. 1989; Luetje and Patrick 1991). There is also good evidence

that the complement of receptor subtypes present on a neuron changes during

development (Moss et al. 1989).

The receptor formed in the oocyte from alpha4 and beta2 subunits is probably a

pentamer comprising three beta subunits and two alpha subunits (Cooper et al.

1991). Although it is clearly possible to form receptors containing more than

one kind of alpha subunit (S. Helekar and J. Patrick, unpublished observations),

the diversity of oligomeric receptor structures formed in the CNS is not known.

Two additional members of the gene family have been identified in the chick

(Couturier et al. 1990; Schoepfer et al. 1990). These new members are alpha7

and alpha8, and the proteins encoded by these clones differ from the other

alpha subunits in several regards. The alpha7 subunit forms an acetylcholine6
gated ion channel in the absence of other added subunits (Couturier et al.

1990). Therefore, alpha7 appears to form homooligomeric receptors. The

functional receptors thus formed are blocked by alpha-bungarotoxin, unlike any

of the other neuronal receptors studied to date. Finally, they are derived from

genes with an intron/exon structure that is different from that of the other

neuronal receptor subunits (Couturier et al. 1990).

There are additional members of the gene family for which a function has

not yet been identified. Alpha5 is homologous to the other alpha subunits in

its overall architecture and in the presence of the two contiguous cysteines

in the extracellular domain (Boulter et al. 1990). However, this protein does

not form a functional receptor when RNA encoding it is injected into oocytes

in combination with any other receptor encoding RNA. It seems unlikely that

these clones fail to encode functional receptors as a consequence of some

cloning artifact that generated an incorrect sequence because the sequence

has been confirmed by analysis of genomic clones. The gene encoding

alpha5 is expressed in the CNS in a precise set of neuronal structures (Wada

et al. 1990). There has not yet been a systematic analysis of the contribution

this subunit might make as a third component of an oligomeric receptor.

Beta3 likewise fails to form functional receptors when injected in pairwise

combinations with the known functional receptors and, like alpha5 is

expressed in a well-defined set of neuronal structures.

In situ hybridization has shown that the members of this gene family are

expressed throughout the CNS (Wada et al. 1989, 1990). The beta2-encoding

RNA is found in almost all brain nuclei examined, and the other alpha- and

beta-encoding RNAs are expressed in specific but generally overlapping

subsets of nuclei. The beta4 subunit was first described as localized to the

medial habenula but is now known to be widely expressed in the central and

peripheral nervous systems (Moss et al. 1989). The alpha subunits are

expressed in discrete but overlapping sets of loci. However, these data do not

show the location of the expressed protein, and it is not yet known which

particular subunit combinations are located on dendrites, cell bodies, or axons.

In summary, molecular biological approaches to neuronal nicotinic receptors

have defined a gene family, documented the expression of this gene family in

the CNS, and demonstrated that various combinations of the proteins derived

from these genes form functionally different receptors. It is not yet known if all

the members of the gene family have been identified. Nor does one know the

full spectrum of combinations of subunits that exist in vivo or the roles that these

different subunit combinations play in the function and/or modification of

synapses.

7
It is the case, however, that neuronal nicotinic receptors differ in their

functional properties depending on which beta subunits are included in the

oligomer. Receptors containing a beta2 and an alpha3 subunit are sensitive to

the neuronal bungarotoxin (Boulter et al. 1987), whereas those in which an

alpha2 replaces the alpha3 subunit are 100-fold less sensitive (Wada et al.

1988). Receptors substituting an alpha4 are intermediate in sensitivity (Boulter

et al. 1987; Luetje et al. 1990b). This is consistent with the view that the ligand

binding site is on the alpha subunit. However, substitution of a beta4 for beta2

renders the alpha3-containing receptor insensitive to this toxin, suggesting that

the beta subunits either contribute to the toxin binding site or modify the

conformation of the alpha subunits (Duvoisin et al. 1989; Luetje et al. 1990b).

Different combinations of alpha and beta subunits are also distinguishable in

their responses to agonists. The relative sensitivities for acetylcholine, nicotine,

dimethylphenylpiperainium, and cytisine were determined for all six receptor

combinations that can be made from alpha2, alpha3, or alpha4 in combination

with either beta2 or beta4. These experiments were done in the Xenopus

oocyte where it was also established that the expressed muscle-type receptor

had a pharmacological profile indistinguishable from the cell line from which the

clones were derived. The results of these studies demonstrate that both the

alpha and the beta subunits contribute to agonist specificity. Receptors

containing a beta2 subunit differ in their response to acetylcholine and nicotine

depending on which alpha subunit is present. Nicotine is about thirtyfold more

effective on receptors containing alpha2 than on receptors containing alpha3,

suggesting that the alpha subunits determine agonist specificity. Cytisine is the

least effective agonist in all receptors containing a beta2 subunit. However,

substitution of the beta4 for the beta2 renders cytisine the best of the four

agonists. Although cytisine is the best agonist on beta4-containing receptors,

competition experiments suggest that cytisine is an antagonist on beta2-

containing receptors. These results demonstrate that both the alpha and

beta subunits contribute to both agonist and antagonist recognition and suggest

that one possible consequence of the expression of different combinations of

receptor subunits in the CNS is altered ligand affinity and specificity (Luetje and

Patrick 1991).

The neuronal nicotinic receptors differ from the muscle nicotinic receptors in

two additional interesting ways. The neuronal nicotinic acetylcholine receptors

are more permeable to calcium and in this property more closely resemble the

N-methyl-D-aspartate type of glutamate receptor than a muscle nicotinic

acetylcholine receptor (Vernino et al. 1992). The neuronal nicotinic receptors

are also modulated by external calcium ions. The author of this chapter and his

colleagues observed that increasing the external calcium ion concentration

resulted in large increases in the current produced by a given concentration of

8
receptors expressed in bovine chromaffin cells. The effect is not seen with

either barium or calcium, is not a consequence of activation of the calciumactivated chloride channel, and is not a consequence of an increased

contribution of calcium to the current but rather a modulation by calcium of

the response of the receptor to agonist. This result distinguishes the neuronal

nicotinic receptors from the muscle nicotinic receptors, which, in contrast, show

a decrease in current in the presence of elevated extracellular calcium and

suggest a different mechanism for regulation of receptor function at synapses

in the CNS. Both the extent of regulation by external calcium and the

magnitude of the calcium permeability are determined by the particular

combination of subunits that make up the receptor. Both of these phenomena

could contribute to cholinergic neurotoxicity and could be particularly relevant

if the receptors were located presynaptically.

DISCUSSION

One consequence of the diversity of receptor subunits and of the receptors

they form is a diversity of responses to different ligands. This observation

suggests that efforts to more carefully define the properties of the ligands

that activate or block different receptors could have important results. It might

prove possible to define classes of agonists or antagonists that are exquisitely

specific for particular combinations of subunits. These ligands might have

several important uses. They might be valuable for dissecting the function

of particular cholinergic systems in the brain by providing the investigator

with sharp tools. They might also be powerful drugs to help overcome

addiction to nicotine. Alternatively, they might prove to be drugs that provide

some of the beneficial aspects of nicotine but with fewer of the harmful results

and prove useful in treating diseases in which misfunction of nicotinic function

is suspected, such as Alzheimer’s disease. This diversity of ligand binding

specificity is clearly a double-edged sword because, although it may allow the

creation of powerful tools, it may (as pointed out above) provide yet additional

drugs with abuse potential.

However, it seems reasonable that there is a window of opportunity in the

pharmacological diversity of the ligand-gated ion channels. There is the

potential for the design of drugs that would provide important access to

function in the CNS. It also seems likely that the magnitude of the window

is currently underestimated. There may be many more aspects of receptor

function that vary with subunit combination, and access to the diversity related

to these aspects may be important. For example, different receptor subunit

combinations might be differentially regulated by peptides or external ions,

allow passage of different combinations of ions, be subject to regulation by

different cytoplasmic mechanisms, or be found in different portions of the

9
neuron. An interesting result in this regard might be the presence of nicotinic

receptors in the presynaptic membrane where they might regulate release of

such neurotransmitters as dopamine or serotonin. The discovery of the

diversity of ligand-gated ion channels is recent, but the idea has been rapidly

assimilated. However, there remains the exploitation of this diversity to better

understand the brain and to design drugs to better deal with the various

diseases that affect the brain.

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M.; Fukuda, K.; and Numa, S. Role of acetylcholine receptor subunits in

gating of the channel. Nature 318:1538-543, 1985.

Schoepfer, R.; Conroy, W.; Whiting, P.; Gore, M.; and Lindstrom, J. Brain abungarotoxin binding protein cDNAs and MAbs reveal subtypes of this

branch of the ligand-gated ion channel gene superfamily. Neuron 5:35-48,

1990.

Sumikawa, K.; Houghton, M.; Smith, J.C.; Bell, L.; Richards, B.M.; and Barnard,

E.A. The molecular cloning and characterization of cDNA coding for the

alpha subunit of the acetylcholine receptor. Nucleic Acids Res 10:5809-

5822, 1982.

Takai, T.; Noda, M.; Mishina, M.; Shimizu, S.; Furutani, Y.; Kayano, T.; Ikeda,

T.; Kubo, T.; Takahashi, H.; Takahashi, T.; Kuno, M.; and Numa, S. Cloning,

sequencing, and expression of cDNA for a novel subunit of acetylcholine

receptor from calf muscle. Nature 315:761-764, 1985.

Tzartos, S.J.; Kokla, A.; Walgrave, S.L.; and Conti-Tronconi, B.M. Localization

of the main immunogenic region of human muscle acetylcholine receptor to

residues 67-76 of the alpha subunit. Proc Natl Acad Sci U S A 85:2899-

2903, 1988.

Tzartos, S.J.; Loutrari, H.V.; Tang, F.; Kokla, A.; Walgrave, S.L.; Milius,

R.P.; and Conti-Tronconi, B.M. Main immunogenic region of Torpedo

electroplax and human muscle acetylcholine receptor: Localization and

microheterogeneity revealed by the use of synthetic peptides. J Neurochem

54:51-61, 1990.

Vernino, S.; Amador, M.; Luetje, C.W.; Patrick, J.; and Dani, J. Calcium

modulation and high calcium permeability of neuronal nicotinic acetylcholine

receptors. Neuron 8:127-134, 1992.

Wada, E.; McKinnon, D.; Heinemann, S.; Patrick, J.; and Swanson, L.W. The

distribution of mRNA encoded by a new member of the neuronal nicotinic

acetylcholine receptor gene family (alpha5) in the rat central nervous system.

Brain Res 526:45-53, 1990.

12
Wada, E.; Wada, K.; Boulter, E.; Deneris, E.S.; Heinemann, P.J.; and Swanson,

L. The distribution of alpha2, alpha3, alpha4, and beta2 neuronal nicotinic

receptor subunit mRNAs in the central nervous system. A hybridization

histochemical study in the rat. J Comp Neurol 284:314-335, 1989.

Wada, K.; Ballivet, M.; Boulter, J.; Connolly, J.; Wada, E.; Deneris, E.S.;

Swanson, L.W.; Heinemann, S.; and Patrick, J. Functional expression of a

new pharmacological subtype of brain nicotinic acetylcholine receptor.

Science 240:330-334, 1988.

AUTHOR

James Patrick, Ph.D.

Professor and Head

Division of Neuroscience

Baylor College of Medicine

One Baylor Plaza

Houston, TX 77030

13
Neurotransmitter and Drug Receptor

Genes

George R. Uhl

INTRODUCTION

Understanding neurotransmitter and drug receptor genes has potential for

enhancing the molecular neurobiology of substance abuse in at least two

fashions. First, understanding the molecular mechanisms whereby drugs

and neurotransmitters affected by drugs interact with their initial biological

targets, their receptors, can enhance understanding of acute drug action.

Structure-function relationships can be approached by modifying both the

ligands and the receptors, enhancing possibilities for development of, for

example, antiabuse medications. Second, since receptor genes likely are

key to the mechanisms underlying substance abuse, they are promising

candidates for the population variants that could explain some of the individualto-individual differences in susceptibility to drug abuse in human populations.

Recent studies of genes encoding a novel -aminobutyric acid (GABA) receptor

and of variant dopamine D2 receptor genes in drug-using and drug-free human

populations clearly demonstrate these points.

NOVEL GABA RECEPTORS: GABA C?

Classical pharmacological, binding. and electrophysiological studies suggest

that GABA produces most of its activities through interactions with two major

receptor classes: (1) GABA A receptors, composed of subunits that form

GABA-gated chloride channels and bind bicuculline, muscimol, barbiturates,

and benrodiazepines and interact with ethanol, and (2) GABA B receptors

that are responsive to baclofen and can alter calcium fluxes. Thus, GABA A

receptors are principal sites for the action of three classes of abused

substances: ethanol, barbiturates, and benzodiazepines.

Studies of cloned GABA A receptor subunits reveal that mixtures of

and -subunits may form functional brain receptors. Variation in subunit

composition can alter the activities of the resultant receptors; the specific

14
receptor subunit profile expressed by a neuron thus determines its differential

cellular responsiveness to GABA or related drugs (Pritchett and Seeburg

1990; Luddens et al. 1990). With the diversity of the possible GABA receptors

that could be formed in vivo, the exact criteria for defining GABA A receptors

have become debatable: (1) Should classical pharmacologic criteria, such

as inhibition by the competitive GABA antagonist bicuculline, define these

receptors, or (2) should structural membership in the family of multimeric

ligand-gated chloride channels defined by cloning studies constitute the

defining feature?

Some GABA responses in visual pathways are mimicked by muscimol and

inhibited by picrotoxin but are insensitive to the competitive GABA A antagonist

bicuculline and to GABA B antagonists (Sivilotti and Nistri 1988, 1989, 1991;

Nistri and Sivilotti 1985). These receptors are also insensitive to barbiturates

and benzodiazepines. Miledi and coworkers have recently shown that GABA

responses conferred by retinal mRNA in the Xenopus oocyte system display

the same bicuculline and baclofen resistance (Polenzani et al. 1991). Based

on the pharmacologic criteria noted above, GABA C responses thus would

be neither GABA A nor GABA B. GABA responses that are insensitive to

both bicuculline and baclofen have been called GABA C by Johnston (1986).

However, electrophysiological features of GABA C responses are consistent

with their mediation by ligand-gated ion channels such as those characteristic

of GABA A receptors (Olsen and Venter 1986): Conceivably, bicucullineinsensitive GABA responses could be conferred by a receptor highly

homologous to other known GABA A receptors.

We have recently cloned a cDNA for a receptor subunit, GABA p-1, whose

mRNA is highly expressed in retina. When expressed as a single subunit in

the Xenopus oocyte system, p-1 mRNA consistently and robustly confers

picrotoxin-sensitive GABA responses whose reversal potential indicates

changed chloride conductance. Although several GABA A receptor subunits

can form functional ligand-gated channels when expressed in various

combinations, such responses are typically variable and inconsistent unless

several subunits are coexpressed (Blair et al. 1988; Khrestchatisky et al.

1989; Shivers et al. 1990; Malherbe et al. 1990; Verdoorn et al. 1990).

GABA p-1 responses are strikingly insensitive to inhibition by bicuculline

(Shimada et al., in press). The GABA binding site of this receptor thus

shows substantial differences from the GABA binding sites on and

ß-homo-oligomeric receptors (Blair et al. 1988; Khrestchatisky et al. 1989;

Shivers et al. 1990; Malherbe et al. 1990; Verdoorn et al. 1990). This site’s

properties fit with those of the “unusual” bicuculline-resistant GABA receptor

in visual pathways, with GABA responses in Xenopus oocytes injected with

15
retina mRNA, and with the GABA C receptor defined by Johnston (Sivilotti

and Nistri 1989; Johnston 1986). Expression of the GABA p-1 cDNA as a

homo-oligomer thus creates a unique GABA binding site. When the primary

structure of this receptor cDNA is compared with other known ligand-gated

channels, its closest homology is with GABA A receptor subunits.

Nevertheless, its sequence is more divergent from the other classes of GABA

receptor subunits than they are from each other. If a GABA A receptor is

defined based on its membership in this family and based on its ability to form a

Iigand-gated channel, then the GABA p-1 receptor belongs in the GABA A

family. To the extent that GABA A receptors are defined based on a

pharmacologic feature, bicuculline sensitivity, this receptor falls into the class of

“unusual GABA receptors” or GABA C receptors (Sivilotti and Nistri 1991;

Johnston 1986).

Could this receptor be expressed as a p-1 homo-oligomer in the retina or

brain? The unique properties of the p-1 receptor are maintained when it is

coexpressed with either an or a ß-GABA A subunit (Shimada et al., in press).

Although studies defining which subunits are coexpressed by neurons are

necessary before the significance of such observations for in vivo receptor

function can be known, these results—and observations that the major

component of GABA responses obtained in Xenopus expression studies of

mRNA isolated from the retina is bicuculline insensitive—are consistent with

the p-1 subunit’s self-association in vivo.

The pharmacologic profile obtained from the expressed p-1 subunit suggests

that the resultant receptors could be termed GABA C. If further evidence

supports a self-associating role for this subunit, this role may be sufficiently

unique to demand this designation. In any case, these receptors’ properties

make them ideal molecular tools with which to investigate the features

necessary to confer ethanol, benzodiazepine, and barbiturate sensitivity on

ligand-gated chloride channel GABA receptors. By making chimeric receptors

containing specific regions of GABA A -subunits spliced onto the p-1

backbone, researchers have the opportunity to produce single subunits that

should express at high levels by themselves and gain sensitivity to barbiturates

and benzodiazepines with the addition of specific -sequences. Such

constructions will allow testing of hypotheses such as that advanced by

Pritchett and Seeburg (1990) that specific N-terminal amino acids are

important molecular features for benzodiazepine actions.

DOPAMINE D2 RECEPTOR ALLELES IN SUBSTANCE ABUSERS

Several substances that share the potential for abuse by humans also share the

ability to enhance dopamine activity in mesolimbic/mesocortical circuits thought

16
to be important for behavioral reward and reinforcement (Lippa et al. 1973;

Di Chiara and Imperato 1988; Wise and Rompre 1989). For example, cocaine’s

ability to inhibit reuptake of dopamine indicates a possible direct action for this

highly reinforcing drug in these dopaminergic circuits (Ritz et al. 1987;

Grigoriadis et al. 1989).

Blum and coworkers (1990) suggested that the “A1” allele of the dopamine

D2 receptor gene may display an association with alcoholism. This allele,

identified by a Taq I restriction fragment length polymorphism (RFLP) of the

human dopamine D2 receptor (Grandy et al. 1989), was present in 69 percent

of alcoholics but only 20 percent of nonalcoholics. However, Bolos and

colleagues (1990) found that the A1 allele frequency was not significantly

higher in 40 alcoholics than in 127 individuals from two other samples not

characterized with respect to alcohol use. A substantial genetic contribution

to susceptibility to alcoholism is supported by family, twin, and adoption

studies (Goodwin 1979; Cloninger et al. 1981; Cloninger 1987). A genetic

component of vulnerability to drug abuse is less clearly documented but

has been suggested in both twin and adoption studies (Cadoret et al. 1987;

Pickens et al. 1991). These considerations led to the examination of whether

subjects with substantial self-reported alcohol, other drug, or nicotine use also

display elevated A1 allelic frequencies.

Examining such an allelic association in drug abusers raises methodological

concerns relating to polysubstance abuse, reliability of subjects, and means

used to categorize factors such as extent of drug use and dependence.

Recognizing these difficulties and the importance of a possible association

between drug use and specific receptor gene alleles, O’Hara and colleagues

(submitted for publication) studied D2 receptor alleles in almost 400 individuals

volunteering for research protocols at the National Institute on Drug Abuse

Addiction Research Center or presenting to the Johns Hopkins hemodialysis

and genetics clinics to provide population controls.

The A1 allele was present at a higher frequency in blacks than in whites

(z=4.37, P<.00001) for all groups combined. The A3 allele was present in

15 of 206 black subjects but absent in the 225 white subjects (z=3.64, P<.001),

providing further evidence for a racial difference in D2 allelic status. No

association between ethnicity and presence of the A1 allele was found in

an analysis of Greek (7 of 25=28 percent A1 present) and Italian (8 of 25=32

percent A1 present) individuals.

Associations between A1 allele presence and drug use were examined

separately in blacks and whites. In whites, a trend toward an association

was found in comparisons of subjects with substantial (++ and +++) vs.

17
minimal (0 and +) total drug use (z=1.57, P<.06). Bartholomew’s test also

showed a trend for an increasing gradient in proportion of subjects with the A1

allele across the four levels of substance use (P<.10). Black subjects displayed

no apparent association for either comparison.

Allelic association was also examined for individual drugs. A1 allele presence

was higher for white heavy users (++ and +++) of most substances compared

with whites with the lowest overall substance use (0 on total use); none of

these differences reached statistical significance. However, since many of

the subjects used multiple substances, the comparisons are not between

independent groups. Black subjects displayed no association between A1

presence and use of any substance class.

The hypothesis that individual differences in substance abuse may be due, in

part, to different alleles of the dopamine D2 receptor gene arose from initial

work in alcoholics (Blum et al. 1990) and was strengthened by a compelling

biological rationale for interactions between abused drugs and brain dopamine

systems (Di Chiara and lmperato 1988; Wise and Rompre 1989).

The lack of strong association between D2 receptor gene alleles and

substance use evident in this study is consistent with estimates of the

heritable components of alcoholism and drug abuse (Devor and Cloninger

1989; Cadoret et al. 1987). One recent study of concordance rates for

alcoholism in twin populations suggests that between 20 and 30 percent of

the vulnerability to abuse or depend on these substances may be genetic in

origin (Pickens et al. 1991). Attempts to link familial alcohol susceptibility to

specific chromosomal markers and patterns of inheritance in families have not

been consistent with a single genetic locus (Gilligan et al. 1987; Aston and Hill

1990). The strong association between a single gene allele and alcoholism

found by Blum and coworkers (1990) would thus fit poorly with this extent of

heritability. The large environmental influences on expression of alcoholism

and Blum and colleagues’ study of unrelated individuals rather than defined

pedigrees also make the strength of their findings surprising.

The associations that Uhl and colleagues (1992) found reach only the margins

of statistical significance and are evident in only one race. Thus, they should

be approached with both interest and caution, although an association of this

magnitude would fit better with previous results concerning heritability of drug

abuse. Striking results (P<0.001) can be obtained when Uhl and colleagues’

(1992) data are combined with other published studies: 31 percent (78 of 253)

of white individuals lacking documented substance abuse and 46 percent (88 of

193) of whites with alcohol abuse/dependence or heavy substance use display

the A1 allele (Blum et al. 1990; Grandy et al. 1989; Bolos et al. 1990; Parsian et

18
al. 1991). Although alternative explanations must be considered (see below),

these results cannot rule out an association between a D2 receptor genotype

and substance use.

In contrast to the finding of a modest possible association between receptor

gene alleles and level of drug use, there was a highly significant effect of race

on allelic frequencies. The 20-percent A1 allele frequency in whites contrasts

with 37 percent for blacks. No white individual had an A3 allele, whereas the

frequency in blacks was 3 percent. These data agree with findings of higher

A1 allele presence in blacks than in whites (Blum et al. 1990) and no A3 alleles

in 167 whites (Bolos et al. 1990).

Gene allelic frequency differences among distinct white populations are well

documented for disease-related genes such as those causing thalassemia

(southern European predominance) and cystic fibrosis (northern European

predominance) (Cystic Fibrosis Genetic Analysis Consortium 1990; Orkin et

al. 1982). Population-to-population differences within Caucasian groups

presumably exist for many anonymous RFLP markers as well, although

analysis comparing Greek and Italian individuals fails to provide support

for such differences in D2 allelic distribution (O’Hara et al., submitted for

publication). The modest possible allelic association with substance use in

whites could result from disproportionate membership of heavy substance users

in ethnic groups with high A1 frequencies, but the broad populations from which

the different groups were drawn and O’Hara and colleagues’ (submitted for

publication) failure to find substantial differences in two Caucasian populations

make this hypothesis less likely.

The results of this study could be extended in several ways. Sequencing genes

cloned from A1 and A2 homozygotes could pinpoint base pair changes at the

polymorphic Taq I sites and address the possibility that other base pair changes

linked to these alleles might produce functional differences. Individuals with

A1, A2, and A3 genotypes could also be tested to identify differing physiological

and psychological responses to administered drugs. Finally, study of the

genotypes of additional white individuals with carefully ascertained ethnicity

and drug use, members of more kindreds displaying striking familial patterns

of substance use, and individuals meeting criteria for drug abuse/dependence

diagnoses could enhance the data presented here. Positive results of such

investigations would strengthen confidence that a D2 receptor allele confers

vulnerability to substance abuse and provides a biological marker allowing

targeted interventions for vulnerable individuals.

19
SUMMARY

Studies of the two neurotransmitter receptor genes described here illustrate the

rich possibilities that this area of molecular neurobiology holds for drug abuse

research. Since many of the 100 to 150 different neurotransmitters could have

multiple receptor genes, as many as 1,000 different genes or 1 percent of the

human genome might encode neurotransmitter receptors. Clearly, as the other

chapters in this monograph also indicate, the field is ripe with possibilities of

discovery with implications for drug action.

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ascertained through a pair of male alcoholics. Am J Hum Genet 46:879-887,

1990.

Blair, L.A.C.; Levitan, E.S.; Marshall, J.; Dionne, V.E.; and Barnard, E.A. Single

subunits of the GABAA receptor form ion channels with properties of the

native receptor. Science 242:577-579, 1988.

Blum, K.; Noble, E.P.; Sheridan, P.J.; Montgomery, A.; Ritchie, T.;

Jagadeeswaran, P.; Nogami, H.; Briggs, A.H.; and Cohn, J.B. Allelic

association of human dopamine D2 receptor gene in alcoholism. JAMA

263:2055-2060, 1990.

Bolos, A.M.; Dean, M.; Lucas-Derse, S.; Ramsburg, M.; Brown, G.L.; and

Goldman, D. Population and pedigree studies reveal a lack of association

between the dopamine D2 receptor gene and alcoholism. JAMA 264:3156-

3160, 1990.

Cadoret, R.J.; O’Gorman, T.; Troughton, E.; and Heywood, E. An adoption

study of genetic and environmental factors in drug abuse. Arch Gen

Psychiatry 43:1131-1136, 1987.

Cloninger, C.R. Neurogenetic adaptive mechanisms in alcoholism. Science

236:410-416, 1987.

Cloninger, C.R.; Bohman, M.; and Sigvardsson, S. Inheritance of alcohol

abuse: Cross-fostering analysis of adopted men. Arch Gen Psychiatry

38:861-868, 1981.

Cystic Fibrosis Genetic Analysis Consortium. Worldwide survey of the WF508

mutation—report from the Cystic Fibrosis Genetic Analysis Consortium. Am

J Hum Genet 47:354-359, 1990.

Devor, E.J., and Cloninger, C.R. Genetics of alcoholism. Annu Rev Genet

23:19-36, 1989.

Di Chiara, G., and Imperato, A. Drugs abused by humans preferentially

increase synaptic dopamine concentrations in the mesolimbic system

of freely moving rats. Proc Natl Acad Sci U S A 85:5274-5278, 1988.

Gilligan, S.B.; Reich, T.; and Cloninger, C.R. Etiologic heterogeneity in

alcoholism. Genet Epidemiol 4:395-414, 1987.

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Goodwin, D.S. Alcoholism and heredity. Arch Gen Psychiatry 36:57-61, 1979.

Grandy, D.K.; Litt, M.; Allen, L.; Bunzow, J.R.; Marchionni, M.; Markham, H.;

Reed, L.; Megenis, R.E.; and Civelli, O. The human dopamine D2 receptor

gene is located on chromosome 11 at Q22-Q23 and identifies a TAQ 1

RFLP. Am J Hum Genet 45:778-785, 1989.

Grigoriadis, E.E.; Lew, A.A.W.; Sharkey, J.S.; and Kuhar, M.J. Dopamine

transport sites selectively labeled by a novel photoaffinity probe: 125l-Deep.

J Neurosci 9(8):2664-2670, 1989.

Johnston, G.A.R. Multiplicity of GABA receptors. In: Olsen, R.W., and Venter,

J.C., eds. Benzodiazepine/GABA Receptors and Chloride Channels.

Receptor Biochemistry and Methodology. Vol. 5. New York: Alan R. Liss,

1986. pp. 57-71.

Khrestchatisky, M.; MacLennan, A.J.; Chiang, M.Y.; Xu, W.; Jackson, M.B.;

Brecha, N.; Sternini, C.; Olsen, R.W.; and Tobin, A.J. A novel a subunit in

rat brain GABAA receptors. Neuron 3:1745-753, 1989.

Lippa, A.S.; Antelman, S.M.; Fisher, A.E.; and Canfield, R.D. Neurochemical

mediation of reward: A significant role of dopamine? Pharmacol Biochem

Behav 1:23-28, 1973.

Luddens, H.; Pritchett, D.B.; Kohler, M.; Killisch, I.; Keinanen, K.; Monyer, H.;

Sprengel, R.; and Seeburg, P.H. Cerebellar GABAA receptor selective for

a behavioral alcohol antagonist. Nature 346:648-651, 1990.

Malherbe, P.A.; Draguhn, A.; Multhaup, G.; Beyreuther, K.; and Mohler, H.

GABAA-receptor expressed from rat brain and ß-subunit cDNAs displays

potentiation by benzodiazepine receptor ligands. Mol Brain Res 8:199-208,

1990.

Nistri, A., and Sivilotti, L. An unusual effect of -aminobutyric acid on synaptic

transmission of frog tectal neurons in vitro. Br J Pharmacol 85:917-921,

1985.

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in dopamine D2 receptor alleles suggest caution for associational studies.

Hum Hered, submitted for publication.

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Chloride Channels. Receptor Biochemistry and Methodology. Vol. 5. New

York: Alan R. Liss, 1986.

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Sexton, J.P.; Waber, P.G.; and Giardina, P.J. Linkage of beta-thalassaemia

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Parsian, A.; Todd, R.D.; Devor, E.J.; O’Malley, K.L.; Suarez, B.K.; Reich, T.;

and Cloninger, C.R. Alcoholism and alleles in the human D2 dopamine

receptor locus. Arch Gen Psychiatry 48:655-663, 1991.

Pickens, R.W.; Svikis, D.S.; McGue, M.; Lykken, D.T.; Heston, L.L.; and

Clayton, P.J. Heterogeneity in the inheritance of alcoholism: A study of

male and female twins. Arch Gen Psychiatry 48:19-28, 1991.

21
Polenzani, L.; Woodward, R.M.; and Miledi, R. Expression of mammalian

aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes.

Proc Natl Acad Sci U S A 88:4318-4322, 1991.

Pritchett, D.S., and Seeburg, P.H. -Aminobutyric acid, receptor -subunit

creates novel type II benzodiazepine receptor pharmacology. J Neurochem

54(5):1802-1804, 1990.

Ritz, M.C.; Lamb, R.J.; Goldberg, S.R.; and Kuhar, M.J. Cocaine receptors on

dopamine transporters are related to self-administration of cocaine. Science

237:1219-1223, 1987.

Shimada, S.; Cutting, G.; and Uhl, G. -Aminobutyric acid A or C receptor?

Aminobutyric acid p1 receptor RNA induces bicuculline-, barbiturate-, and

benzodiazepine-insensitive -aminobutyric acid responses in xenopus

oocytes. Mol Pharmacol, in press.

Shivers, B.D.; Killisch, I.; Sprengel, R.; Sontheimer, H.; Kohler, M.; Schofield,

P.R.; and Seeburg, P.H. Two novel GABAA receptor subunits exist in distinct

neuronal subpopulations. Neuron 3:327-333, 1990.

Sivilotti, L., and Nistri, A. Complex effects of baclofen on synaptic transmission

of the frog optic tectum in vitro. Neurosci Lett 85:249-254, 1988.

Sivilotti, L., and Nistri, A. Pharmacology of a novel effect of -aminobutyric acid

on the frog optic tectum in vitro. Eur J Pharmacol 164:205-212, 1989.

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system. Prog Neurobiol 36:35-92, 1991.

Uhl, G.R.; Persico, M.D.; and Smith, S.S. Current excitement with D2 dopamine

receptor gene alleles in substance abuse. Arch Gen Psychiatry 49:157-160,

1992.

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Functional properties of recombinant rat GABAA receptors depend upon

subunit composition. Neuron 4:919-928, 1990.

Wise, R.A., and Rompre, P.P. Brain dopamine and reward. Annu Rev Psychol

40:191-225, 1989.

AUTHOR

George R. Uhl, M.D., Ph.D.

Chief

Laboratory of Molecular Neurobiology

Addiction Research Center

National Institute on Drug Abuse

Associate Professor of Neurology and Neuroscience

Johns Hopkins University School of Medicine

P.O. Box 5180

Baltimore, MD 21224

22
The Diversity of the Dopamine

Receptors

Olivier Civelli, James R. Bunzow, Qun-Yong Zhou, and David K.

Grandy

INTRODUCTION

Among all the neuronal pathways, the dopamine system is thought to have the

leading role in modulating drug addiction. This system relies on the interaction

of one neurotransmitter, dopamine, with several receptors.

Classically, dopamine was thought to exert its effects by binding to only two

G protein-coupled receptors, known as the D1 and D2 receptors (Kebabian

and Calne 1979). These receptors have been differentiated pharmacologically,

biologically, and physiologically and also by their anatomical distribution.

These two receptors exert their biological actions by coupling to and activating

different G protein complexes. The D1 receptor interacts with the Gs complex

to activate adenylyl cyclase, whereas the D2 interacts with Gi to inhibit cyclic

adenosine monophosphate (CAMP) production.

CLONING OF THE D2 DOPAMINE RECEPTOR

The cloning of the D2 dopamine receptor resulted from the use of a strategy

based on the sequence homology expected to exist among G proteincoupled receptors. The ß2-adrenoreceptor coding sequence was used as

a hybridization probe to screen a rat genomic library under low-stringent

hybridization conditions. By screening the equivalent of three genomes,

90 positive clones were identified, from which 20 were characterized and

partially sequenced. This allowed the characterization of the clones encoding

the rat ß1-adreno (Machida et al. 1990), the serotonin 1a (Albert et al. 1989),

the muscarinic 4 (J.R. Bunzow and O. Civelli, unpublished observation), and

another clone, RGB-2, which, as described below, encodes the dopamine D2

receptor (Bunzow et al. 1988).

The RGB-2 clone was used to screen a rat brain cDNA library. One positive

clone, containing a 2.5-kb insert, was sequenced, and its corresponding

23
peptide sequence was determined. This clone encodes a 415-amino acid

protein with all the expected characteristics of a G protein-coupled receptor:

It has seven hydrophobic domains, 21 amino acid residues that are conserved

among all cloned G protein-coupled receptors and potential glycosylation and

phosphorylation sites, and a significant degree of sequence similarity with the

other receptors in this gene family.

The full-length RGB-2 cDNA was cloned into a plasmid containing the

metallothionein promoter, and this construct was cotransfected with pRSVneo

(a selectable marker conferring resistance to the antibiotic neomycin) into

mouse Ltk-cells. Stable transfectants were prepared and analyzed for their

ability to bind dopamine ligands (Bunzow et al. 1988). L-RGB2Zem-1

membranes bound D2 dopamine agonists and antagonists with the same

pharmacological profile as do rat striatal membranes. These studies used

the antagonist [3H] spiperone, whose binding was shown to be saturable and

of high affinity. [3H] spiperone binding to L-RGB2Zem-1 membranes was

displaced by several antagonists with the stereospecificity expected of a D2

receptor and with the same Kis as determined in rat striatal membranes.

D2 receptors are present on lactotroph cells of the anterior pituitary where

they regulate prolactin (PRL) secretion. The somatomammotroph cell line

GH4C1 is derived from a rat pituitary tumor, is devoid of endogenous dopamine

receptors, and is known to secrete PRL. GH4C1 cells were transfected with

the RGB-2 cDNA metallothionein construction. One clone, GH4ZR7, was

found to express high levels of RGB-2 mRNA (Albert et al. 1989). Since

the D2 receptor is expected to inhibit CAMP levels, vasointestinal peptide

(VIP) was used to stimulate endogenous CAMP production. Dopamine

inhibited both basal and VIP-stimulated CAMP levels in media from GH4ZR7

cells. Intracellular CAMP levels were also inhibited. The stereospecificity of

these inhibitions was demonstrated using isomers of sulpiride: The active

enantiomer (-)sulpiride blocked the inhibition, whereas (+)sulpiride had no

effect. To demonstrate that the changes in CAMP levels were the result of

an inhibition of adenylate cyclase, dopamine was added to membranes of

VIP- or forskolin-stimulated GH4ZR7 cells, and adenylate cyclase activity was

measured. Dopamine inhibited adenylate cyclase activity in a stereoselective

manner. Finally, the inhibition of PRL secretion by dopamine was assayed

in GH4ZR7 cells. VIP and thyrotropin-releasing hormone (TRH) are known

to enhance PRL release by a cAMP-dependent and a cAMP-independent

mechanism, respectively. Dopamine was able to inhibit PRL secretion

stimulated by both hormones. These inhibitions were reversed by the active

antagonist (-)sulpiride but not by (+)sulpiride. Therefore, the RGB-2 cDNA

encodes a D2 dopamine receptor that is functional.

24
CLONING OF THE DOPAMINE D1 RECEPTOR

The success of the homology approach in D2 receptor cloning opened the

door for the cloning of other dopamine receptors, in particular the D1 receptor.

The authors and colleagues took advantage of the polymerase chain reaction

(PCR)-based approach, which had been developed to clone several thyroid

G protein-coupled receptors (Libert et al. 1989). This approach consists of

synthesizing two sets of synthetic oligonucleotides that correspond to two

highly conserved regions among all the G protein-coupled receptors (found

generally in transmembrane domains III and VI). These oligonucleotides

are used as primers in a PCR reaction for specific amplification of cDNAs

containing complementary sequences. The cDNAs used for the D1 receptor

cloning were synthesized from rat striatum. To direct the PCR approach

toward the specific cloning of the D1 receptor, we added another technical

step. Because it is known that Gs-coupled catecholamine receptors have a

putative third cytoplasmic loop of 52 to 78 amino acids (Zhou et al. 1990), our

total population of PCR products was size fractionated, and products in the

expected range were sequenced. Of 24 PCR products, 7 encoded potential G

protein-coupled receptors, one of which showed structural features expected to

belong to Gs-coupled catecholamine receptors. This clone was used to screen

a rat cDNA and human and rat genomic libraries since most catecholamine

receptor genes lack introns in their coding regions (Lefkowitz et al. 1988).

The isolated clone was sequenced and shown to contain all the characteristics

expected of G protein-coupled receptors (i.e., share the highest degree of

similarity and prototypical structural features of the catecholamine receptors).

The absence of a glutamic residue found in the third transmembrane

domains of all the ß-adrenoreceptors and the size of the third cytoplasmic

loop suggested that the cloned receptor could be a Gs-coupled dopamine

receptor, namely the D1 receptor.

The demonstration that the cloned receptor was the D1 receptor was

accomplished by expressing the corresponding gene. First, the putative

D1 receptor human gene was expressed by transient expression in COS-7

cells. Membrane proteins from the transfected cells were tested for their

ability to bind D1 receptor ligands. The specific antagonist SCH 23390 was

found to have the highest affinity for the cloned receptor, and the overall

pharmacological profile was that of a D1 receptor binding site. The biological

activity of the cloned receptor was studied upon transient transfection in human

kidney 293 cells and analysis of dopamine stimulation of adenylyl cyclase

activity. The cloned receptor was shown to stimulate adenylyl cyclase activity

according to a pharmacological profile expected for the D1 receptor. Therefore,

we concluded that we had cloned the D1 dopamine receptors.

25
DIVERSITY OF THE DOPAMINE RECEPTORS

As discussed above, pharmacological analyses had agreed on the existence of

only two dopamine receptors (Hess and Creese 1987; Creese 1986; Leff and

Creese 1985). With the cloning of these two receptors, new tools were at hand

to further understanding of the dopamine system. In 2 years, several studies

using molecular biological approaches have shown that the classical view of the

dopamine system was incomplete.

Two Forms of the D2 Receptor

The discovery that not one but two dopamine D2 receptor forms exist was

reported in 1989 (Selbie et al. 1989; Grandy et al. 1989; Dal Toso et al. 1989;

Giros et al. 1989; Monsma et al. 1989; Chio et al. 1990; Miller et al. 1990;

O’Malley et al. 1990) and showed that the two D2 receptor forms exist in

human, rat, and bovine cells. These two forms differ in 29 amino acid residues

located in the putative third cytoplasmic loop of the receptor. The short form is

the one originally cloned (as described above); the long form is new and was

discovered either by screening cDNA libraries or by PCR analyses.

Several data were obtained about the 29 amino acid addition. First, the

additional residues do not modify the affinity or the profile of the D2 receptor

for antagonists (Grandy et al. 1989; Giros et al. 1989). Second, they do not

affect significantly the ability of the receptor to inhibit CAMP production (Dal

Toso et al. 1989) as could have been expected from their location in the third

cytoplasmic loop. Third, it was found that the 29-amino acid addition contains

two potential glycosylation sites, but thus far, nothing is known about their

importance (Grandy et al. 1989). Fourth, it was found that although the two

forms of the D2 receptor coexist in all tissues analyzed, their ratio varies. The

short form is the least abundant; its concentration is very low in the pituitary but

represents about half of the D2 receptor mRNA in the pons or medulla (Giros et

al. 1989; O’Malley et al. 1990). Fifth, the generation of the two forms of D2

receptor was shown to be the result of an alternative splicing event occurring

during the maturation of the D2 receptor pre-mRNA (Grandy et al. 1989; Dal

Toso et al. 1989; O’Malley et al. 1990), which was demonstrated by the

discovery of an 87-bp exon encoding the additional amino acid residues.

These studies also led to the description of the organization of the D2 receptor

gene, with the coding part of the D2 receptor encoded by seven exons, one of

which (exon 5 in Grandy et al. 1989) is alternatively spliced.

In summary, the two D2 receptor forms have not been shown to differ in their

pharmacological or biological activities. They are generated by alternative

splicing and coexist in a tissue-specific ratio. Any differences in their biological

significance have yet to be demonstrated.

26
New Dopamine Receptors

Probably the most striking discovery to emerge from the cloning of G proteincoupled receptors is their diversity. Every class of G protein-coupled receptor

studied by recombinant DNA techniques has been proven more complex than

had been characterized pharmacologically (Bonner et al. 1987; Schwinn et al.

1990; Emorine et al. 1989). In view of the diversity of physiological responses

modulated by dopamine receptors, there was ample reason to believe that the

dopamine receptor class would also follow this trend. The success of the

homology approach in cloning the D2 receptor and the availability of dopamine

receptor probes led to the search for new, undescribed dopamine receptors.

D3 Receptor. Through the combination of cDNA library screening, PCR

extension, and genomic library screening, a cDNA was isolated that encodes

for a novel receptor related to the D2 receptor, thereafter coined the D3

receptor (Sokoloff et al. 1990). This receptor shares 75 and 41 percent of its

putative transmembrane sequences with the D2 and D1 receptors, respectively.

Moreover, it is encoded by a gene that contains five introns in its coding region

and whose organization is similar to that of the D2 receptor. The D3 receptor

structure is also highly similar of that of the D2 receptor, in that it contains a

large third cytoplasmic loop of similar size to that of the long form of the D2

receptor. However, the organization of the gene in this loop does not allow for

alternatively spliced forms. The D3 receptor also contains the residues found

to be important for catechol and amine groups recognition in the catecholamine

receptors. Altogether, the structure of the D3 receptor suggested a close

relationship to the D2 receptor, which was confirmed by pharmacological

analyses.

When expressed in eucaryotic cells (COS-7 or CHO cells), the D3 receptor

was shown to have a pharmacological profile reminiscent of that of the D2

receptor. It binds D2 ligands (not D1 or other catecholamine ligands), although

its affinity to most neuroleptics was 10- to 100-fold less than that of the D2

receptor. However, the D3 receptor was found to bind two particular

antagonists, (+)AJ76 and (+)UH232, with three to five times more affinity

than the D2 receptor. These antagonists are thought to have a higher

specificity for the dopamine presynaptic receptors or autoreceptors.

Interestingly, the binding of dopamine to the D3 receptor was not affected

by the addition of guanylnucleotides, which block G protein-coupled receptors

in their high affinity state and are used to measure G protein coupling. This

result might be explained by the absence of suitable G proteins in the

transfected cells (COS-7 and CHO cells) or by a low modulation of dopamine

binding by guanylnucleotides at the D3 receptor. The latter was not shown to

modulate CAMP formation in CHO cells, in contrast to the other dopamine

receptors.

27
The tissue distribution of the D3 receptor was determined by Northern blot and

in situ hybridization analyses. It not only was found to overlap but also to differ

from that of the D2 receptor. The D3 receptor is absent in the pituitary and is

expressed at low levels in the neostriatum, whereas high densities of the D2

receptor are present in both. The distribution of the D3 receptor overlaps with

the D2 receptor in the olfactory tubercules and the hypothalamus. Moreover,

the D3 receptor is expressed at high levels in the islands of Calleja and in the

nucleus accumbens, regions that are part of the limbic system and where the

concentrations of D2 receptor are relatively lower. In addition, the D3 receptor

was found to colocalize with the D2 receptor in presynaptic cells that produce

dopamine in the substantia nigra and in the ventrotegmental areas.

D4 Receptor. By analyzing the mRNA population of the neuroepithelioma

SK-N-MC cells with D2 receptor cDNA probes under conditions of low

stringency, the existence of a D2-related mRNA was detected (Van Tol et al.

1991). The corresponding cDNA and gene were sequenced and found to

encode another novel dopamine receptor, the D4 receptor. In its putative

transmembrane domains, the D4 receptor is 41, 52, and 51 percent identical

to the D1, D2, and D3 receptors, respectively, and it contains the residues

necessary for catecholamine recognition. Its putative third cytoplasmic loop

is shorter than that of the D2 receptor. At the genomic level, the D4 coding

sequence is separated by four introns that are positioned similarly to those

of the D2 receptor. In addition, a 52-bp repeat borders both sides of the third

intron. This unusual intron-exon junction does not contain conventional splice

sites and allows for potentially variable alternative splicing events without

changes in protein sequence.

The D4 receptor was expressed in COS-7 cells, and its pharmacological

profile was determined. Most of the tested agonists and antagonists displayed

affinities for the D4 receptor similar to or lower than that for the D2 receptor.

However, the D4 receptor binds one particular antipsychotic, clozapine, with

an affinity tenfold higher than either the D2 or D3 receptors.

Clozapine is a particularly interesting antipsychotic agent whose action is not

associated with the motor control side effects that plague other neuroleptics.

However, clozapine has its own side effects. Most deleterious is that it causes

agranulocytosis in a few cases, which has prevented its general use. In any

case, the discovery of a dopamine receptor specific for a neuroleptic that

might not affect the centers for motor control in the central nervous system is

important. A preliminary analysis of the D4 receptor tissue location indicated

that the D4 receptor is expressed in the mesocorticolimbic system rather than in

the nigrostriatal systems (Van Tol et al. 1991), adding credit to the hypothesis

that stimulation of this receptor has little impact on control of movement. These

28
observations suggest that the D4 receptor is most likely the primary target

mediating the antipsychotic action of clozapine.

Other Dopamine Receptors. There are indications that the diversity in

dopamine receptors does not stop at the D4 receptor. The existence of

another D2-like receptor has been reported (Todd et al. 1989). This receptor

was expressed upon transfection of rat genomic DNA into mouse fibroblasts

and was identified by its ability to bind iodinated spiperone. This receptor

has a pharmacological profile closely resembling that of the cloned D2 receptor,

yet it has a sequence different from that of the cloned D2 receptor as shown by

the inability of its mRNA to hybridize to three sets of oligonucleotides specific to

the D2 sequence. Stimulation of this receptor by dopamine leads to an

increase in intracellular calcium concentrations that appears not to be mediated

through G protein coupling.

Other D1-like receptors have also been described. D1-like receptors have

been detected in renal tissue (Felder et al. 1989). These are linked to the

activation of phosphatidylinositol-specific phospholipase C. These D1-like

receptors are different from the cloned D1 receptor because the latter is not

expressed in renal tissue (Zhou et al. 1990; Dearry et al. 1990). A D1-like

receptor that couples to inositol phosphate production has also been detected

by expression in Xenopus oocytes (Mahan et al. 1990). This receptor is

encoded by a mRNA found in the rat striatum but is of different size than the

one encoding the cloned D1 receptor.

There are also indications that yet other dopamine receptors might exist.

Pharmacological and biological data discussing the existence of putative

receptors have been discussed (Andersen et al. 1990). Moreover, several

laboratories have recently cloned a new D5 or D1b receptor (D.G. Grandy,

personal communication, 1991; H.H.M. Van Tol, personal communication,

1991; M.G. Caron, personal communication, 1991).

CONCLUSIONS AND PERSPECTIVES

Based on pharmacological studies, the dopamine receptors have been

classified into two subtypes, the D1 and D2 receptors. The cloning of the

dopamine receptors has revealed that, although the number of dopamine

receptors is larger than expected, they can still be classified under the

broad categories of D1- and D2-like receptors. The D1-like receptors are

encoded by genes that have no intron in their coding sequence, share more

than 50 percent identity with the D1 receptor in the sequences part of the

transmembrane domains, have a pharmacological profile that resembles that

of the D1 receptor and that binds efficiently the antagonist SCH 23390, and

29
stimulate adenylyl cyclase activity. The D2-like receptors have similar

characteristics when compared with the D2 receptor—they efficiently bind

spiperone and inhibit adenylyl cyclase activity.

The complexity of the dopamine receptor family is not surprising in view of the

complexity of the other receptor families that are part of the G protein-receptor

supergene family (Bonner et al. 1987; Schwinn et al. 1990; Emorine et al.

1989). How many dopamine receptors will be found is unknown. However, as

presently understood, there are two widespread and quantitatively predominant

dopamine receptors, the classical D1 and D2 receptors. The other dopamine

receptors are present in significantly lower amounts in restricted localizations,

and they can be related to the two major dopamine receptors through their

pharmacological profile. Therefore, most of what has been known about

dopamine agonists’ and antagonists’ actions has to be reevaluated in view of

the existence of the different dopamine receptors. This underscores the impact

that the cloning of the D2 receptor will have on the field of dopamine receptors.

Finally, the discovery of new dopamine receptors is also interesting from an

evolutionary point of view. One neurotransmitter interacts with a variety of

receptors, suggesting that nature has spent a large effort in developing the

targets in mechanism of synaptic transmission, a fact that could increase

understanding of higher brain function.

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ACKNOWLEDGMENT

This work was supported by National Institute of Mental Health grant

MH-45614.

AUTHORS

Olivier Civelli, Ph.D.

Associate Professor

Department of Cell Biology and Anatomy

Scientist

Vollum Institute for Advanced Biomedical Research

James R. Bunzow, M.S.

Senior Research Associate

Vellum Institute for Advanced Biomedical Research

32
Qun-Yong Zhou, Ph.D.

Department of Molecular Biology and Biochemistry

David K. Grandy, Ph.D.

Senior Research Associate

Vollum Institute for Advanced Biomedical Research

Oregon Health Sciences University

Portland, OR 97201

33
Cloned Dopamine Receptors: Targets

in Therapy of Drug Abuse

Philip Seeman

INTRODUCTION

Most self-rewarding behaviors operate through the brain dopamine system.

This generalization also holds for drug abuse. For example, many common

drugs of abuse, including amphetamine, cocaine, LSD, nicotine, ethanol, and

opiates, promote the release of dopamine in vivo (Di Chiara and lmperato

1988). Moreover, the acute overdoses of some of these drugs of abuse (e.g.,

amphetamine, cocaine, or LSD, but not ethanol or opiates) often are treated

by dopamine receptor-blocking medications such as haloperidol or other

neuroleptics.

EFFECTIVE THERAPY TARGETS: TRANSPORTERS OR RECEPTORS?

In clinical psychiatry and neurology, most medications that are used to alleviate

various illnesses block neurotransmitter transporter sites or receptor sites. In

general, the more effective medications are those that block receptors directly.

Medications that block the transmitter uptake sites (or transporter sites),

although useful, generally have been less effective clinically than those that

block or stimulate receptors directly.

For example, imipramine or chlorimipramine are used in the treatment of

clinical depression. These compounds inhibit the uptake or transport of

norepinephrine into nerve terminals. These medications generally require

about 2 or 3 weeks before clinical improvement occurs. Moreover, the

magnitude of the overall efficacy of these medications is unclear, because

many studies indicate that admitting a depressed patient into a hospital

alleviates depression in 60 percent of patients. This compares with about

70 percent improvement when using both medication and hospitalization of

depressed patients.

The mechanism thought to explain the action of these antidepressant drugs is

that, by inhibiting the uptake of norepinephrine, the drugs increase or prolong

34
the release of the transmitter, which in turn leads to a reduction of

ß-adrenoceptors in the postsynaptic neurones. Thus, the ß-adrenoceptors

are indirectly and slowly affected by the transporter-inhibiting drugs.

A more rapid and effective antidepressant action may be obtained by

stimulating the ß-adrenoceptors directly by means of ß-adrenoceptor agonists.

Although this agonist-type strategy has been found experimentally to be

rapid and effective in alleviating depression, the cardiovascular side effects

currently preclude routine treatment by agonist therapy.

However, fluoxetine is an important example of a successful transporter

blocker that alleviates depression relatively rapidly in about one-third of

depressed patients. The effect becomes apparent within 1 week of treatment.

A second example of presynaptic therapy affecting the transporter site is in

Parkinson’s disease. Dopamine uptake inhibitors, including benztropine,

are no longer used in treating this illness. However, agonists that stimulate

dopamine D1 or D2 receptors are effective in Parkinson’s disease.

Presynaptic receptor therapy is also less effective clinically. For example,

compounds such as BHT 920 and 3-PPP act on presynaptic dopamine

receptors to inhibit the release of dopamine. Although such inhibition

does confer neuroleptic-like effects clinically (e.g., against psychosis or

schizophrenia), the clinical action alleviates only about 40 percent of psychotic

patients, compared with an improvement of 35 percent of such patients without

neuroleptics, and approximately 70- to 90-percent improvement with dopamine

receptor-blocking neuroleptics.

GOAL: TARGETING DIFFERENT DOPAMINE RECEPTORS IN BRAIN

PSYCHOMOTOR REGIONS

Although receptors are effective clinical targets, recent medications are not

sufficiently receptor selective to yield clinical actions free of unwanted side

effects. The recent discoveries of multiple receptors for each transmitter now

permit the design and development of medications selective for a specific

subtype of receptor.

Thus, designing a neuroleptic that selectively targets D2-like receptors in

nonmotor brain regions will obviate motor side effects of such a neuroleptic in

psychotic patients. This appears to be the case with clozapine, a neuroleptic

that is selective for the D4 dopamine receptor (Van Tol et al. 1991).

35
TYPES OF DOPAMINE RECEPTORS

The first type of dopamine receptor reported, now termed D1, was identified by

its ability to respond to dopamine and to stimulate adenylate cyclase (Kebabian

et al. 1972). The DNA for this receptor has been cloned (Sunahara et al. 1990;

Zhou et al. 1990; Dearry et al. 1990); the amino acid sequence of D1 is shown

in figure 1.

The second type of dopamine receptor, now termed D2, was identified by its

affinity for nanomolar concentrations of antipsychotic drugs (Seeman et al.

1974, 1975a, 1975b, 1976, 1984, 1987; Titeler et al. 1978; Seeman and

Niznik 1990). The DNA for D2 has been cloned (Bunzow et al. 1988; Grandy

et al. 1989; Martens et al. 1991); the amino acid sequence for D2 is shown in

figure 1.

Additional dopamine receptors have been found recently by homology probing,

that is, by probing genomic DNA or cDNA with oligonucleotides containing

bases similar or identical (homologous) to D1, D2, and related catecholamine

receptors.

This approach yielded short and long forms of D2 (Giros et al. 1989; O’Dowd

et al. 1990), a D3 dopamine receptor (Sokoloff et al. 1990; Giros et al. 1990),

a D4 dopamine receptor (Van Tol et al. 1991), and a D5 dopamine receptor

(Sunahara et al. 1991).

In addition, this approach yielded truncated forms of these receptors, such as

a D3 receptor with its third transmembrane portion deleted and a D3 receptor

with its second outer (or extracellular) portion deleted (Giros et al. 1991). The

sequences of these truncated D3 receptors, named D3 (TM-del) and D3 (O2-

del), respectively, are shown in figure 1.

Moreover, the method of homology probing has revealed at least two human

pseudogenes of D5 (Nguyen et al. 1991).

Of the amino acid sequences shown in figure 1, 13 amino acids are

homologous in at least 75 various membrane-located receptors (for various

amines, peptides, and hormones) that are all GTP sensitive. These 13 amino

acids, which are common to all the G-linked receptors, are illustrated by arrows

in figure 2 and by solid black triangles in figure 1.

36
FIGURE 1. Amino acid sequences of dopamine receptors. D1 human is from Sunahara et al. 1990; D1 rat from

Zhou et al. 1990; D2 human from Grandy et al. 1989 and Dal Toso et al. 1989; D2 rat from Bunzow et al.

1988; D2 frog from Martens et al. 1991; D3 human from Giros et al. 1990; D3 rat from Sokoloff et al.

1990; D3 rat (with transmembrane segment 3 deleted) and D3 rat (with extracellular segment 2 deleted)

from Giros et al. 1991; D4 human from Van Tol et al. 1991; and D5 human from Sunahara et al. 1991.

The solid black triangles indicate the 13 amino acids that are found to be identical (homologous) in more

than 75 G-linked receptors for various amines, peptides, and hormones.
FIGURE 1. continued
FIGURE 1. continued
FIGURE 2. Illustrating the rat dopamine D2 (short) receptor within the

membrane. The arrows indicate the 13 amino acids that are

homologous in more than 75 G-linked receptors for amines,

peptides, and hormones. Dopamine is assumed to attach its

hydroxyl groups to the serine residues in the fifth transmembrane

segment.

CLONED DOPAMINE RECEPTOR PHARMACOLOGY

The sensitivities of the various dopamine receptors to agonists and antagonists

are listed in table 1. For each of these sites to be termed a dopamine receptor,

it is essential that dopamine be the most potent endogenous transmitter to

inhibit binding; otherwise, the site is not a dopamine receptor, by definition.

All the binding sites shown in the table meet this criterion, because epinephrine,

norepinephrine, and serotonin were all weaker than dopamine in inhibiting the

binding of each radioligand.

40
TABLE 1. Sensitivities of dopamine receptors to agonists and antagonists

Various

Human Species Rat Human Rat Human

Site or Clone D1 Tissue D2 D2 short D2 long D2 short D2 long D3 D4 hybrid D5

Tissue/cell Cos-77,13,14 Striatum Ltk-1,8 CHO 293 cell Ltk-LT8,12 CHO Cos-7 Cos-714

ant. pit.6 GH4ZR72.5 Cos-79

Ligand *Sch239827

*Spip

*Sch2339013,14

K, nM

Agonists

K, nM

Cos-711 29310

*Spip1.2 †l-Sul *Spip *Dom8 †

I-Sul *Spip12 *Sch2339014

*Spip5

*Spip12

*Dom8

*Rac9

†

I-Sul11 *Spip10

K, nM K, nM K, nM K, nM K, nM K, nM K, nM

ADTN-(+)

Apomorphine

Bromocriptine

Dopamine

Dopamine+GN

Epinephrine

Fenoldopam

(-)Norepinephrine

NPA

Pergolide

PHNO-(+)

Quinpirole-(-)

Serotonin

SKF 38393

SKF 76783

4,60014

2107

2,34014

>55,0007

20;60

50,00013

1,81614

1,36314

14,0007

9,69014

877

64514

Hi:1.76

Hi:~26

1.96

Hi:7.56

Hi:2.86

~6,0006

Hi:0.46

243 2509

145

5.3312.612

203

7.43

Hi:7311 Hi:2.811 24,8009253

17,00011,7053

273

Hi:1.26

Hi:4.86

~10,0006

Hi:1576

2130.623

57635.13

9,5603

5,0003

Hi:24 909

Hi:4.1 363

340 454

Hi:28 228

Hi:49-450

321 15

1,760 12,000

6.5 1,136

918

79

46 >20,000

4,180 3,000

1,800 100

530
TABLE 1. continued

Site or Clone

Antagonists

Butaclamol-(+)

Chlorpromazine

Clozapine

Eticlopride

Fluphenazine

Haloperidol

*lodosulpnde

Ketanserin

Pimozide

Prochlorperazine

Radopride

*Radopride

Remoxipride

SCH 23390

SKF 83566

Spiperone

*Spiperone

Sulpiride-S

Do.

Sulpiride-R

Thioridazine

YM-09151-2

*YM-09151-2

Human

D1

Various

Species Rat

Tissue D2 D2 short D2 long

0.9;3

7314

14114

18,00014

2114

2714

1907

>72,0007

0.1;0.4

0.314

2207

36,00014

20,45414

10014

0.96

3

6

100±204

0.096

1±0.55

1.5±0.54

2086

4

6

2

4

1.94

300±904

1,6906

0.076

0.056

186

15±54

0.066

0.950.812

2.83

1.59

168556313812

0.8-3 0.5-0.8

0.613

>1,0001

2.43

4.73

1.65

1.83

7148

0.09620.0693

0.095

5.511 9.23

15.954.811

11511 10311

3.33

0.095

Human Rat Human

D2 short D2 long D3 D4 hybrid D5

1

8

10.512

3.29

9138

0.0538

05510 .0510

469

3112

0.0912

6.13

1803

9.83

1.23

3.73

353

3.53

0.613

253

4223

7.83

40 27

37 133

9 250

2.1 19,000

46 14

5.1 48

148

43

2,500

237

3,690

3,560

0.06

0.08

52

0.3

0.4

4,500

77,270

12

0.09

28,636

300
TABLE 1. continued

KEY: *=tritium; †l-Sul=[1251]iodosulpiride; ant. pit.=anterior pituitary; GN=guanine nudeotide; Dom=domperidone; Halo=haloperidol; Hi=highaffinity state of D2; hybrid=hybrid of gene and cDNA from SKNMC neuroblastoma; NPA=N-propylnorapomorphine; Rac=raclopride;

Spip=spiperone

1Bunzow et al. 1988

2Albert et al. 1990

3Sokoloff et al. 1990

4Seeman 1990

5P. Seeman, unpublished data

6Seeman and Niznik 1988

7Dearry et al. 1990

8Grandy et al. 1989

9Stormann et al. 1990

10Dal Toso et al. 1989

11Giros et al. 1989

12Van Tol et al. 1991

13Sunahara et al. 1990

14Sunahara et al. 1991
The D1-like receptors are D1 and D5. They are approximately equally sensitive

to all agonists except dopamine. The dopamine dissociation constant (K value)

shown in the table for D1 is 2,340 nM and for D5 is 228 nM. Both these values

refer to the low-affinity state of D1 and D5, because under these experimental

conditions the Cos-7 cells did not reveal the high-affinity state. Because D5

is approximately 10 times more sensitive to dopamine, this suggests that D5 is

more readily activated than D1 in vivo. Thus, D5 may provide the major

background tone in the nervous system, or it may be recruited during massive

discharges of dopamine from nerve terminals, as might occur during episodes

of drug abuse.

Although the expressed D1 and D5 receptors did not reveal the high-affinity

state in tissue culture under these conditions, it is known that the high-affinity

state of D1 in brain tissue has a K of about 0.7 nM for dopamine, compared

with a value of about 10 nM for the high-affinity state of D2 for dopamine. Thus,

the D1/D5 receptors are about one order more sensitive to dopamine than the

D2-like receptors (that is, D2, D3, and D4) (see also Gehlert et al. 1992). The

reason for emphasizing this point is that the high-affinity state of these receptors

is the functional state (George et al. 1985).

As exemplified for clozapine in the table, this drug has about a tenfold higher

affinity for D4 than for D2 or D3. This supports the principle of developing

receptor-selective medications in the future.

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Giros, B.; Martres, M.P.; Pilon, C.; Sokoloff, P.; and Schwartz, J.C. Shorter

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Giros, B.; Martres, M.P.; Sokoloff, P.; and Schwartz, J.C. Clonage du gene du

recepteur dopaminergique D3 humain et identification de son chromosome.

C R Acad Sci Paris 311:501-508, 1990.

Giros, B.; Sokoloff, P.; Martres, M.P.; Riou, J.F.; Emorine, L.J.; and Schwartz,

J.C. Alternative splicing directs the expression of two D2 dopamine receptor

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Frothingham, L.; Fischer, J.B.; Burke-Howie, K.J.; Bunzow, J.R.; Server,

A.C.; and Civelli, O. Cloning of the cDNA and gene for a human D2

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ACKNOWLEDGMENT

This chapter was prepared with support from National Institute on Drug Abuse

grant DA-07223-01.

46
AUTHOR

Philip Seeman, M.D., Ph.D.

Professor

Departments of Pharmacology and Psychiatry

University of Toronto

Medical Sciences Building

Eight Taddlecreek Road

Toronto, Ontario M5S 1A8

CANADA

47
Cannabinoid Receptors: Which Cells,

Where, How, and Why?

Lisa A. Matsuda, Tom I. Bonner, and Stephen J. Lolait

INTRODUCTION

In both laboratory animals and humans exposed to marijuana or

cannabimimetic compounds, the majority of effects appear to be mediated

by the central nervous system. Although -tetrahydrocannabinol -THC)

has been known to be the primary psychoactive component of marijuana

since the mid-1960s (Gaoni and Mechoulam 1964), a receptor-mediated

mechanism for marijuana’s actions has been recognized only within the past

several years (Howlett et al. 1990). The existence of a cannabinoid receptor

implies that an endogenous “cannabinoid” ligand also must exist. However,

the identity of this ligand is unknown. In addition, a lack of readily available

cannabinoid receptor antagonists has further hindered the elucidation of the

physiological and pathological significance of cannabinoid receptors. Recent

cloning and identification of a cDNA that encodes a G protein-coupled

cannabinoid receptor not only provides solid evidence for a cannabinoid

receptor protein but also provides nucleic acid sequence information that

can be used to localize the mRNA for this receptor (Matsuda et al. 1990).

By using the technique of in situ hybridization histochemistry (ISHH), the

neuroanatomical localization of cannabinoid receptor mRNA identifies

populations of brain cells that potentially could mediate cannabinoid-induced

effects. Furthermore, comparing the localization of the mRNA for the receptor

with the localization of cannabinoid receptor proteins (Herkenham et al. 1991a)

reveals clues regarding the circuitry of the cells expressing these receptors.

LOCALIZATION OF RECEPTOR mRNA IN RAT BRAIN

The expression of the cannabinoid receptor gene was visualized in coronal

tissue sections of adult rat brain using ISHH with a 35S-tailed 48-base

oligonucleotide probe, SKR6-1. This probe complements sequence from the

rat cDNA, SKR6 bases 349-396 (Matsuda et al. 1990). In transfected cells,

this cDNA encodes a receptor that mimics the cannabinoid receptor previously

characterized in both neural cell lines and brain tissues in vitro (Howlett et al.

48
1986; Bidaut-Russell et al. 1990). In response to -THC and CP 55940,

a potent synthetic nonclassical cannabinoid analog, cyclic AMP (CAMP)

production in SKR6-transfected cells decreases in a dose-dependent,

stereoselective, and pertussis toxin-sensitive manner (figure 1) (Matsuda et al.

1990). Moreover, the gene for the human homolog of this receptor encodes a

protein that binds 3H-CP 55940, a potent, synthetic cannabinoid analog, in a

saturable and specific manner (Felder et al. 1991).

In sections from adult rat brain, a regionally specific and unique localization

pattern for the cannabinoid receptor mRNA was found (Matsuda et al.,

submitted for publication). In general, labeling intensities were highest in

forebrain regions (olfactory areas, caudate nucleus, hippocampus) and in

FIGURE 1. Cannabinoid-induced inhibition of calcitonin gene-related peptide

(CGRP, 250 nM)-stimulated CAMP production. Intact Chinese

hamster ovary cells stably expressing the SKR6 CDNA were

exposed to various concentrations of CP 55940 or -THC for 5

minutes. Data points represent the average percent inhibition

of CAMP (±SEM) from three experiments, each performed in

triplicate. Chemical structures of CP 55940 and -THC are

shown. -THC was obtained from the National Institute on Drug

Abuse; CP 55940 was donated by the Pfizer Research Corp.

49
the cerebellar cortex (figure 2). Although the localization of cannabinoid

receptor mRNA was quite similar to that of the cannabinoid receptor protein

(Herkenham et al. 1991a), numerous discrepancies were evident. In most

cases, mismatches between cannabinoid receptors and receptor mRNA likely

resulted from the expression of receptor proteins in the axons and/or terminals

of projection neurons. Although receptor and mRNA discrepancies could

result from comparing ISHH labeling with binding data obtained with a ligand

that recognizes multiple cannabinoid receptor subtypes, the ligand used to

localize cannabinoid receptors (

3H-CP 55940) appears to recognize a single

cannabinoid receptor subtype (Devane et al. 1988; Herkenham et al. 1990).

Indeed, Herkenham and colleagues (1991b, 1991c) have reported experimental

evidence for the localization of cannabinoid receptors to projection target areas

of both striatal and cerebellar neurons.

In the midbrain and hindbrain, ISHH labeling generally was uniform (of similar

intensity) among all labeled cells within a given region or anatomical nucleus,

and intensities varied from moderate to very low for different regions (data not

shown). In the forebrain, the intensity of ISHH labeling varied from very low to

very high. Several forebrain areas displayed a nonuniform labeling pattern in

which heavily labeled cells were present with cells labeled to a more moderate

extent. This nonuniform labeling was clearly evident in the hippocampus, where

cells displaying very high intensity labeling were scattered throughout Ammon’s

horn and in a monolayer subjacent to the granular layer of the dentate gyrus;

moderately labeled cells included those in the pyramidal cell layer and those

in the hilar region of the dentate gyrus (figure 3). Other forebrain regions—

anterior olfactory nuclei (figure 2, panel a), nucleus of the lateral olfactory tract,

basolateral amygdaloid nucleus (data not shown), horizontal limb of the nucleus

of the diagonal band (figure 3, panel b), cerebral cortex (figure 2, panels b and

c)—also displayed cells that were labeled to a high intensity. Although the

physiological significance of high levels of mRNA likely will depend on the

density of cannabinoid receptors on these cells, the mechanism(s) involved

in this range of gene expression would be of interest.

Possible explanations for high amounts of receptor mRNA include an increased

rate of transcription of the receptor gene and/or a decreased rate of transcript

degradation. If transcription rate varies (degradation rates remaining constant),

high amounts of mRNA could result from different promoters and cell-specific

induction mechanisms. Although splicing variants in 5’-untranslated sequences

have been identified by comparisons of the human cannabinoid receptor

gene (T.I. Bonner, M.J. Brownstein, C.C. Felder, C. Chen, and L.A. Matsuda,

unpublished manuscript), the SKR6 cDNA, and a human cannabinoid receptor

cDNA (Gerard et al. 1990), more detailed information concerning the sites

that regulate cannabinoid receptor gene expression is not yet available. In the

50
FIGURE 2. ISHH labeling of cannabinoid receptor mRNA in adult rat brain. Panels a through dare negative film

images of coronal sections taken at various levels through the brain; labeling by the SKR6-1 probe

appears as white on a dark background. Abbreviations: Fr, frontal cortex; AO, anterior olfactory

nucleus; Cg, cingulate cortex; Sl, intermediate septal nucleus; C, caudate nucleus; MS, medial

septal complex; DB, nucleus of the diagonal band; Pir, piriform cortex; Hi, hippocampus; PrC,

precommissioned nucleus; Sth, subthalamic nucleus; Cb, cerebellum; Sol, nucleus of the solitary

tract; IO, inferior olive.
FIGURE 3. Expression of the cannabinoid receptor gene in areas associated

with memory. Images were produced in a manner similar to those

pictured in figure 2 but were taken at higher magnifications.

Panel a: hippocampus, CA1, pyramidal cell layer of field CA1 of

Ammon’s horn; CA3, pyramidal cell layer of field CA3 of Ammon’s

horn: DG, dentate gyrus (arrow features the hilar region of the

dentate gyrus). Panel b: medial septal complex, MS, medial

septum; VDB, vertical limb of the nucleus of the diagonal band;

HDB, horizontal limb of the nucleus of the diagonal band. Panel

c: mammillary body, LM, lateral nucleus.

52
situation where mRNA stablity may vary (constant rates of transcription), less

mRNA would accumulate in cells in which the cannabinoid receptor mRNA is

more readily degraded. Conclusive information regarding this possibility is also

not available; however, cDNA clones with different lengths of 3’-untranslated

sequence have been described and appear to be the result of two distinct

polyadenylation sites (Matsuda et al. 1990). This finding may be relevant

since motifs in sequences 3’ of the coding region appear to influence message

stability in many eukaryotic genes (Shaw and Kamen 1986; Brawerman 1987).

Clearly, additional research is needed to determine the mechanism(s) by which

certain subpopulations of cells express high levels of cannabinoid receptor

mRNA.

In animals, cannabimimetic activity is routinely assessed by drug discrimination,

specific motor responses (dog ataxia, rodent catalepsy) (Razdan 1986), or

an increase in pain thresholds (Johnson and Melvin 1986). High levels of

receptor mRNA in the caudate nucleus and cerebellum (figure 2) are

consistent with the marked effects that cannabinoids exert on the motor

behavior of laboratory animals. Potential sources for cannabinoid-induced

analgesia include numerous regions that displayed low-to-moderate amounts

of receptor mRNA, including the cingulate cortex (moderate), intralaminar

thalamic nuclei (moderate), central grey (low), nucleus raphe mangus (low),

pontine reticular formation (low), and paragigantocellular reticular nucleus

(moderate) (data not shown).

In humans, memory deficits are some of the most consistently reported

effects of marijuana (Miller and Branconnier 1983). Clear labeling observed

in the rat forebrain suggests several potential sites in the human brain that

could mediate this effect, including the hippocampus, medial septal complex,

lateral nucleus of the mammillary body (figure 3), and the amygdaloid complex

(data not shown)—regions that traditionally have been associated with various

aspects of memory functions. Similarly, labeling was detected clearly in rat

forebrain regions that correspond to those that could mediate marijuanainduced effects on human appetite and mood (hypothalamus, amygdaloid

complex, anterior cingulate cortex [data not shown]).

In contrast to the effects on mood and cognition, marijuana-induced

autonomic effects in humans are mild (Hollister 1986). However, in animals,

cannabimimetics decrease respiration, heart rate, and blood pressure

(Dewey 1986). These cardiovascular and respiratory responses are likely

due to the moderate levels of cannabinoid receptor mRNA that were found

in numerous midbrain and hindbrain regions (the pontine nucleus, the

paragigantocellular reticular nucleus, the lateral reticular nucleus, the area

postrema [data not shown], nucleus of the solitary tract [figure 2]). Although

53
the cannabinoid-induced effects on autonomic functions are rarely lethal in

humans, further study of the cells that express the cannabinoid receptor gene

in the midbrain and hindbrain may reveal an important physiological role(s)

for the cannabinoid receptor and its endogenous ligand.

SUMMARY

Localization of the mRNA for this receptor has identified many regions of the

rat brain in which the gene for this receptor is active. Several of these regions

are consistent with the cannabinoid- or marijuana-induced effects that occur

in both laboratory animals and humans. However, other labeled regions are

not easily associated with well-known effects of marijuana (Matsuda et al.,

submitted for publication). Although great progress has been achieved in

elucidating the mechanism of action of cannabis in recent years (Howlett et al.

1990), much remains to be discovered about the expression of cannabinoid

receptors in the brain and exactly how this receptor influences numerous brain

functions.

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29:307-313, 1986.

Johnson, M.R., and Melvin, L.S. The discovery of nonclassical cannabinoid

analgetics. In: Mechoulam, R., ed. Cannabinoids as Therapeutic Agents.

Boca Raton, FL: CRC Press, 1986. pp. 121-145.

Matsuda, L.A.; Bonner, T.I.; and Lolait, S.J. Localization of cannabinoid

receptor mRNA in rat brain. J Comp Neurol, submitted for publication.

Matsuda, L.A.; Lolait, S.J.; Brownstein, M.J.; Young, A.C.; and Bonner, T.I.

Structure of a cannabinoid receptor and functional expression of the cloned

cDNA. Nature 346:561-564, 1990.

Miller, L.L., and Branconnier, R.J. Cannabis effects on memory and the

cholinergic limbic system. Psychol Bull 93:441-456, 1983.

Razdan, R.K. Structure-activity relationships in cannabinoids. Pharmacol Rev

38:75-149, 1986.

Shaw, G., and Kamen, R. A conserved AU sequence from the 3’ untranslated

region of GM-CSF mRNA mediates selective mRNA degradation. Cell

46:659-667, 1986.

ACKNOWLEDGMENTS

The work described in this chapter was supported in part by grants provided

by the National Alliance for Research on Schizophrenia and Depression and

the Stanley Foundation, USA (SJL). M.J. Brownstein synthesized the

oligonucleotide probes. W.S. Young III, M. Herkenham, and C.R. Gerfen

provided technical guidance and also assisted in confirming identified brain

regions.

AUTHORS

Lisa A. Matsuda, Ph.D.

Assistant Professor

Department of Psychiatry and Behavioral Sciences

55
Medical University of South Carolina

171 Ashley Avenue

Charleston, SC 29425

Tom I. Bonner, Ph.D.

Research Biologist

Stephen J. Lolait, Ph.D.

Guest Researcher

Laboratory of Cell Biology

National Institute of Mental Health

National Institutes of Health

Building 36, Room 3A17

Bethesda, MD 20892

56
Studies on μ-Opioid-Binding Sites With

Peptide Antibodies

Eric J. Simon, Theresa L. Gioannini, Yi-He Yao, and Jacob M.

Hiller

PURIFICATION OF A μ-OPIOID-BINDING PROTEIN AND ITS

CHARACTERIZATION

The purification to homogeneity of a μ-opioid-binding protein (OBP) from

bovine striatal membranes and its characterization by its ability to bind

opioid antagonists saturably, reversibly, and with high affinity was reported

previously by Gioannini and colleagues (1985). Briefly, the purification

involved two major steps: (1) affinity chromatography on a then-novel

derivative of naltrexone, 6-desoxy-aminoethylaminonaltrexone, coupled to the

carboxyl side chain of CH-Sepharose, followed by (2) lectin chromatography

on wheat germ agglutinin agarose. A single band of apparent molecular weight

(mol wt) of 65 kD was obtained on SDS-polyacrylamide gel electrophoresis

(SDS-PAGE). The purification achieved was 60,000- to 70,000-fold, close to

the theoretical value for a protein of this size with a single binding site. The

purified OBP binds opioid antagonists with high affinity, but it binds against

agonists with low affinity, presumably because it is uncoupled from the Gi

protein to which it is normally coupled. The evidence that OBP is a μ-binding

opioid protein is as follows:

1. OBP is isolated on an affinity matrix that contains a ligand that binds

μ-receptors preferentially.

2. Binding of opioid antagonists is displaced by the μ-selective ligand D

Ala2Gly-I5 enkephalin (DAGO), albeit at high concentrations, but not by

selective -ligands.

3. OBP binds the μ-specific antagonist 3H-cyprodime (Schmidhammer et

al. 1989). At saturating concentrations of ligand, OBP binds as much

cyprodime as bremazocine does, suggesting that all the binding to OBP

of bremazocine, a universal ligand, is to μ-sites.

57
4. Finally, the molecular weight of OBP (65 kD) is the same as that observed

when 125l-ß-endorphin is cross-linked to opioid-binding sites in tissues and

a cell line, which were shown to be μ-sites by specific displacement with

μ-ligands (Howard et al. 1985, 1986).

The presence of disulfide bridges (-S-S-) that contribute to the secondary

structure of OBP is indicated by the difference in mobility of OBP in SDS-PAGE

under nonreducing vs. reducing conditions (Gioannini et al. 1985). As shown

in figure 1, treatment of OBP with increasing concentrations of reducing

reagent dithiothreitol (DTT) produced a stepwise shift from an apparent

molecular weight of 53 to 65 kD (Gioannini et al. 1989). The importance

of disulfide bonds, not only for secondary structure but also for function, is

reflected in the sensitivity of opioid ligand binding to inhibition by DTT. The

major opioid receptor types differed in their sensitivity to DTT as follows:

μ> with -sites being virtually resistant to even very high concentrations

of DTT (Gioannini et. al. 1989). The inhibition produced by DTT is reversible,

was observed to a much lesser degree with antagonist ligands than with

agonists, and was due to a reduction in affinity (increase in kD) rather than in

the number of receptor sites (Bmax). The possibility that these disulfide bridges

may have a role in receptor activation by agonist ligands is being explored.

MICROSEQUENCING OF PURIFIED OBP AND GENERATION OF

ANTISERA AGAINST PEPTIDE SEQUENCES

The availability of pure OBP encouraged the authors to attempt to determine

a portion of its primary sequence. Direct sequencing of OBP proved

unsuccessful, indicating that OBP is an N-terminally blocked protein and

must be fragmented to obtain peptides for amino acid sequencing, which

greatly increases the quantities of purified protein required. Two peptide

fragments, 21 and 13 amino acids in length, respectively, were generated by

chemical cleavage of OBP with CNBr followed by isolation of the peptides on

reverse phase high-performance liquid chromatography. The amino acid

sequences obtained were not found in databases of known protein sequences.

Polyclonal antibodies have been generated against portions of the peptides by

injection of rabbits with the synthetic peptides coupled to thyroglobulin, followed

by appropriate booster injections. The interaction of these antibodies with

purified OBP, bovine brain regions, and several cell lines are discussed below.

INTERACTION OF PEPTIDE ANTIBODIES WITH PURIFIED OBP

The peptide fragments used for antibody production and the antibodies

generated against each are indicated in table 1.

58
FIGURE 1. The effect of increasing DTT concentrations on the apparent

molecular weight of purified μ-OBP. Purified μ-OBP was

radioiodinated and separated from free iodine. Samples were

treated with the indicated concentrations of DTT and subjected

to SDS- PAGE.

SOURCE: Gioannini et al. 1989. Copyright 1989 by John Wiley & Sons, Ltd.

Reprinted by permission of John Wiley & Sons, Ltd.

All the antibodies were able to immunoprecipitate a major portion of 125llabeled purified OBP after incubation with the antibody followed by incubation

with protein A. The amount precipitated after correction for background

(radioactivity precipitated by normal rabbit serum [NRS] or irrelevant immune

sera) ranged from 40 percent for the weakest antibody (Ab 161) to more than

60 percent for the strongest, Ab 162 and Ab 165, at dilutions of 1:200.

Background of the assay accounted for 5 to 7 percent of added radioactivity.

Ab 162 and Ab 165 can immunoprecipitate 30 percent of 125l-OBP even at a

1:1,000 dilution. The protein precipitated by these antibodies was dissociated

from the complex and subjected to SDS-PAGE. Autoradiography of the gel,

59
TABLE 1. Antibodies generated against peptide sequences derived from

purified OBP

Antibody Fragment

165, 166, 6639 N-terminal 12 amino acids of peptide 1

163 C-terminal 7 amino acids of peptide 1

161, 162 N-terminal 10 amino acids of peptide 2

carried out under reducing conditions, showed a radioactive band with an

apparent molecular weight of 65 kD (i.e., the molecular weight of OBP).

Sequential treatment of OBP with antibodies, derived from the two different

peptides, indicated that initial treatment with either antibody removed all

immunoprecipitable antigen (i.e., no further immunoprecipitation occurred with

the second antibody). These results confirm that the two peptides isolated and

sequenced from fragmented OBP are derived from the same protein.

The interaction of OBP with the antipeptide antibodies also was examined in

immunoblots. An immunoreactive protein corresponding to a 65 kD mol wt

was detected with each antibody examined. The strongest signal was

produced with antibodies against the N-terminal portion of peptide 1 (Ab 165,

166, 6639) at a 1:100 dilution. The 65-kD band obtained in immunoblots with

OBP can be blocked by preincubation of the antisera with the appropriate

peptide (100 μM), but not by other peptides. No signal was detected when

OBP was immunoblotted against nonimmune serum. It is noteworthy that no

signal was detected unless OBP was reduced with DTT. Apparently, the

presence of the disulfide bridges and the resulting secondary structure of the

protein prevent access of the antibodies to the epitopes.

IMMUNOBLOTS OF TISSUES AND CELL LINES WITH ANTISERA AGAINST

OBP PEPTIDES

The authors decided to determine whether the peptide antibodies were capable

of producing positive immunoblots with extracts of tissues containing high

concentrations of μ-opioid receptors. The first tissue examined was an extract

of bovine striatal membranes, the source of purified OBP. lmmunoblotting with

peptide antisera gave a band at the appropriate molecular weight of 65 kD.

The positive signal at 65 kD mol wt seen in immunoblots with both digitonin

and CHAPS extracts of bovine striatal membranes prompted us to examine

cell lines and other bovine brain tissues for positive signals in immunoblots

60
with the antipeptide antibodies. lmmunoblots of SDS-solubilized bovine tissues

with the various antibodies indicated the presence of immunoreactive protein

(65 kD mol wt). The signal could be blocked by preincubation of antisera with

the appropriate peptide (50 to 100 μM). The tissues that reacted with antibody,

in addition to striatum, were frontal cortex, hippocampus, and thalamus, all

regions known to have moderate-to-high levels of μ-opioid receptors. Pons

and white matter produced no or a barely detectable response, which correlates

with their low levels of opioid receptors. The sensitivity of Ab 165, which

produces the strongest signal, is evidenced by its ability at a dilution of 1:100

to detect μ-opioid-binding material equivalent to 0.001 percent of the 30 to 50

μg of protein loaded per sample (300 to 500 pg of OBP) in an immunoblot.

lmmunoblots with the cell line SK-N-SH, which contains predominantly

μ-binding sites, produced a strong positive reaction at a position comparable

to that seen with purified OBP and brain tissue extracts (65 kD). The

immunoreactive protein detected in NG1O8-15 cells, a cell line that is reported

to contain only -receptors, migrated to a position slightly lower than that seen

in the SK-N-SH cells or with purified OBP (apparent molecular weight of

approximately 58 kD). In both cell lines, the response could be blocked by

preincubation of the serum with 100 μM concentration of peptide. Two

negative control cell lines, HELA cells and C6 glioma cells, produced no

detectable response. The detection of a response with the NG108-15 cells

suggests cross-reactivity of the antibodies with -receptors, although the

presence of small amounts of μ-receptors, hitherto not detected by binding

assays, could not be ruled out.

The ability of the antipeptide antibodies to react with native receptors was

investigated by examining the effect on opioid ligand binding and by evaluating

the extent to which active receptors can be removed from solution by

immunoprecipitation. None of the antibodies inhibited binding of opioid ligands

to either membrane-bound or soluble receptors. No depletion of receptors

was detected in the supernatants after immunoprecipitation with any of the

antibodies. It was concluded that the antibodies recognize only denatured

receptors. Not unexpectedly, the short amino acid sequences to which the

antibodies were made may not be accessible to the antibody in the native

receptor or may assume a secondary structure not recognized by the antibody.

All the above results, with antisera generated against microsequence from the

purified OBP, support the authors’ contention that we have purified an opioidbinding site—in particular, the immunoblots of tissues and cell lines that give

bands of the appropriate molecular weight. Moreover, there is good correlation

between positive signals and levels of opioid receptors in the tissues or

cultures.

61
CROSS-REACTIVITY OF PURIFIED OBP WITH ANTISERA GENERATED

AGAINST BOVINE RHODOPSIN

Biochemical and physiological evidence indicates that all three major types of

opioid receptors, μ, negatively modulate adenylate cyclase and,

therefore, are coupled to guanine nucleotide regulatory proteins (G proteins).

This suggests that opioid receptors belong to the large family of receptors for

hormones, neurotransmitters, and peptides that effect signal transmission by

activating a G protein. Analysis of the amino acid sequences of many proteins

of this class has revealed some significant structural features common to all

members of this family, the most striking of which is the presence of seven

hydrophobic domains thought to span the cell membrane. The following results

further support the hypothesis that OBP belongs to this class of G-coupled

proteins.

Antibodies generated against membrane-associated rhodopsin and against five

specific amino acid sequences in rhodopsin were used. In immunoblots against

OBP, two antibodies, one against membrane-associated rhodopsin and one

against a sequence in the carboxyl terminal tail (CT1), reacted strongly,

whereas an antibody against the 1-2 loop (first cytoplasmic loop between

transmembrane domains 1 and 2) reacted weakly. Weiss and colleagues

(1987) had previously studied the interaction of purified ß-adrenergic receptor

from S49 lymphoma cells with this same series of antibodies under identical

reaction conditions. The pattern of reactivity of these antibodies with OBP

was the same as that previously reported by Weiss and coworkers (1987)

for purified ß-adrenergic receptor. These researchers had shown that

preincubation of antiserum CT1, an antiserum against a sequence in the

C-terminal tail of rhodopsin, with the peptide (rhodopsin 325-343) used to

generate it, was able to diminish greatly the signal obtained in immunoblots of

the ß-adrenergic receptor. This supports the specificity of the immune crossreaction. A rhodopsin peptide (324-348), which contains the sequence (325-

343) used to generate the CT1 antiserum, was made available to the authors’

laboratory. This peptide, when preincubated with antiserum CT1 at 100 μM

concentration, completely abolished the positive signal in immunoblots with

OBP. A rhodopsin peptide (rhodopsin sequence 331-348), which lacked the

first six amino acids of the CT1 sequence, was ineffective in reducing the

intensity of the 65-kD signal.

To verify that rhodopsin antibodies recognize the same protein as the peptide

antibodies, OBP was immunoprecipitated by Ab 165. An immunoblot of the

proteins remaining in the supernatant after immunoprecipitation showed a

strong diminution of the signal (65-kD band) obtained with the rhodopsin

antibody CT1, relative to a control supernatant from “immunoprecipitation” with

62
NRS. The protein immunoprecipitated with Ab 165 was eluted from the antigenantibody complex and examined in an immunoblot against CT1 . A positive

signal was observed at 65 kD mol wt, whereas no signal was detected with the

NRS control. This experiment indicates that the protein recognized by the

rhodopsin antibodies is the same as that recognized by OBP-derived peptide

antibodies.

The results reported here, in conjunction with those of Weiss and colleagues

(1987), indicate that the three proteins—bovine rhodopsin, S49 lymphoma

ß-adrenergic receptor, and OBP purified to homogeneity from bovine striatal

membranes—share common epitopes. Since there seems to be relatively little

amino acid sequence homology in the areas used for antibody production (at

least between rhodopsin and the ß-adrenergic receptor), structural features,

perhaps at the level of secondary and/or tertiary structure, along with limited

amino acid homology, may be responsible for the immunological crossreactivity. Weiss and coworkers (1987) have reported evidence that three

amino acids, K, N, and P in positions 325-327, may be important for recognition

of antibody CT1 by rhodopsin and by ß-adrenergic receptor. Current data

suggest that this also may be true for OBP.

The results presented constitute evidence, beyond that previously obtained,

that μ-opioid-binding sites are members of the family of G protein-coupled

receptors and are likely to show the typical seven membrane-spanning domain

structure of these proteins, when their complete amino acid sequence becomes

known. It should be noted that similar results have been obtained by W.A. Klee

and coworkers (personal communication, October 1991). These researchers

found that -receptors labeled with the affinity label 3H-FIT and purified from

NG108-15 cells in culture were immunoprecipitated by an antiserum generated

against a peptide sequence from the C-terminal tail of rhodopsin. This finding

lends credence to ours and suggests that μ- and -opioid receptors both share

antigenic sites with rhodopsin.

UPDATE ON ATTEMPTS TO CLONE THE μ-OBP

Oligonucleotides have been prepared based on the amino acid sequence of the

isolated peptides. Bovine brain and striatal cDNA libraries have been screened

with the labeled oligonucleotide probes, and clones have been obtained. Partial

sequencing revealed that the clones did not contain the full probe used to

detect them; that is, they were false-positive clones. This is undoubtedly due to

the considerable degeneracy of the authors’ sequences, which contain many

amino acids coded by four or six codons. Screening is currently in progress

using different oligonucleotides. Clones are being sequenced to see if they

contain the sequence present in the probe. If so, sequencing of the complete

63
cDNA will be carried out. The resulting structure should give an idea whether it

is the opioid receptor, although proof must await transfection of suitable cells

and expression of an active binding site with the appropriate ligand specificity.

REFERENCES

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.

Gioannini, T.L.; Liu, Y.-F.; Park, Y.-H.; Hiller, J.M.; and Simon, E.J. Evidence

for the presence of disulfide bridges in opioid receptors essential for ligand

binding. Possible role in receptor activation. J Mol Recognition 2(1):44-48,

1989.

Howard, A.D.; de la Baume, S.; Gioannini, T.L.; Hiller, J.M.; and Simon, E.J.

Covalent labeling of opioid receptors with radio-iodinated human betaendorphin. J Biol Chem 260:10833-10839, 1985.

Howard, A.D.; Gioannini, T.L.; Hiller, J.M.; and Simon, E.J. Identification of

distinct binding site subunits of mu and delta opiate receptors. Biochemistry

25:357-360, 1986.

Schmidhammer, H.; Burkard, W.P.; Eggstein-Aeppli, L.; and Smith, C.F.C.

Synthesis and biological evaluation of 14-Alkoxymorphinans. (-)-NCyclopropylmethyl-414-dimethoxy-morphinan-6-one, a selective μ-opioid

receptor antagonist. J Med Chem 32:418-421, 1989.

Weiss, E.R.; Hadcock, J.H.; Johnson, G.L.; and Malbon, C.C. Antipeptide

antibodies directed against cytoplasmic rhodopsin sequences recognize the

beta-adrenergic receptor. J Biol Chem 262:4319-4323, 1987.

ACKNOWLEDGMENTS

The work described in this chapter was supported by National Institute on Drug

Abuse grant DA-00017 (EJS).

Dr. Gary Johnson (National Jewish Center for Immunology and Respiratory

Medicine, Denver, CO) and Dr. Ellen Weiss (University of North Carolina,

Chapel Hill, NC) supplied the rhodopsin antisera. Dr. Anatol Arendt and Dr.

Paul Hargrave (University of Florida) supplied the rhodopsin peptides for the

blocking experiments. Dr. Catherine Strader (Merck Sharp and Dohme,

Rahway, NJ) isolated the peptides from purified OBP. Dr. Lawrence Taylor, Dr.

Huda Akil, and Dr. Stanley Watson (University of Michigan Medical School)

generated antisera against the peptides.

64
AUTHORS

Eric J. Simon, Ph.D.

Professor

Departments of Psychiatry and Pharmacology

Yi-He Yao, Ph.D.

Assistant Research Scientist

Department of Psychiatry

Jacob M. Hiller, Ph.D.

Research Associate Professor

Department of Psychiatry

New York University Medical Center

550 First Avenue

New York, NY 10016

Theresa L. Gioannini, Ph.D.

Research Assistant Professor

Department of Psychiatry

New York University Medical Center

Associate Professor

Department of Natural Sciences

Baruch College

City University of New York

New York. NY 10010

65
Molecular Cloning and Characterization

of Neurotransmitter Transporters

Randy D. Blakely

INTRODUCTION

For precise chemical signaling between neurons and target cells, the

magnitude and duration of neurotransmitters in synaptic spaces must be

tightly regulated. Two principal and distinct mechanisms are classically

recognized as being responsible for rapid transmitter inactivation. Transmitter

can be either enzymatically metabolized, as with acetylcholinesterase hydrolysis

of acetylcholine (Taylor 1991), or actively accumulated into presynaptic terminals

and/or surrounding glia (Iversen 1967; Schousboe 1981; Hendley 1984; Cooper

et al. 1986; Horn 1990; Nicholls and Atwell 1990). First demonstrated in the

periphery by Axelrod and colleagues while studying the fate of intravenous

radiolabeled catecholamine (Axelrod 1971), rapid transport across neuronal

and glial membranes has been extensively characterized for many central

neurotransmitters. In addition to norepinephrine (NE), the neurotransmitters

dopamine (DA), serotonin (5-HT), L-glutamic acid (Glu), -aminobutyric acid

(GABA), and glycine (Gly) each can be actively transported across brain

membranes in a region-dependent manner consistent with the localization

of neuronal terminals releasing these compounds. Choline availability is the

rate-limiting process in acetylcholine biosynthesis. Therefore, it is no surprise

that even cholinergic neurons utilize an active transport process to actively

recapture synaptic choline following acetylcholine hydrolysis (Kuhar and Murrin

1978). The physiologic importance of transmitter inactivation by reuptake

carriers is particularly apparent when examining the clinical and societal impact

of agents with a high selectivity for blocking monoamine transport, including

cocaine, amphetamines, and many antidepressants (Ritz et al. 1987; Galloway

1988; Richelson 1990). Furthermore, the ability of synaptic transporters to

accumulate neurotransmitter-like toxins, including 6-hydroxydopamine, 1-methyl4-phenylpyridinium (MPP+), 5,6-dihydroxytryptamine, and AF64A, among

others, suggests that these proteins have a role in the establishment of

selective neuronal vulnerability to exogenous agents. Thus, neurotransmitter

transporters represent critical targets for both therapeutic and pathologic

alterations of synaptic function.

66
Active transport of neurotransmitters (and choline) is driven by the

transmembrane Na+ gradient (Kanner and Schuldiner 1987), established

by Na+/K+ adenosinetriphosphatase (ATPase), in a process analogous to

the active transport of glucose across brush border epithelia (Stein 1986).

However, unlike intestinal glucose transport, additional ions are required for

transport of many neurotransmitters, such as intracellular K+ and extracelluar

Cl-. Membrane vesicle (Kanner and Schuldiner 1987) and whole cell patch

clamp (Nicholls and Atwell 1990) studies indicate that the effects of these ions

on transport are unrelated to their effects on transmembrane potential (Vm),

but rather indicate that there is parallel movement of these ions during each

translocation cycle. These energetic properties, along with clear pharmacologic

differences, distinguish these transporters from the intracellular vesicular

carriers used to package neurotransmitters for exocytosis (Cooper et al. 1986;

Kanner and Schuldiner 1987). Specific radiolabeled antagonists, including

[

3H]nisoxetine for the NE transporter (Tejani-Butt et al. 1990), [3H]GBR12909

and [

3H]GBR12935 for the DA transporter (Anderson 1989), [3H]citalopram

for the 5-HT transporter (D’Amato et al. 1987), and [

3H]hemicholinium-3 for

the choline transporter (Sandberg and Coyle 1985), have been used to label

transporters, both in broken cell preparations and in brain sections, permitting

both pharmacologic and anatomic analyses. These tools have confirmed the

enrichment of transporter sites on presynaptic terminal membranes and, at

least for the choline transporter (Saltarelli et al. 1987), have revealed alterations

in carrier density on plasma membranes in response to depolarization.

Despite decades of kinetic and pharmacologic studies on the behavior of

neurotransmitter transporters in membrane preparations, structural data

relevant to questions of mechanism and regulation have been difficult to obtain.

One initial assumption is that all transporters utilizing cotransported Na+ ions,

from Escherichia coli (E. coli) to man, are derived from a common ancestral

gene, with sequence identity illustrative of common tasks such as Na+ binding

or translocation, and sequence divergence responsible for additional ionic and

pharmacologic specificities. Homologous, facilitated glucose transporters

(which lack the ability to move sugar uphill against a concentration gradient)

are found throughout phylogeny as members of an extended gene family

(Henderson 1990). The intestinal Na+/glucose (Hediger et al. 1987, 1989)

and the E. coli Na+/proline (Nakao et al. 1987) transporters are encoded by

single genes and bear sequence similarities absent from the family of facilitated

carriers, revealing the presence of a distinct gene family encoding Na+/

symporter proteins. One possibility was that neurotransmitter transporters

would bear detectable sequence conservation with this latter gene family.

However, attempts to use the limited homology of the Na+/glucose and Na+/

proline transporters to identify novel carriers expressed in the rodent central

nervous system met with no success (R.D. Blakely, unpublished data),

suggesting a novel gene family might encode neurotransmitter transporters.

67
EXPRESSION OF NEUROTRANSMlTER TRANSPORTERS IN XENOPUS

LAEVIS OOCYTES

In the absence of sequence information suitable for molecular cloning of

transporter cDNAs by conventional hybridization strategies, Blakely and

colleagues (1988, 1991a) turned to the Xenopus laevis oocyte expression

system to characterize and perhaps clone transporter mRNAs. Following

the injection of poly(A)+RNA, Xenopus laevis oocytes actively accumulated

many different transmitter substrates, including Glu, GABA, Gly, 5-HT, DA,

and choline (Blakely et al. 1988). Transport of these substrates was timeand temperature-dependent, and uptake was abolished with the removal of

extracellular Na+. Regional enrichment of transporter mRNAs followed well

the distribution of soma predicted to synthesize each carrier. In addition,

pharmacologic sensitivities of oocyte-expressed transporters were similar

(Blakely et al. 1991a) if not identical to those observed in brain membrane

preparations (table 1). Using mRNA prepared from different brain regions

during development, Blakely and colleagues (1991a) revealed a postnatal

rise in Glu, GABA, and Gly transporter transcript abundance paralleling the

postnatal rise in brain transport activity for these substrates. Most important

in the consideration of expression cloning strategies was that mRNAs encoding

Glu, GABA, and Gly carriers could be size-fractionated on sucrose density

gradients, with minimal loss of activity relative to unfractionated mRNA and

with a narrow distribution of peak activities (figure 1, example of Glu transporter

size fractionation). These data supported the assumption that neurotransmitter

transporters were likely to be encoded by single, separable mRNAs and,

therefore, could be cloned utilizing expression as a functional screen for the

presence of a single transporter clone in a cDNA library.

MOLECULAR CLONING AND EXPRESSION OF A COCAINE- AND

ANTIDEPRESSANT-SENSITIVE NOREPINEPHRINE TRANSPORTER

DERIVED FROM A HUMAN NEUROBLASTOMA (SK-N-SH) CELL LINE

Despite the demonstrated ability of the Xenopus laevis oocyte expression

system to reconstitute neurotransmitter transporters, there were several

drawbacks precluding its use for the cDNA cloning of these carriers. Seasonal

variability in oocyte viability and expression levels, coupled with an inability

to produce a high “signal-to-noise” ratio for monoamine transporters (Blakely

et al. 1988), dictated the design and implementation of an alternative strategy

(figure 2). This method, based on the ability of COS cells to replicate episomal

copies of transfected plasmids bearing SV40 replication origins (Gluzman

1981), has been used in various forms to clone cDNAs for several membrane

proteins, including several peptide receptors (Aruffo and Seed 1987; Sims et

al. 1988; D’Andrea et al. 1989; Munro and Maniatis 1989). Pacholczyk and

68
TABLE 1. Inhibitor sensitivity of oocyte uptake activities induced by adult rat

Substrate

L-Glu

mRNAs

RNA Source

Forebrain

Cerebellum

GABA

Gly

Choline

DA

Inhibitor Concentration Percent Inhibition

Dihydrokainate 1 mM 61.0 ± 6.9

Dihydrokainate 100 mM 16.0 ± 5.3

D-aspartate 100 μM 91.7 ± 15.5

Nipecotic acid 100 μM 10.0 ± 1.1

Spinal cord Dihydrokainate 1 mM 63.0 ± 8.1

Cerebellum D-aspartate 100 μM 8.2 ± 1.4

Nipecotic acid 100 μM 81.2 ± 9.0

Spinal cord Nipecotic acid 100 μM 75.7 ± 10.1

Spinal cord Nipecotic acid 100 μM 0.3 ± 0.1

Spinal cord Hemicholinium-3 10 μM 82.2 ± 4.0

Midbrain Nomifensine 10 μM 98.3 ± 12.3

Adrenal Nomifensine 10 μM 79.4 ± 6.8

NOTE: Xenopus laevis oocytes were injected with 40 ng poly(A+) RNA and

incubated at 18 °C for 2 to 3 days prior to assay. Uptake assays (22 °C)

with different [3H]-labeled substrates were performed as described in

Blakely and colleagues (1991a) with or without inhibitors at the

concentrations indicated. Mean transport inhibition (±SEM) relative to

assays conducted with substrates alone is presented.

SOURCE: Blakely, R.D.; Clark, J.A.; Pacholczyk, T.; and Amara, S.G. Distinct,

developmentally regulated brain mRNAs direct the synthesis of

neurotransmitter transporters. J Neurochem 56:860-871, 1991a.

Copyright 1991 by Raven Press, Ltd. (New York).

colleagues (1991) reasoned that the uptake of an iodinated analog of NE,

meta-iodobenzylguanidine (mlBG), could be visualized in cells successfully

transfected with the NE transporter (NET). Prior to embarking on this

method, they characterized the transport of mlBG in SK-N-SH cells, an

adrenergic neuroblastoma cell line possessing a high-affinity NE uptake

system (Richards and Sadee 1986), and found the substrate to be

69
FIGURE 1. Size-fractionation of Glu transporter mRNA

SOURCE: Blakely, R.D.; Clark, J.A.; Pacholczyk, T.; and Amara, S.G.

Distinct, developmentally regulated brain mRNAs direct the

synthesis of neurotransmitter transporters. J Neurochem 56:860-

871, 1991a. Copyright 1991 by Raven Press, Ltd. (New York).

accumulated in a desipramine-sensitive manner as reported (Smets et al.

1989) and imageable autoradiographically. Because the technique could

be performed on large tissue-culture pans, Pacholczyk and colleagues (1991)

70
FIGURE 2. COS cell expression system for cDNA cloning of Na+/L-NET. AV

MLP-adenovirus major late promoter.

were able to screen large pools of cDNA library plasmids derived from SK-N-SH

mRNA. Plasmid DNA derived from scraped cells overlying “hot spots” on the

film was extracted by the Hirt lysis procedure, and the method was repeated

several times on a smaller scale. Finally, a single plasmid was isolated, which

was capable of producing a functional mlBG transporter in transfected

COS cells, with transport activity abolished when autoradiographic uptake

71
experiments were conducted in the presence of the NE transport-specific

inhibitor desipramine (T. Pacholczyk, unpublished data).

To demonstrate that the single cloned cDNA directed the synthesis of a

transporter with the expected ionic and pharmacologic sensitivities of the native

human NET (hNET), Pacholczyk and colleagues (1991) cloned the cDNA

insert downstream of a T7 RNA polymerase promoter in pBluescript SKII(-)

and transfected the construct into transformed human fibroblasts (HeLa)

infected with a vaccinia virus encoding T7 RNA polymerase (Fuerst et al.

1986). Blakely and coworkers (1991b) had demonstrated that this expression

method gave faithful, rapid, and high-level expression of cloned Na+/glucose

and Na+/GABA transporters, thus providing a convenient transient expression

method for analysis of the putative hNET. Assays performed on HeLa cells

12 hours after transfection revealed the generation of a high level of Na+-

dependent NE transport activity; analysis of substrate-dependence revealed

the induction of a single, saturable activity with an NE Kt of 457 nM (figure 3).

Assays conducted with a wide range of transporter and receptor antagonists

revealed the NE uptake (1) to be markedly sensitive to selective NE transport

inhibitors as compared with DA or 5-HT transport inhibitors, (2) to be potently

antagonized by both cocaine and D-amphetamine, and (3) to be insensitive to

inhibitors of either amino acid and sugar transport or to adrenergic and

adrenergic receptor antagonists (figures 4a and 4b, table 2). As expected,

reserpine, an inhibitor of the vesicular NE transport inhibitor (Kanner and

Schuldiner 1987), also failed to block NE uptake in transfected cells, ruling out

any possibility for ectopic expression of vesicular carrier. Thus, all the readily

testable pharmacological features of Uptake 1 (Iversen 1967) appear to be

encoded by this single cDNA. Given that multiple RNAs are not required to

synthesize a functional carrier, these findings also suggest that the membraneimbedded transporter may be monomeric in composition, as has been argued

for the transporter Lac permease (Costello et al. 1987), or may exist as a

homomultimer, as envisaged with the Na+/glucose transporter (Stevens et al.

1990).

Sequence analysis of the isolated NET cDNA reveals a large open reading

frame encoding a highly hydrophobic 617-amino acid peptide with a predicted

molecular weight of ~69 kD. Hydrophobicity analysis (Kyte and Doolittle 1982)

reveals ~12 regions capable of forming transmembrane domains. Although this

motif of 11 to 13 transmembrane domains superficially resembles the pattern

observed with both the Na+/glucose and E. coli Na+/proline symporters,

sequence analysis fails to reveal significant conservation in primary sequence.

Rather, shared sequences evident with the cloned rat and human Na+/GABA

transporters (GAT1) (Guastella et al. 1990; Nelson et al. 1990) reveal the

presence of a new transporter gene family, united by the function of their

72
FIGURE 3. Transport of L-NE in transfected HeLa cells

SOURCE: Pacholczyk et al. 1991. Reprinted by permission from Nature

350:350-354. Copyright © 1991 Macmillan Magazines Ltd.

(London).

substrates as neurotransmitters. Alignment of the predicted amino acids for

GABA and NE carriers reveals 46-percent identity, which rises to 68 percent,

allowing for conservative substitutions. Sequences predicted to lie within or

adjacent to predicted transmembrane regions contain many of the conserved

residues. One striking stretch, lying between putative transmembrane domains

1 and 2, possesses 19 out of 20 residues absolutely conserved. A comparison

of their virtually superimposable hydrophobicity profiles gives a clear indication

that the GABA and NE transporters are likely to assume similar secondary

structures in the plasma membrane (figure 5). As with GAT1, a large

hydrophilic loop bearing three consensus sites for N-linked glycosylation is

observed between transmembrane domains 3 and 4. When paired with the

absence of a detectable signal sequence for membrane insertion, this feature

suggests an initial model with both NH2- and COOH-termini in the cytoplasm,

as depicted in figure 6.

73
FIGURE 4. Inhibitor sensitivity of transfected Na+/L-NET

SOURCE: Pacholczyk et al. 1991. Reprinted by permission from Nature

350:350-354. Copyright © 1991 Macmillan Magazines Ltd.

(London).

74
TABLE 2. Inhibitor sensitivity of L-NE uptake in pNET-transfected HeLa cells

Inhibitor KI

 (nM) Hill (nH)

Mazindol 1.36

Desipramine 3.88

Nomifensine 7.68

Nortriptyline 16.5

D-amphetamine 56.1

lmipramine 65.4

Amitriptyline 100

GBR 12909 133

DA 139

Cocaine 140

Paroxetine 312

Benztropine 822

Citalopram >1,000

Propranolol 10,000

5-HT >10,000

Yohimbine >10,000

Prazosin >10,000

Hemicholinium-3 >10,000

Nipecotic acid >10,000

Phloridzen >10,000

Reserpine >10,000

0.98

1.22

1.17

0.95

1.00

1.08

0.75

1.06

0.86

1.25

0.75

0.82

—

—

—

—

—

—

—

—

—

NOTE: HeLa cells (200,000 to 300,000/well) infected with a T7 RNA

polymerase-containing vaccinia virus were transfected with pNE

(100 ng) and incubated with 20 nM [2,5,6,3H]-L-NE (New England

Nuclear) ±inhibitors for 15 minutes at 37 °C. KI values and Hill

coefficients reflect mean estimates from triplicate determinations

of complete uptake inhibition curves, adjusting for substrate

concentration after Cheng and Prusoff (1973).

SOURCE: Adapted from Pacholczyk et al. 1991. Reprinted by permission

from Nature 350:350-354. Copyright © 1991 Macmillan Magazines

Ltd.

High-affinity, antidepressant-sensitive NE uptake activity is enriched in

projections of brain stem noradrenergic neurons, particularly from the locus

coeruleus, as well as in terminals of noradrenergic sympathetic neurons and

in the neural crest-derived adrenal medulla. Northern hybridizations with

labeled NET reveal a 5.8-kb RNA localized to rat brain stem, rat adrenal gland,

75
FlGURE 5. Hydrophobicity comparison of GABA and NET proteins encoded

by cloned GAT1 and hNET CDNAS. The transformation is as

described by Kyte and Doolittle (1982) with a window of 19

residues. Hydrophobic values are positive in this display with

shaded regions indicating the 12 putative transmembrane

regions.

rat PC-12 cells, and the human SK-N-SH cells (Pacholczyk et al. 1991). A

more widespread 3.6-kb band is also observed in brain regions and cell lines

and may represent a splicing variant of NET or a related and cross-hybridizing

transcript. Because desipramine-sensitive NE uptake has been reported in

primary cultures of neonatal rat astrocytes, this latter species may also

represent a more broadly distributed glial carrier. Also, although both PC-12

and SK-N-SH cells exhibit many noradrenergic traits, their phenotypes can

be quite heterogeneous depending on culture conditions, and thus, multiple

transcripts may also reflect the mixed character of these cells.

In summary, a single 1,983-bp cDNA clone encoding a 617-amino acid

protein is sufficient to transfer upon nonneuronal cells the ability to accumulate

NE with high affinity, in a Na+-dependent manner, and with marked sensitivity

76
FIGURE 6. Hydrophobicity-based structural model of the human Na+

/L-NET

SOURCE: Adapted from Pacholczyk et al. 1991. Reprinted by permission

from Nature 350:350-354. Copyright © 1991 Macmillan Magazines

Ltd.

to specific antidepressants, cocaine, and amphetamine. As these latter agents

have profound effects on human behavior, exhibiting both therapeutic and

abuse potential, identification of a clone for this transporter may provide a

direct route to the rational design of novel therapies. In addition, the availability

of a cDNA encoding the NET should allow for an examination of the degree to

which structural alterations in the NE carrier gene underlie hereditary patterns

of major affective disorders, hypertension, and drug abuse.

EVALUATION OF DIVERSITY WITHIN THE NEUROTRANSMITTER

TRANSPORTER GENE FAMILY

The presence of conserved amino acid sequences between GAT1 and NET

suggests that other neurotransmitter transporters, such as those for DA, 5-HT,

Glu, Gly, and choline, are likely to be members of this family. Indeed, given the

77
major pharmacologic differences between the transporters encoded by GAT1

and NET, it is likely that the observed 46-percent identity between these two

proteins represents the lower limit of sequence similarity to be observed as

new members are uncovered. This would seem true for the monoamine

transporters, where considerable antagonist overlap exists among NE, DA,

and 5-HT uptake systems (Hendley 1984; Cooper et al. 1986; Richelson 1990).

One conventional approach to the identification of related gene products is the

screening of cDNA libraries by hybridization at reduced stringency. Using this

approach, however, Nelson and colleagues (1990) were unable to identify

homologs of GAT1 , suggesting that additional family members might exhibit

too little overall identity with GAT1 to permit elucidation in this manner.

Recently, with the introduction of the polymerase chain reaction (PCR) (Saiki

et al. 1988), it became possible to use short stretches of dispersed sequence

identity to identify and clone novel members of an extended gene family (Gould

et al. 1989; Libert et al. 1989; Kamb et al. 1989). In the implementation of this

strategy, Peek and colleagues (1991) have designed degenerate, inosinesubstituted oligonucleotides based on the sequence identity present between

GAT1 and NET in the regions connecting transmembrane domains 1 and 2

as well as that observed in transmembrane 6. With these oligonucleotides

as primers, Peek and colleagues (1991) amplified rat and human cDNAs and

subcloned products of a size similar to that found between these sequences

in NET. Sequence analysis of the cloned amplification products revealed

multiple, distinct gene products all bearing sequence identity to NET and

GAT1. Several of these products bear considerable sequence identity to NET

(60 to 80 percent), and others exhibit only the level of identity (40 to 50 percent)

observed when comparing NET and GAT1 over these regions. Although these

amplification fragments represent partial cDNA clones, they can be used to

determine the anatomical localization of the endogenous transcripts by Northern

and in situ hybridizations and can be used as high-stringency probes for the

identification of their full-length functional sequences by conventional library

hybridization. Northern analysis of each of these products revealed several

that are broadly distributed across the rodent central nervous system and

perhaps difficult to associate with the distribution of a particular neurotransmitter

system. However, several fragments hybridize in a regionally selective manner.

For example, one clone is enriched in forebrain regions, whereas another is

highly concentrated in thalamic, midbrain, and brain stem regions. Particularly

striking is the hybridization of two clones selectively to midbrain and brain stem

regions. Peek and coworkers (1991) performed in situ hybridization analysis of

these clones and found one of these to be localized to the substantia nigra and

ventral tegmental area, whereas the other is confined to the midbrain and brain

stem raphe complex, identifying these two clones as likely candidates for the

DA and 5-HT transporter cDNAs, respectively.

78
Utilizing oligonucleotides derived from the 5’-end of the PCR fragment

encoding the 5-HT transporter candidate, Blakely and colleagues (1991c)

isolated a series of overlapping clones from a rat brain stem cDNA library.

Sequence analysis of one of these clones reveals the presence of a large

open reading frame in register with that of NET. After subcloning the

bacteriophage insert into a T7-promoter-bearing plasmid, Blakely and

colleagues (1991c) transfected this cDNA into HeLa fibroblasts infected

with T7 polymerase-vaccinia virus. After 8 hours, transfected fibroblasts

exhibit marked expression of 5-HT transport, which can be abolished by the

selective 5-HT transport antagonist fluoxetine (figure 7). Thus, like NET, the

5-HT transporter (SERT) appears to be encoded by a single RNA (Blakely

et al. 1991c). The availability of a cDNA clone encoding this carrier should

permit a refined analysis of the structural basis of antidepressant interactions

with nerve terminals. Sequence comparisons with NET and GAT1 reveal

FIGURE 7. Expression of 5-HT transport in HeLa cells transfected with SERT

cDNA

79
highly conserved residues likely to be important for common tasks, including

substrate translocation and Na+ binding. In addition, with regulation of the

5-HT carrier reported to be altered in depressed humans (Paul et al. 1981;

Meltzer et al. 1981), the availability of a cDNA clone encoding this carrier

should lead to a direct search for structural changes associated with the

human SERT homolog in affective disorders. Recently, the first cloning of

a functional rat brain DA transporter cDNA was also achieved (Kilty et al.

1991; Shimada et al. 1991), bearing sequence identity to the substantia nigra

localized PCR species described above. Together, the elucidation of the

5-HT and DA transporters broaden understanding of the structural basis for

neurotransmitter transporter diversity and provides new tools for the molecular

analysis of transporter gene expression and regulation in brain disorders and

drug abuse.

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AUTHOR

Randy D. Blakely, Ph.D.

Assistant Professor and Woodruff Neuroscience Investigator

Department of Anatomy and Cell Biology

Emory University School of Medicine

Atlanta, GA 30322

83
Regulation of Gene Expression by

Dopamine: Implications in Drug

Addiction

Orla M. Conneely, Ronan F. Power, and Bert W. O’Malley

INTRODUCTION

Cocaine is thought to produce pleasure by increasing dopamine transmission

in mesocortical and mesolimbic dopaminergic tracts. Increased dopaminergic

transmission results from an inhibition of the dopamine reuptake transporter

by cocaine, which also inhibits reuptake of both serotonin and norepinephrine

(Ritz et al. 1988). The reward-seeking behavior produced by cocaine selfadministration is similar to that produced by electrical self-stimulation of

dopaminergic pathways. The observation that chronic cocaine administration

decreases electrical self-stimulation reward indices suggests that a downregulation of dopaminergic reward areas may accompany addiction (Gawin

and Ellinwood 1988).

The molecular mechanisms underlying long-term neuroadaptive changes that

accompany chronic cocaine abuse have yet to be established. However, it

is becoming increasingly evident that neuronal adaptation to repeated

perturbation of neurochemical signals involves changes in gene expression.

Examples of such genomic regulation of neuronal adaptation include the

regulation of gene expression by serotonin during long-term facilitation

(Barzilai et al. 1989) and the development of kindling by repeated electrical

stimulation of the hippocampus (Jonec and Wasterlain 1979; Shin et al. 1990).

Therefore, it seems plausible that the effects of chronic cocaine abuse would

be reflected at a level of altered gene expression in dopaminergic and/or

dopaminoceptive cells. In this case, elucidation of the mechanisms by which

dopamine transduces a neurotransmitter signal to changes in gene expression

would be expected to yield valuable information on the molecular nature of

neuroadaptation to repeated cocaine administration. To this end, the authors

have identified a class of transcription factors that are activated by dopamine.

Activation of these factors results in a dopamine-dependent induction of

transcription of specific target genes. The activated factors are members

of the steroid/thyroid receptor superfamily of transcription factors.

84
THE STEROID/THYROID RECEPTOR FAMILY OF TRANSCRIPTION

FACTORS

The steroid/thyroid receptor family constitutes a subclass of transcription

factors that regulate and coordinate complex events in development,

differentiation, and homeostasis. These proteins control behavioral and

physiological responses by regulating the expression of specific gene networks

in response to diverse stimuli (for a review, see Evans 1988). Members of

the above family of transcription factors contain three conserved regions of

amino acid sequence (McDonnell et al. 1987). The conserved regions consist

of a DNA-binding domain responsible for specific binding of the transcription

factor to upstream regulatory DNA sequences within the promoter region

of responsive target genes (Evans 1988) and two additional regions of

conservation located in the ligand-binding domain of the ligand-activated

members of this family (McDonnell et al. 1987). The steroid receptors were

the first characterized members and serve as prototypes for this class of

transcription factors (Hollenberg et al. 1985; Greene et al. 1986; Conneely

et al. 1987). These receptors are soluble proteins that upon specific binding

to their cognate steroid hormone become active transcription factors capable

of regulating the expression of specific target genes (Evans 1988). In addition

to the steroid receptors, the ligand-activated members of this family include

receptors for retinoic acid (Giguere et al. 1987), vitamin D3 (Baker et al.

1988), and thyroid hormone (Weinberger et al. 1986); more than 20 additional

members have been identified. They have been termed “orphan receptors”

since no ligand has been identified that binds to these proteins. However,

recent evidence has demonstrated that several members of the steroid/

thyroid receptor superfamily can be activated in the absence of ligand by

phosphorylation (Glineur et al. 1990; Denner et al. 1990). This observation

raises the possibility that the orphan receptors may be activated by direct

ligand binding and/or by phosphorylation via indirect signaling pathways.

One such orphan receptor, chicken ovalbumin upstream promoter-transcription

factor (COUP-TF) (Wang et al. 1989), participates in the regulation of several

genes, including the chicken ovalbumin gene (Wang et al. 1987), the rat insulin

II gene (Hwung et al. 1988), and the proopiomelanocortin gene (Drouin et al.

1989). Although COUP-TF is widely distributed in tissues throughout the body,

including the brain, at least one of its functions appears to be the regulation of

retinal cell development since the Drosophila counterpart of this protein is

required for the determination of photoreceptor cell fate (Mlodzik et al. 1990).

The present study was initiated in an attempt to identify chemical signals that

would lead either directly or indirectly to the activation of COUP-TF. In the

context of the studies described below, functional COUP-TF expression is

measured as COUP-TF-dependent activation of a specific target gene.

85
DOPAMINE REGULATES COUP-TF-DEPENDENT GENE TRANSCRIPTION

To search for compounds that activate COUP-TF, the authors replaced the

DNA-binding domain of COUP-TF with that of the tissue-specific progesterone

receptor (figure 1). Since the enhancer DNA specificity is provided by the

DNA-binding domain, the resulting chimera is expected to direct COUP-TFdependent transcription of progesterone-responsive target genes (Beato

1991). This strategy was employed to prevent competition in the assay from

endogenous COUP-TF in transfected cells. Expression plasmids containing

the chimeric COUP-TF construct (pADFCOUP) and a progesterone-responsive

target gene (PRETKCAT) were cotransfected into a progesterone receptor

negative monkey kidney cell line, CV1. The target gene used in these studies

contained two copies of a progesterone-responsive enhancer element (PRE)

located upstream of the herpes simplex virus thymidine kinase promoter linked

to a chloramphenicol acetyl transferase (CAT) gene.

FIGURE 1. Strategy for generation of the chimeric COUP protein.

Schematic representations of cPR and COUP-TF are shown.

Prog designates the progesterone-binding domain of cPR. The

chimeric COUP-TF (FCOUP) contains the NH2 and COOHterminal domains of COUP-TF fused to the DNA-binding domain

of cPR. The cPR cDNA (Conneely et al. 1987) was digested

with HindIII at nucleotide 1801, repaired, and ligated to Bsmldigested, blunt-ended human COUP-TF cDNA (hCOUP) to fuse

the cPR DNA-binding domain to the COOH-terminus of COUPTF. A polymerase chain reaction was used to generate a Sacl

site in the COUP-TF cDNA immediately 5’ to the DNA-binding

domain. Sacl-digested COUP-TF cDNA then was ligated to Sacldigested PR cDNA to fuse the N-terminus of COUP-TF to the PR

DNA-binding domain.

SOURCE: Data from Power et al. 1991a

86
More than 150 compounds and tissue extracts were tested for their ability to

activate COUP-dependent CAT gene expression. Cells were maintained in

serum-free media, and individual compounds were added immediately after

transfection. The cells were harvested after 48 hours, and extracts were

assayed for expression of CAT activity. The results of these experiments

demonstrated that the neurotransmitter, dopamine, was capable of stimulating

COUP-TF-dependent activation of CAT expression. The data shown in figure 2

demonstrate that concentrations of dopamine as low as 3 mM were capable of

stimulating CAT gene activity fivefold to tenfold (lanes 2 to 7). Dopamine did

not stimulate CAT expression in cells transfected with a parent expression

plasmid that lacked the chimeric COUP-TF coding sequences (lane 12). This

finding indicated that activation of gene expression did not result from a general

increase in transcriptional activity in transfected cells but was dependent on the

expression of a chimeric COUP transcription factor.

The activation of COUP-TF by dopamine does not result from direct ligand

binding because dopamine binding assays demonstrated no direct binding

of COUP-TF to the neurotransmitter. This observation suggested that the

activation of COUP-TF resulted from an indirect signaling pathway. Dopamine

action is mediated by its interaction with several membrane-bound dopamine

receptors. These G-coupled receptors are of two subtypes, D1 and D2,

distinguished by their ability to stimulate, D1, and inhibit, D2, adenylyl cyclase,

respectively (Kebabian and Calne 1979). Analysis of the ability of selective

agonists of these receptor subtypes to activate COUP-dependent gene

expression demonstrated that the dopamine receptor agonist -ergocryptine

mimicked dopamine in its ability to activate CAT expression (lanes 8 to 11).

These data suggested that a dopamine-receptor-mediated signaling pathway

was responsible for the activation of the COUP-TF chimera.

CV1 CELLS POSSESS A DOPAMINE-SENSITIVE ADENYLYL CYCLASE

The presence of dopamine receptors in renal tissue has been documented

(Felder et al. 1989). To determine whether a dopamine-dependent signaling

pathway of the D1 receptor subtype is operative in CV1 cells, the authors

analyzed these cells for expression of a dopamine-sensitive adenylyl cyclase.

The results are shown in figure 3. Dopamine elicited a twofold increase in cyclic

AMP (CAMP) levels in membranes prepared from these cells. Maximum

stimulation was achieved with 100 mM dopamine. Thus, a dopaminergic

responsive cyclase system is intact in CV1 cells.

COUP-TF IS ACTIVATED BY PHOSPHORYLATION

The above data taken together suggested that the activation of COUP-TF by

dopamine may be a phosphorylation-mediated event. Phosphorylation has

87
FIGURE 2. Induction of CAT gene expression by dopamine (D), and

ergocryptine (E). pADFCOUP (5 μg) and PRETKCAT (5 μg)

were cotransfected into CV1 cells as described (Conneely et al.

1987). Cells were cultured for 2 days in serum-free media

supplemented with Nutridoma (Boehringer Mannheim:

Indianapolis, IN) in the absence (lane 1) or presence of varying

concentrations of dopamine (lanes 2 to 7) and -ergocryptine

(lanes 8 to 11). The media was replaced after 24 hours, and

fresh compounds were added. As a control, cells were

transfected with P91023 (B), which lacked COUP-TF coding

sequences, and with PRETKCAT and cultured in the presence

of 100 μM dopamine (lane 12). CV1 cell extracts were prepared

for CAT assays as described (Conneely et al. 1987), and the

assays were performed for 12 hours with 50 μg of protein extract

The results shown are representative of at least six separate

experiments in which duplicate points were performed. The

variation in signals between duplicate points in any one

experiment was not more than 5 percent. In all experiments the

positions of [

14C]chloramphenicol (C) and the 1- and 3-acetylated

forms of [

14C]chloramphenicol are indicated (1AC and 3AC,

respectively).

SOURCE: Data from Power et al. 1991a

88
FIGURE 3. Dopamine-sensitive adenylyl cyclase in CV1 cells. Production of

CAMP in homogenates from untransfected CV1 cells cultured in

Nutridoma-supplemented media was measured as a function of

increasing dopamine concentration. Adenylyl cyclase assays

were performed in duplicate for each point. Homogenization of

ceils and adenylyl cyclase assays were performed exactly as

described (Toro et al. 1987).

SOURCE: Data from Power et al. 1991b

89
previously been shown to play a role in activation of several transcription

factors, including members of the steroid/thyroid receptor family (Glineur et al.

1990; Denner et al. 1990). Therefore, we examined the ability of 8-bromo-cyclic

adenosine monophosphate (8-Br-cAMP) and the protein phosphatase 1 and 2A

inhibitor, okadaic acid, to mimic dopamine in the activation of CAT gene

expression. The result shown in figure 4 demonstrates that these agents also

stimulate COUP-TF-dependent transcription in the authors’ assay system and

thus support the hypothesis that the dopamine activation of COUP-TF is a

phosphorylation-mediated process.

DOPAMINE STIMULATES LIGAND-INDEPENDENT ACTIVATION OF

SEVERAL MEMBERS OF THE STEROID/THYROID RECEPTOR FAMILY

The ability to activate COUP-TF by a signaling pathway stimulated by dopamine

prompted an examination of the classical steroid receptor members of the same

family to determine whether they can be activated by dopamine in the absence

of ligand. The results of these assays are shown in figure 5.

The authors selected several members of this family and found that the

progesterone (cPRA, cPRB), estrogen (hER), and vitamin D3 (hVDR) receptors

also were activated by dopamine and okadaic acid. Again, CV1 cells were

transfected with the appropriate receptor expression vector and a specific

reporter gene (HREtKCAT) containing the appropriate receptor responsive

enhancer element (HRE) located upstream of the thymidine kinase promoter

and a CAT gene. The level of activation attained with dopamine was

comparable to that attained with the natural ligand for these receptors (figure

5, panels A to D). Interestingly, not all steroid receptors were activated by

this neurotransmitter under our test conditions. CV1 cells cotransfected with a

vector for expression of the human glucocorticoid receptor pRShGRa (Giguere

et al. 1986) and the PRE/GRETKCAT reporter plasmid were transcriptionally

responsive to dexamethasone but were unresponsive to either dopamine or

okadaic acid (panel E). Likewise, cells transfected with a vector for expression

of the human mineralocorticoid receptor pRShMR (Arriza et al. 1987) showed

the expected increase in CAT gene expression in response to aldosterone

and dexamethasone but showed only a marginal response to dopamine and

okadaic acid (panel F). It has been shown that the progesterone receptor can

be activated by 8-Br-cAMP in the absence of hormonal ligand (Denner et al.

1990). The data above are consistent with the notion that the activation of

several members of this family by dopamine occurs through phosphorylation

of these transcription factors via a dopamine-dependent intracellular signaling

pathway. However, confirmation of this mechanism will require direct

identification of a dopamine-dependent phosphorylated site on these

transcription factors.

90
FIGURE 4. Activation of COUP-TF-mediated transcription by

phosphorylation. Cells transfected with pADFCOUP (5 μg)

and PRETKCAT (5 μg) were either untreated (basal) or treated

with 8-Br-cAMP (10-3M) or okadaic acid (OA) (5x10-8M) as

described (Denner et al. 1990).

SOURCE: Data from Power et al. 1991a

91
FIGURE 5. The effect of dopamine on the transcriptional activity of steroid

hormone receptors. Expression vectors (5 μm) for the chicken

progesterone receptor A and B forms (cPRA, cPRB) (Conneely

et al. 1987), human glucocorticoid receptor (hGR) (Giguere et al.

1986), and human mineratocorticoid receptor (hMR) (Felder et

al. 1989) were cotransfected with the reporter plasmid PRE/

GRETKCAT (5 μm) (Jantzen et al. 1987). The human estrogen

receptor (hER) expression construct (2 μm) was cotransfected

with an estrogen receptor-responsive reporter, ERE E1bCAT

(5 μm). The human vitamin D3 receptor construct (1 μm)

(McDonnell et al. 1989) was cotransfected with a reporter

92
plasmid VDRETKCAT (4 μm) containing three copies of the

vitamin D receptor response element fused to the herpes simplex

virus thymidine kinase promoter and the bacterial CAT gene.

Transfected cells were either untreated (basal) or treated with

10-7M of the relevant ligands for each receptor; progesterone

(P4), estradiol (E2), 1,25(OH)2D3 (VITD3), dexamethasone (DEX),

and atdosterone (ALDO). Cells also were treated with the

indicated concentrations of dopamine (D) and okadaic acid

(OA; 5X10-8M). CAT activity was determined 42 hours

posttransfection.

SOURCE: Data from Power et al. 1991b

DOPAMINE REGULATION OF GENE EXPRESSION IS REGULATED BY A

RECEPTOR OF THE D1 SUBTYPE

To examine the role of D1 and D2 receptors in the mediation of dopaminedependent activation of gene expression, we tested the ability of selective

D1 and D2 agonists to mimic the dopamine activation of progesterone

receptor-dependent gene transcription. CV1 cells, transfected with the

cPRA expression vector and the reporter plasmid PRETKCAT, were

treated with either progesterone, dopamine, the selective D1 receptor agonist

SKF38393, or the selective D2 receptor agonist quinpirole. The relative

amounts of CAT activity expressed after treatment of the cells with each

compound are shown in figure 6. The D1-selective agonist SKF38393

stimulated CAT-gene expression to a level even greater than progesterone

or dopamine. However, no stimulation of CAT activity was obtained afler

treatment with the selective D2 receptor agonist quinpirole. Thus, the

activation of the steroid receptors by dopamine appears to result from a

signaling pathway that is mediated by a D1-adenylyl-cyclase-linked receptor.

DISCUSSION

The authors have demonstrated that the neurotransmitter dopamine can

stimulate an intracellular signaling pathway that results in activation of

members of the steroid/thyroid receptor family of transcription factors.

The ability of dopamine D1 receptor agonists to selectively mimic dopamine

in the activation of these factors, together with the demonstration of a

dopaminergic-responsive cyclase system in CV1 cells, suggests that the

pathway is mediated by a dopamine receptor of the D1 subtype. Furthermore,

the activation of COUP-TF and the steroid receptors examined in this study

appears to be mediated by phosphorylation. Receptors activated by dopamine

93
FIGURE 6. Regulation of progesterone receptor-mediated gene transcription

by dopamine receptor agonists. CV1 cells were transfected with

the cPRA expression vector and the PRETKCAT reporter as

described in the figure 2 legend. Transfected cells were either

untreated (basal) or treated with progesterone (P4; 10-7M),

dopamine (400 μM), the selective D1 dopamine receptor agonist

SKF38393 (100 μM), or the selective D2 dopamine receptor

agonist quinpirole (Quin; 20 μM). CAT activity was determined 42

hours posttransfection. Each result represents a mean value

obtained from duplicate experiments.

94
also are activated by 8-BR-cAMP and the phosphatase inhibitor okadaic acid,

whereas other members of the same family of receptors that are not activated

by dopamine also are not activated by 8-Br-cAMP and okadaic acid.

The data obtained in this study provide the first demonstration of a link between

a dopamine-stimulated signaling pathway and transcription factor activation.

The activation of members of the steroid/thyroid receptor family of transcription

factors by dopamine provides a means by which dopamine can regulate gene

expression in dopaminoceptive cells. Furthermore, the data suggest that the

same signaling pathways that mediate short-term cellular responses to

dopamine stimulation also may mediate long-term neuroadaptive responses

to dopamine by reprograming genomic expression. In this regard, additional

studies will be required to substantiate the physiological relevance of this

pathway, to identify which members of the steroid/thyroid receptor family are

expressed in dopaminoceptive cells, and to identify the specific target genes

that are regulated by dopamine.

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AUTHORS

Orla M. Conneely, Ph.D.

Assistant Professor

Ronan F. Power, Ph.D.

Research Associate

Bert W. O’Malley, M.D.

Professor and Chairman

Department of Cell Biology

Baylor College of Medicine

Texas Medical Center

One Baylor Plaza

Houston, TX 77030

97
Regulation of Opioid Gene Expression:

A Model To Understand Neural

Plasticity

Michael J. Comb, Linda Kobierski, Hung Ming Chu, Yi Tan,

David Borsook, Karl Herrup, and Steven E. Hyman

PLASTICITY OF OPIOID GENE EXPRESSION: A MODEL TO ANALYZE

MECHANISM

Over the past several years, it has become clear that neurotransmitter

regulation of gene expression is a critical mechanism underlying neural

plasticity (Golet et al. 1986; Montarolo et al. 1986; Goodman 1990; Sheng

and Greenberg 1990; Morgan and Curran 1991; Comb et al. 1987). The role

of peptide neurotransmitters in this process has received much attention

because they act as important regulators of neural signaling and their synthesis

and expression is dependent on the transcription of precursor genes in the

nucleus. This direct biosynthetic dependence on gene transcription endows

peptide transmitters with special “adaptive” properties that distinguish them

from classical small-molecule neurotransmitters (Comb et al. 1987). Perhaps

the most interesting and best studied example of this process in brain is the

activity-dependent switch in opioid peptide expression in hippocampus (Gall

1988; White et al. 1987; White and Gall 1987; Morris et al. 1988). Electrical

or chemical stimulation of the rodent perforant pathway, the major input to

hippocampus from entorhinal cortex, turns on expression of proenkephalin

and turns off expression of prodynorphin in granule cells of the dentate gyrus.

These changes in opioid precursor result in dramatic and longlasting changes in

proenkephalin and prodynorphin peptides (Gall 1988; White et al. 1987) in

mossy fibers and mRNAs (White et al. 1987; White and Gall 1987; Morris et

al. 1988; Sonneberg et al. 1989) in granule cell bodies. Because enkephalins

have a profound excitatory effect on target hippocampal pyramidal cells, this

activity-driven switch may exert major control over hippocampal excitability

and may be intimately involved in the molecular mechanisms underlying

plastic changes in hippocampal signaling.

98
As opioid peptides are critical regulators of neural pathways mediating pain,

reward, motivation, and hormone release, understanding how their synthesis

and expression is regulated takes on great practical as well as theoretical

significance. With the realization that neurotransmitters regulate endogenous

opioid biosynthesis has come the understanding that drugs that interfere with

or alter neurotransmission also may influence the expression of endogenous

opioids. Hence, neurotransmitters and drugs of abuse acting through synaptic

connections in brain may alter gene expression in the postsynaptic neuron.

Such drug-induced changes in gene expression may underlie important

components of addiction, withdrawal, and drug-seeking behaviors, which

have been particularly difficult to understand at a mechanistic level.

IMMEDIATE EARLY GENE REGULATION IN BRAIN

Seizures and electrical stimulation also trigger a rapid and transient RNA

induction of several different immediate early genes (IEGs) within granule cells

of the dentate gyrus. For example, RNA encoding different components of the

AP-1 transcription factor complex: c-Fos, c-Jun, and JunB are coinduced

(White and Gall 1987; Sonneberg et al. 1989; Saffen et al. 1988; Cole et al.

1989; Wisden et al. 1990) together with proenkephalin mRNA in hippocampal

granule cells by seizure and electrical stimulation of the perforant pathway.

The time course of IEG induction precedes the induction of proenkephalin

RNA (White and Gall 1987; Sonneberg et al. 1989). These observations have

led to the hypothesis that IEGs may act as “third messengers” to initiate and

coordinate programs of gene expression by regulating the expression of

appropriate “target” genes, for example, proenkephalin. This argument is

strengthened by the observation that binding sites for many of these factors

have been found within a compact region of the proenkephalin promoter

(figure 1) known to mediate synaptic and second-messenger regulation (Comb

et al. 1986, 1988; Hyman et al. 1988; Nguyen et al. 1990; Chu et al. 1991;

Kobierski et al. 1991). In addition, these proteins are known transcription

factors; the authors show herein that different components of the AP-1 complex

have different effects on proenkephalin transcription. Regulation of transcription

by the AP-1 complex at the proenkephalin-inducible enhancer represents the

first neural model system to understand IEG target gene regulation and

promises to outline general mechanisms that may regulate the synthesis

of other key neural signaling molecules. The recent discovery that drugs

of abuse such as cocaine (Young et al. 1991), amphetamine (Graybiel et al.

1990), and morphine (Chang and Harlen 1990) also activate the expression

of IEGs suggests that this process may represent an adaptive response of

the brain to drugs of abuse.

99
FIGURE 1. Human proenkephalin gene promoter sequence
TRANSSYNAPTIC REGULATION OF PROENKEPHALIN TRANSCRIPTION

Studies over the past 5 years have defined a short 50-base-pair region of

the human proenkephalin promoter that is necessary and sufficient for

transcriptional regulation by adenosine 3':5'-cyclic phosphate (CAMP)-,

12-O-tetradecanoyl-phorbol ester (TPA)-, and CA++-dependent intracellular

signaling pathways. Extreme conservation of this sequence between human

and rat genes reinforces its critical role in proenkephalin expression and

regulation. Two functional elements were localized within this region and

shown to interact synergistically to mediate second-messenger regulation

(Comb et al. 1986, 1988; Hyman et al. 1988; Nguyen et al. 1990; Chu et al.

1991; Kobierski et al. 1991). Previous studies have identified and characterized

transcription factors that interact with these DNA elements. NF-1 (Chu et

al. 1991) proteins interact with the CRE-1 element, and AP-1 and AP-4

interact with the CRE-2 element (Comb et al. 1988; Hyman et al. 1988;

Nguyen et al. 1990; Chu et al. 1991; Kobierski et al. 1991). Recent

identification and characterization of the CREB/ATF transcription factors

(Hai et al. 1989; Gonzalez and Montminy 1989) prompted the authors to

examine the role of these factors in the regulation of proenkephalin

transcription. DNA binding studies (in vitro) suggested that these factors

interact poorly with the proenkephalin-inducible enhancer as homodimers

and focused our attention on the AP-1 (Fos/Jun) family of transcription factors,

which bind the enhancer with high affinity (Golet et al. 1986).

Work from the authors’ laboratory (Comb et al. 1988; Kobierski et al. 1991)

and Tom Curran’s laboratory (Sonneberg et al. 1989) has demonstrated

that the AP-1 transcription factor binds the proenkephalin CRE-2 element

and that in vitro-translated c-Jun and c-Fos bind the CRE-2 element as Fos/

Jun heterodimers. Our laboratory has also shown that two other Jun

members, JunD and JunB, bind the CRE-2 element and have opposite

effects on proenkephalin transcription (Kobierski et al. 1991). In this chapter,

discussion iS restricted to the role of the AP-1 factors mediating secondmessenger regulation of proenkephalin transcription at the CRE-2 element.

Previous studies have also demonstrated that CAMP rapidly activates

proenkephalin transcription, with increases in steady-state mRNA levels

apparent within 15 minutes after the addition of forskolin to C6 glioma cells

(Kobierski et al. 1991). RNA levels peak at 1.5 to 2 hours following forskolin

addition and then rapidly decline to undetectable levels at between 4 and 8

hours. Activation is rapidly followed by a refractory period during which the

proenkephalin gene is unresponsive to further stimulation of the CAMP pathway.

The forskolin-dependent rapid activation is not affected by protein synthesis

inhibitors: however, protein synthesis inhibitors block the decline in mRNA

101
levels leading to super induction (Kobierski et al. 1991). In addition, forskolin

treatment of C6 glioma cells leads to rapid induction of c-Fos and JunB mRNA

and a reduction in the level of JunD RNA.

CHARACTERIZATION OF JUND, JUNB, AND C-JUN INTERACTIONS WITH

CRE-2

Three observations have focused the authors’ efforts on identifying members

of the AP-1 complex that bind the CRE-2 element and mediate inducible

expression: (1) Multiple copies of this element reconstitute the most essential

features of CAMP-, TPA-, and Ca++-inducible regulation; (2) ATF/CREB

molecules bind this element poorly if at all; and (3) AP-1 complexes bind the

CRE-2 element with high affinity. To test the ability of Fos/Jun molecules to

stimulate or repress proenkephalin transcription, we have developed expression

vectors and a cotransfection assay to determine the effect of expression of

various Fos/Jun molecules on proenkephalin expression. Full-length cDNAs

encoding c-Jun, JunB, c-Fos, and JunD have been introduced into a Rous

sarcoma virus expression vector. Expression vectors are then cotransfected

with pENKAT-12 into F9 cells in the presence or absence of a plasmid

expressing high levels of the cAMP-dependent protein kinase (PKA).

Results of this type of analysis have focused our attention on two different

Jun molecules, JunD and JunB, which have opposite effects on proenkephalin

transcription. JunD strongly transactivates proenkephalin transcription in a

manner that is totally dependent on the catalytic subunit of the CAMPdependent PKA (figure 2). JunB has no effect in the presence or absence

of PKA and acts to repress JunD-dependent stimulation of proenkephalin

transcription (figure 3). Finally, c-Jun strongly activates proenkephalin

transcription in a constitutive fashion that is not influenced by PKA (see

figure 2). Activation and repression by JunD, JunB, and c-Jun map to the

CRE-2 element by cotransfection using plasmids containing multicopy elements

and point mutations. We have extended these observations to several other

cell lines, including C6 glioma and SK-N-MC cells, that express proenkephalin.

These findings clearly demonstrate that transactivation of proenkephalin by

JunD is completely dependent on the catalytic subunit of the cAMP-dependent

PKA. In addition, as each of these molecules is expressed in the granule cell of

the hippocampus, JunD constitutively (Wisden et al. 1990) and JunB and c-Jun

in a highly inducible fashion (Saffen et al. 1988; Cole et al. 1989; Wisden et al.

1990), it is likely that these molecules play a critical role in the activation and

repression of proenkephalin gene expression in the nervous system.

102
FIGURE 2. Different effects of Jun proteins on proenkephalin transcription.

Plasmids (5 μg each) expressing JunD (RSVJunD), JunB

(RSVJunB), and c-Jun (RSVc-Jun) were cotransfected into

F9 cells together with a proenkephalin/CAT fusion gene

(pENKAT-12 5 μg) in the presence or absence of a plasmid

expressing the catalytic subunit of the cAMP-dependent PKA

(pMTCalpha 5 μg). In addition, a plasmid, pRSVßGal (10 μg),

was also included in each transfection, and ß-Gal expression

was used to normalize CAT expression between transfection

experiments. Data presented are from one experiment but are

representative of at least five different experiments.

CHARACTERIZATION OF JUND AND JUNB BINDING AT THE

PROENKEPHALIN ENHANCER

To examine the binding of JunD and JunB to the CRE-2 element, RNA

encoding JunD, JunB, and c-Fos was transcribed and then translated

individually and in combinations as described below in rabbit reticulocyte

lysates. Control lysates show no specific binding to a 30-base-pair

oligonucleotide, spanning the proenkephalin enhancer from -110 to -80.

However, lysates programed with JunD RNA produce a shifted band,

which is specifically self-competed but not competed by AP-2 or AP-4

oligonucleotides and is very weakly competed by a CRE-1 oligonucleotide.

Both TRE and CRE oligonucleotides compete effectively for JunD DNA

103
FIGURE 3. JunB represses PKA-dependent activation of proenkephalin by

JunD. Plasmids were cotransfected into F9 cells as described in

figure 2. In each case, 5 μg of DNA was used except where

indicated for pRSVJunB.

binding. In addition, mutations within the 3’-end of CRE-2, which have the

most profound effect on second-messenger regulation of proenkephalin

transcription (Comb et al. 1988), also produce the most severe effect on

JunD binding.

In contrast, binding of in vitro-translated JunB to the enhancer is not

observed. Because JunB is known to dimerize with JunD and c-Fos (Ryder

et al. 1989; Nakabeppu et al. 1988), we also analyzed the interaction of these

complexes with the CRE-2 element. Consistent with previous reports using

AP-1 oligonucleotides, binding of JunB and JunD to the CRE-2 element is

dramatically stimulated in the presence of c-Fos.

CLASSIC CRE AND TRE ELEMENTS ALSO MEDIATE PKA-DEPENDENT

TRANSCRIPTION VIA JUND

Because the proenkephalin enhancer contains a hybrid TRE/CRE-like element,

CRE-2, we tested the ability of JunD to transactivate “classic” TRE and CRE

elements in a PKA-dependent fashion by transiently transfecting F9 cells

with plasmids containing the somatostatin CRE, or the collagenase TRE,

104
together with pRSVJunD and pMTCaneo. JunD strongly transactivates the

somatostatin CRE element and transactivates the collagenase TRE to a

somewhat lesser extent. These results indicate that JunD can act at both

CRE and TRE elements to stimulate transcription in a PKA-dependent fashion.

ANALYSIS IN TRANSGENIC ANIMALS

To extend the analysis of mechanisms underlying activity-dependent regulation

of proenkephalin transcription to the intact nervous system, we have made

transgenic animals expressing proenkephalin/LacZ fusion constructs. The

first construct analyzed contains 3 Kb of human proenkephalin 5'-flanking

sequences and 1.5 Kb of 3’-flanking sequences driving expression of the

LacZ reporter. Expression of this construct has been examined in three

independent transgenic lines. This construct also contains the wellcharacterized CAMP-, TPA-, and Ca++-inducible enhancer located within

the first 110 bases of 5’-flanking DNA (Comb et al. 1986) and is shown to

direct correct tissue-specific expression to all (with only two known exceptions,

striatum and olfactory tubercle) sites within the adult reproductive (Borsook

et al., in press) and nervous systems. Although some variation between

founder animals is apparent, there is a remarkable degree of tissue-specific

transgene expression observed in the adult mouse brain.

In brain, as in the reproductive system, ectopic expression of the transgene

is not seen. As illustrated in table 1, expression on the transgene in various

brain regions appears to be remarkably precise as determined by X-Gal

staining. LacZ expression has been most carefully examined in founder

#3 where X-Gal staining is seen in the spinal cord, thalamus, hypothalamus,

hippocampus, entorhinal cortex, cerebral cortex, amygdala, dorsal horn of

the periaqueductal gray matter, ventral tegmental area, pons and medulla

oblongata, and many other brain regions. As expected, the major sites

of hypothalamic transgene expression are the paraventricular nucleus

and ventromedial nucleus. High-level expression is also seen in the

central nucleus of the amygdala in excellent agreement with previous

immunohistochemical studies of cholchicine-treated rats. In addition,

correct tissue-specific expression is seen in laminae I and II (substantia

gelatinosa) of the dorsal horn of the spinal gray matter and is also observed

in scattered cells throughout laminae IV and V-VII. A comparison between

LacZ expression and previously reported expression of proenkephalin peptides

and mRNA is also shown in table 1. Taken together, these remarkable results

suggest that the 30 Kb proenkephalin/LacZ fusion gene directs expression of

LacZ to the vast majority of diverse sites where proenkephalin is normally

expressed in the adult. The one anomaly in brain expression is striatum.

Little or no expression of LacZ is seen in striatum of each of the three

independent founder lines examined. The missing or low-level expression

105
TABLE 1. Comparison of transgene LacZ expression vs. proenkephalin

mRNA (in situ) and peptide immunoreactivity

Structure ß-Gal* In situ lmmunoreactivity

Telencephalon

Olfactory bulb

Cingulate cortex

Entorhinal cortex

Olfactory tubercle

Caudate-putamen

Lateral septum

Bed n. stria termin

Diagonal band Broca

Preoptic area

Amygdala central

Medial

Cortical

Anterior

Lateral

Dentate gyrus

Hippocampal pyramidal

Diencephalon

Hypothalamus anterior

Perifornical

Lateral

Suprachiasmatic

Paraventricular

Ventromedial

Thalamus lateral geniculate

Periventricular

Mesencephalon and

rhombencephalon

Periaqueductal gray

Ventral tegmental

Locus coeruleus

Pontine reticular

Raphe magnus

Gigantocellular reticular formation

Raphe pallidus

Cerebellum (Golgi II)

Spinal cord (I, II, V, X)

++

—

+++

—

—

++

+

++

+

++++

+++

+++

++

+

+

+

++

+++

++

—

++

++++

+++

+

+++

++

+

++

+++

+++

+++

+

—

ND

+++

+++

++++

++++

++

+

+

+

++++

+++

+++

++

+

+

+

++++

+++

++

ND

++

++++

++

+

+

+++

+

+

+++

++

+

+++

++

+++

+++

+++

+++

++

++

++

+

+++

+

+++

+

+

+

+

++

+++

+

+/-

++

+

++

+

++

+++

+

+

+++

++

+

—

+++

KEY (relative intensity of expression): -=none detected; ND=not determined; and

increasing intensity=from + to ++++

*ß-Gal staining in mouse; in situ data and immunohistochemical data from rat,

adapted from Fallon and Leslie (1986)

106
in striatum and olfactory bulb suggests that additional element(s) may be

necessary for expression at these sites. Alternatively, these elements

may be present, yet are more sensitive to the site of chromosomal integration.

To further investigate this possibility, the authors are producing and analyzing

additional independent founder animals.

SUMMARY AND CONCLUSION

The recent finding that neurotransmitters and drugs that affect

neurotransmission have important influences on gene expression suggests

that drug-induced alterations in gene expression may underlie many

long-term effects of addictive drugs, for example, dependence and

drug-seeking behaviors. These long-term adaptive responses to opiate

drugs have been particularly difficult to understand at a mechanistic level.

Data presented here indicate that the gene encoding the opioid precursor

proenkephalin is highly regulated by neural activity, second-messenger

pathways, and PKA. These observations raise the possibility that drugs

of abuse (e.g., opiates acting through opiate receptors) may act at the

genetic level to modulate the expression of endogenous opiates and that

these effects may underlie one component of the brain’s long-term adaptive

response to exogenous opiates. The transgenic animals described above

can be used to investigate opiate drug-induced changes in proenkephalin

gene expression, allowing rapid analysis of changes in proenkephalin gene

expression in highly restricted populations of neurons in a fashion previously

impossible. In addition, by analyzing the effects of specific enhancer mutations

on tissue-specific and transsynaptic regulation of proenkephalin expression,

transgenic models will permit mechanistic investigations within the intact

nervous system that cannot otherwise be undertaken.

Investigation of mechanisms underlying this process requires the analysis of

intracellular signaling pathways, responsive DNA regulatory elements, and the

transcription factors transducing synaptic signals into gene regulation. In the

studies described herein, we demonstrate that AP-1 complexes consisting of

different Jun proteins differentially regulate proenkephalin transcription at the

CRE-2 element. c-Jun constitutively activates proenkephalin transcription,

whereas JunD activates in a fashion completely dependent on the activation of

second-messenger pathways and the cAMP-dependent PKA. JunB alone has

no effect on proenkephalin gene expression, yet this molecule effectively blocks

activation mediated by JunD and, hence, may act as a repressor. These data

are consistent with a model (figure 4) in which preexisting JunD mediates the

rapid cAMP-dependent activation of the proenkephalin enhancer, whereas IEGs

such as JunB or c-Fos mediate the protein synthesis-dependent inactivation.

Because c-Jun activates proenkephalin transcription constitutively, induction

107
FIGURE 4. Model for activation and repression of proenkephalin transcription by AP-1 complexes.

Neurotransmitters or drugs acting through neurotransmitter receptors activate second-messenger

pathways and PKA. Activated PKA rapidly dissociates to the nucleus and phosphorylates substrates,

leading to activation of preexisting JunD/CREB-like transcription factors, which in turn rapidly activate

proenkephalin transcription. These factors also activate IEGs such as c-Jun and JunB, which may

further stimulate or repress proenkephalin transcription.
of c-Jun may lead to a further and prolonged activation of proenkephalin gene

expression. Hence, the ratio of c-Jun to JunB induction may determine whether

proenkephalin is repressed or further activated.

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ACKNOWLEDGMENTS

The work described in this chapter was supported in part by U.S. Public Health

Service grants DA-05706 and DA-00151 (MJC).

AUTHORS

Michael J. Comb, Ph.D.

Assistant Professor

Yi Tan, M.D.

Research Fellow

Laboratory of Molecular Neurobiology

Massachusetts General Hospital

Charlestown, MA 02129

Linda Kobierski, B.A.

Graduate Student

Hung Ming Chu, Ph.D., M.D.

Graduate Student

Program in Neuroscience

Harvard Medical School

Boston, MA 02129

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

Instructor

Department of Anesthesia

Massachusetts General Hospital

Harvard Medical School

Boston, MA 02129

111
Karl Herrup, Ph.D.

Associate Professor

Department of Developmental Neurobiology

E.K. Shriver Center

200 Trapelo Road

Watham, MA 02254

Steven E. Hyman, M.D.

Assistant Professor

Laboratory of Molecular Neurobiology

Department of Psychiatry

Massachusetts General Hospital

Charlestown, MA 02129

112
Cellular and Molecular Analysis of

Opioid Peptide Gene Expression

Cynthia T. McMurray, Karen M. Pallock, and James Douglass

INTRODUCTION

The molecular mechanisms underlying behavioral and physiological events

associated with the intake of addictive drugs such as cocaine, amphetamine,

and opiates are poorly understood. The problem of physical dependence to

and withdrawal from these drugs is challenging to approach experimentally,

as the establishment of these states likely involves a complex cascade of

biochemical and electrical events. Such events involve the diverse and

widely distributed array of central nervous system (CNS) neurotransmitters,

receptors, ion channels, and transporters and extend to complex autonomic

and sensorimotor neural networks. In addition, various events associated

with drug-seeking behavior and addiction are also influenced by genetic,

species, and environmental factors, which only serve to further complicate

experimentation in this area.

There is growing evidence that specific changes in neuronal transcription

patterns result from the administration of narcotic drugs. For example, it

has been reported that the administration of morphine significantly

decreases striatal levels of proenkephalin mRNA in the rat (Uhl et al. 1988),

and chronic naloxone treatment increases expression of both proenkephalin

and protachykinin in the rat striatum (Tempel et al. 1990). In striatum,

prodynorphin gene expression is increased following chronic administration

of the indirect dopamine antagonists methamphetamine and cocaine (Sivam

1989; Hanson et al. 1987). Thus, it appears that administration of narcotic

drugs can induce localized alterations in the synthesis of mRNA encoding

striatal peptide neurotransmitters. More recently, evidence has been presented

suggesting that the aforementioned transcriptional alterations may be mediated

by rapid, transient changes in expression of specific immediate early genes,

such as c-fos. For example, amphetamine and cocaine can induce drugspecific activation of the c-fos gene in striosome-matrix compartments and

limbic subdivisions of the striatum (Graybiel et al. 1990). Furthermore, this

induction appears to involve the D1 dopamine receptor system (Young et al.

113
1991). Thus, to continue to gain insight into the nuclear events associated

with substance abuse, researchers must understand in greater detail the basic

mechanisms that regulate the expression of pertinent transcriptionally active

genes in the CNS, such as those mentioned above.

REGULATED EXPRESSION OF THE RAT PRODYNORPHIN GENE

Distribution and Physiological Effects of Dynorphin Peptides

The prodynorphin precursor encodes the dynorphin family of opioid peptides

(Kakidani et al. 1982; Civelli et al. 1985). Northern blot and in situ histochemical

analysis have determined that the prodynorphin gene is transcriptionally active

in a wide variety of cell types within the central and peripheral nervous systems

(Akil et al. 1984; Civelli et al. 1985). Such regions include the hypothalamus,

striatum, hippocampus, midbrain, brain stem, and cerebral cortex, as well as

neurons within the spinal cord and gut. Prodynorphin transcripts are also

present at relatively high levels in endocrine tissues, such as the anterior

pituitary, adrenal, testis, ovary, and uterus.

The opioid receptor subtype exhibiting specificity for prodynorphin-derived

peptides is the -opioid receptor (for review, see Mansour et al. 1988). The

receptor belongs to the G protein-linked family of receptors and is also coupled

to calcium conductances. This latter characteristic serves to biochemically

distinguish the -receptor from the μ- and -opioid receptor subtypes, which

are coupled to potassium conductances. Studies utilizing radiolabeled ligands

specific for -receptor binding have determined that this species of opioid

receptor is widespread throughout the CNS and endocrine system.

As with the opioid peptides derived from the proopiomelanocortin and

proenkephalin precursors, it has been difficult to assign precise physiologically

relevant functional roles to those peptides derived from the prodynorphin

precursor. However, numerous studies have suggested that the dynorphin

peptides serve to regulate a wide variety of physioIogical and behavioral

responses (Mansour et al. 1988). These roles include the mediation of

visceral analgesia, effects on appetite suppression, mediatron of hypotensive

cardiovascular responses, inhibition of vasopressin secretron and possibly

additional renal functions, involvement in modulation of motor seizure

thresholds and intensity and involvement in recovery from spinal cord injury

and stroke. A role for dynorphin peptides in narcotic tolerance mechanisms

also has been sugested.

114
Transcriptional Regulation of the Rat Prodynorphin Gene In Vivo

The cloning of rat prodynorphin cDNA and genomic DNA (Civelli et al. 1985;

Douglass et al. 1989) has afforded the opportunity to study regulation of

prodynorphin gene expression in distinct neuronal cell types following specific

surgical or pharmacological manipulations. The spinal cord and hippocampus

represent those neural systems in which the most dramatic alterations of

prodynorphin gene expression have been observed to date.

In the spinal cord, prodynorphin transcripts and peptides are localized to

laminae I-II and V-VI, suggestive of a functional role in endogenous pain

recognition and control. Following the onset of unilateral inflammation of the

hindlimb, prodynorphin biosynthesis is substantially elevated in the region of

the spinal cord receiving sensory input from the affected limb (ladarola et al.

1988; Naranjo et al. 1991). Prodynorphin mRNA levels rise substantially within

the first 24-hour period, and maximal stimulation (eightfold to ninefold increase)

is observed between 3 and 5 days after the onset of inflammation. By day 14,

prodynorphin mRNA levels have approached control values. This time course

of induction and subsequent decline closely parallel that of hindlimb edema and

hyperalgesia. Spinal cord DynA1-8 levels also rise approximately threefold

during the inflammatory period, consistent with an increase in both the rate of

synthesis and release of dynorphin peptides from spinal cord neurons. These

data suggest the active participation of dynorphin-containing spinal cord

neurons in the modulation of sensory afferent input during peripheral

inflammatory pain states.

Opioid peptides derived from the prodynorphin precursor are also present

at high levels in the rat hippocampus. In this brain structure, dynorphin

immunoreactivity exhibits a restricted distribution, being localized to the

granule cell/mossy fiber axonal system (McGinty et al. 1983). Over the past

several years, numerous groups have documented striking changes in the

levels of hippocampal prodynorphin mRNA following the onset of seizure

activity. In one study, the acute effects of kainic acid (KA) administration were

examined (Douglass et al. 1991). Following a single subcutaneous injection

of KA (8 mg/kg), hippocampal prodynorphin mRNA levels rose thirteenfold to

fourteenfold within 3 hours, began to decline at 12 hours, and by 48 hours were

at or below control values. Although mRNA levels were dramatically stimulated,

hippocampal DynA1-8 levels remained at values significantly below control at all

time points monitored. This observation suggests that KA treatment also results

in a prolonged stimulation of release of dynorphin peptides from hippocampal

neurons.

115
Interestingly, other paradigms that induce seizure activity, such as

electroconvulsive shock (Xie et al. 1989a), prepyriform cortical kindling (Xie et

al. 1989b), and hippocampal kindling (Morris et al. 1987), serve to significantly

reduce hippocampal prodynorphin mRNA levels to 25 to 40 percent of control

values. Thus, it appears that the prodynorphin gene is capable of being both

positively and negatively transcriptionally regulated in the granule cells of the

dentate gyrus.

Characterization of the Rat Prodynorphin Gene

The studies described above document changes in prodynorphin mRNA

levels in various neuronal cell types and systems. Presumably, these changes

are the result of alterations in the rate of transcription of the prodynorphin

gene. The authors have isolated and characterized the rat prodynorphin gene

(Douglass et al. 1989) to gain an understanding of the molecular events that

mediate transcriptional regulation of the gene. Such analysis has led to the

identification of the rat prodynorphin mRNA CAP site and promoter element

and to the initial characterization of putative transcripitional regulatory elements.

As diagramed in figure 1, the rat prodynorphin gene is composed of four

exonic domains. Exons 1 and 2 encode 5’ untranslated regions of the mRNA,

whereas exon 3 encodes 15 bases of 5’ untranslated region and the first 23

amino acids of the prohormone. Exon 4 encodes the remaining 204 amino

acids of the prohormone, including the three leucine-enkephalin moieties, as

well as the entire 3’ untranslated region. In the CNS, exons 1 through 4 are

spliced together to generate a mature 2,400-base transcript. However, in the

testis (and perhaps adrenal, as well), exon 2 is not contained within the smaller

2,300-base transcript (Garrett et al. 1989). Thus, the 5’ untranslated region

beginning 15 bases from the translational start site is unique in the two species

of prodynorphin mRNA. Sucrose gradient polysome analysis of striatal and

testicular prodynorphin mRNA reveals that both species of mRNA are present

on high molecular weight polysomes, suggesting that the mRNA is efficiently

translated in both tissues (Garrett et al. 1989). The functional significance of

this alternate splicing event involving prodynorphin mRNA is currently unknown.

Analysis of the Rat Prodynorphin Gene Promoter

In vivo studies have documented that rat prodynorphin mRNA levels in specific

neuronal cell types can be dramatically altered following neural stimulation.

These changes presumably reflect alterations in the rate of synthesis of the

prodynorphin transcript. Thus, signals received by prodynorphin-expressing

neurons must be relayed to the cell nucleus, resulting in altered levels of

transcription of the prodynorphin gene. Such transcriptional effects are

116
FIGURE 1. Structure of the rat prodynorphin gene. Alternate splicing of rat prodynorphin precursor mRNA occurs to

generate different forms of mature mRNA in the brain and testis.
presumably mediated through precise interactions between specific DNA

binding proteins (i.e., trans-acting factors) and their target recognition sequence

elements near the prodynorphin promoter region (i.e., cis-acting elements). As

a first step in characterizing such interactions, the authors have determined the

nucleotide sequence of approximately 3.8 kb of rat genomic DNA located 5’ to

the prodynorphin mRNA CAP site. Figure 2 diagrams the location of potential

DNA regulatory elements near the promoter region of both the rat and the

human (Horikawa et al. 1983) prodynorphin genes, as identified by computer

sequence analysis.

For the rat prodynorphin gene, the nucleotide sequence TAGCGTCAG is

present in what constitutes the 5’ untranslated region of the transcript at

position +62. This element is highly homologous to the cyclic AMP (CAMP)

response element present in several neuroendocrine peptide genes (Goodman

1990). At position -102 is the sequence GCCAAT, representing the consensus

binding site for the enhancer protein, EBP20 (for review of trans-acting protein

factors, see Jones et al. 1988). Two potential AP-2 binding sites (AP-2

site=CCCCAGGC) are located at positions -973 and -1826; AP-2 is a 52-kD

protein that has been shown to mediate both phorbol ester and CAMPdependent transcriptional effects. The sequence GGGGCGG at position -1281

represents a potential binding site for the transcriptional activator protein, SP1.

Last, the sequence TGCGTCAG at position -1543 represents a sequence

identical to that found within the human proenkephalin promoter, referred to as

ENKCRE-2. This element is essential for both basal and regulated enhancer

function and binds the AP-1 complex with high affinity (Comb et al. 1988). The

same sequence element is also present at the human c-fos promoter, mediating

cell type-specific transcriptional properties (Velcich and Ziff 1990). The human

prodynorphin promoter also contains consensus binding sequences for AP-2,

SP1, and EBP20, although the sites are not present at the same location

relative to the mRNA CAP site when compared with the rat gene. However,

the nucleotide sequence from approximately -500 to +100 is highly conserved

between rat and human, suggesting that uncharacterized regulatory sequence

elements may be localized to this region.

To begin to determine if any of the aforementioned sequence elements are of

functional significance, various regions of the rat prodynorphin promoter have

been placed 5’ to the bacterial chloramphenicol acetyl transferase (CAT)

coding region and expressed transiently in CV1 cells. Analysis of several

different plasmid constructs is shown in figure 3. A minimal promoter fragment

representing nucleotides -122/+135 has relatively weak promoter activity, and

low levels of CAT activity are produced. This activity serves as a relative

reference point for analysis of other prodynorphin promoter/CAT plasmid

constructs. When the promoter region is extended in the 5’ direction to -598

118
FIGURE 2. Comparison of rat and human prodynorphin promoter regions. Pertinent restriction enzyme sites

are noted. Also noted are the locations of nucleotide sequences that have perfect homology with

characterized binding sites of specific DNA binding proteins. CAP sites, TATA elements, 5’ ends of

characterized cDNA clones, and exon/intron junctions are also shown for both genes. Lastly, the region

exhibiting the greatest degree of nucleotide sequence conservation between the two species is shaded.
FIGURE 3. Basal prodynotphin promoter CAT activity levels.

Characterization of the ability of rat prodynorphin genomic

DNA fragments to serve as transient transcriptional promoters.

Nucleotides representing 5’ and 3’ ends of rat prodynorphin

genomic DNA fragments (containing the mapped CAP site at

position +1, and the TATAAA promoter element at -28) ligated

proximal to the CAT structural gene are shown. The resulting

plasmids were transfected into CV1 cells, and cellular CAT

activity levels measured. Following standardization, CAT activity

levels were normalized against those for the minimal promoter

construct. The sequence and location of potential regulatory

elements are also noted.

or -930, again, low levels of CAT activity are observed. However, when the

promoter region is extended to -1250, -1504, or -1860, relative levels of CAT

activity are induced, 10-, 35-, and 60-fold, respectively. These data suggest

that positive transcription sequence elements, such as those described above,

are located in this region of genomic DNA. In addition, a positive transcription

sequence element appears to be present in the 5’ untranslated region of the

120
prodynorphin transcript between nucleotides +60 and +135, as there is an

approximately sixfold reduction in CAT activity when comparing the -1860/+135

promoter region to the -1860/+60 fragment. This positive effect may be

mediated through the cAMP-responsive element (CRE)-like element present

at +62. Thus, positive sequence elements both 5’ and 3’ to the CAP site may

act in concert to strengthen transcription from the promoter.

To begin to determine if positive-acting elements in the 5’ region of the promoter

can function outside of their natural context, the -1860/-1504 and -1504/-1250

fragments were placed in front of the minimal -122/+135 promoter. Surprisingly,

relative CAT activity levels were approximately 600- and 300-fold elevated,

respectively, when compared with -122/+135 CAT levels. Two possible

explanations for this observation are that negative transcription sequence

elements are present in the region of genomic DNA from -1250 to -122 or that

the spacing of the positive transcription elements relative to the promoter plays

a role in their efficacy.

HAIRPIN FORMATION WITHIN THE ENHANCER REGION OF THE HUMAN

PROENKEPHALIN GENE

The proenkephalin gene encodes the polyprotein precursor to the enkephalin

family of opioid peptides. The human proenkephalin gene promoter has been

extensively characterized (Comb et al. 1988), and a wide variety of DNA binding

proteins are able to interact with high degrees of affinity and specificity with the

promoter region from nucleotides -110 to -70. Site-specific mutational analysis

has determined that two specific sequence elements designated as ENKCRE-1

(-104 TGGCGTA -98) and ENKCRE-2 (-92 TGCGTCA -86) play a critical role

in the transduction of signals transmitted from cell surface receptors to the

proenkephalin nuclear transcription complex (Comb et al. 1988). Analysis of

the promoter region containing ENKCRE-1 and ENKCRE-2 reveals that these

elements are contained within a 23bp imperfect palindrome and that each

strand has the potential to form not only a duplex structure but also hairpin

structures (figure 4). A unique feature of these hairpin structures is that each

would form with mismatched base pairs: the top strand (GT) forming two GT

pairs and the complementary bottom strand (AC) forming two AC pairs.

The sequence shown in figure 4 was chemically synthesized to investigate

hydrogen bonding properties within each strand as well as the duplex

(McMurray et al. 1991). Melting experiments employing the single-stranded

oligonucleotides demonstrated that increases in temperature result in a highly

cooperative increase in absorbance, indicative of the large degree of base

unstacking found in hydrogen-bonded structures upon melting. At neutral

pH, 45 °C was the temperature at the midpoint for transition (tm) of the GT

121
FIGURE 4. Potential secondary structures formed by the human enkephalin

enhancer region. For the duplex structure, the position of

nucleotides relative to the endogenous CAP site are noted. The

shaded boxed sequences represent elements CRE-1 and CRE-2,

which constitute the cAMP-inducible enhancer. For the GT and

AC hairpin structures, the shaded boxes indicate the positions of

the mismatched base pairs.

SOURCE: McMurray et al. 1991. Hairpin formation within the enhancer region

of the human enkephalin gene. Copyright 1991 by Cynthia T.

McMurray (Rochester, MN).

strand, 49 °C for the AC strand, and 63 °C for the duplex. In addition, the

hyperchromicity for the melting of each strand was roughly half the value for

the melting of the duplex. Thus, in the absence of their partner strand, the

individual strands are capable of forming stable hairpin structures, despite

the fact that each hairpin forms with two mismatched base pairs.

122
To determine the possibility of induction of structural transitions within the

human enkephalin enhancer, the conformational state of the native enhancer

duplex has been examined under a variety of conditions. A G-50 column

chromatography/nondenaturing PAGE system has served as the means

of analysis of column-purified, radiolabeled oligonucleotides (McMurray et

al. 1991), as shown in figure 5. At a solution pH of 7.0, the double-stranded

enhancer duplex (first lane noted as D) is easily distinguished from the

single-stranded forms (the AC hairpin is sample AC; the GT hairpin is sample

GT) by its greatly reduced mobility. In addition, the single-strand forms are

observed as doublets, with the faster migrating form corresponding to the

hairpin conformation and the slower migrating form corresponding to the

linear strand conformation (C.T. McMurray, unpublished results). In solution,

the duplex form of the 23bp human enkephalin enhancer is able to dissociate

into the AC and GT single-strand forms by altering the pH; lane E represents

radiolabeled enhancer duplex in pH 5.5 buffer analyzed on a pH 7.0

polyacrylamide gel. Under conditions in which the solution pH is raised to

7.0, the oligonucleotides once again migrate as a double-stranded duplex form,

as shown in sample lane D. This pH-dependent conformation/migration pattern

is presumably due to the ability to switch from double-stranded duplex to

single-stranded hairpin conformations, as a nonpalindromic 23mer continues

to migrate as a duplex under pH 5.5 conditions (sample lane C). Thus,

incubation of the enkephalin enhancer oligonucleotide duplex in pH 5.5

buffer results in complete conversion of the duplex form to the single-strand

forms. Furthermore, the process is reversible, as the single-stranded forms

can convert to the duplex form by adjusting the solution pH to 7.0. It is also

noteworthy that the lowest pH condition employed in the study is well above

that where significant base protonation generally occurs and is well within the

pH range where nucleic acid duplexes are stable.

To further examine the nature of this pH effect, the stability of the enhancer

duplex, as well as both of the individual strands that constitute the duplex,

was measured as a function of pH (figure 6) (McMurray et al. 1991). Stability

was determined by observing the midpoint of the thermal transition (t m) for

each conformation. For the native enhancer duplex (open squares), thermal

stability varies only 5 °C over the pH range from 5.5 to 9.0. The duplex exhibits

maximal stability in the pH range from 7.0 to 9.0. The GT hairpin is also

quite stable over the same pH range; from pH 5.0 to 9.0 only a 5 °C change in

tm is observed. In marked contrast, however, the stability of the AC hairpin

displays the opposite pH-dependent stability profile relative to both the duplex

and the GT strand. The AC strand undergoes a 30 °C increase in stability as

the pH decreases from 9.0 to 5.5. At pH 9.0, conditions under which both

the duplex and the GT strand are most stable, the AC strand is essentially

denatured. Between pH 6.0 and 5.5, the stability of the AC hairpin is actually

123
FIGURE 5. PAGE analysis of G-50 column fractions for the GT and AC

strand, a nonpalindromic 23mer duplex, and the human

enkephalin enhancer duplex. In the left three lares are standards

representing the duplex (D), the AC hairpin (AC). and the GT

hairpin (GT). Lane A, GT strand in pH 5.5 buffer analyzed on a

pH 7.0 gel (1x10-8M base pairs); lane B, AC strand in pH 5.5

buffer analyzed on a pH 7.0 gel (1.5x10-8M base pairs); lane C, a

nonpalindromic 23mer duplex in pH 5.5 buffer analyzed on a pH

5.5 gel (1.3x10-8M base pairs); lane D, the human enkephalin

enhancer duplex in pH 7.0 buffer analyzed on a pH 7.0 gel

(1.8x10-8M base pairs); lane E, the human enkephnlin enhancer

duplex in pH 5.5 buffer analyzed on a pH 7.0 gel (1.8x10-8M base

pairs): lane F, the human enkephalin enhancer duplex in pH 5.5

buffer analyzed on a pH 5.5 gel (1.8x10-4M base pairs).

SOURCE: McMurray et al. 1991. Hairpin formation within the enhancer region

of the human enkephalin gene. Copyright 1991 by Cynthia T.

McMurray (Rochester, MN).

124
FIGURE 6.

SOURCE:

pH dependence of melting transitions for the human enkephalin

enhancer duplex the GT strand and the AC strand

T°C represents the temperature at the midpoint for transition.

McMurray et al. 1991. Hairpin formation within the enhancer region

of the human enkephalin gene. Copyright 1991 by Cynthia T.

McMurray (Rochester, MN).

125
greater than that of the double-stranded duplex. Thus, under slightly acidic

conditions, the energy difference between the duplex and both of the hairpin

forms is small, and under low concentration conditions (data not shown), the

hairpin conformation becomes the favored form.

The dramatic pH-dependent increase in stability of the AC strand arises in

part from protonation (figure 7), with additional hydrogen bonding presumably

occurring from protonation of the N1 of adenine within the AC mismatched pair.

From the pH titration curve shown in figure 6, the midpoint for protonation of

the AC strand is near physiological pH of 7.2. The importance of measuring

the pKa is that it allows identification of the relevant range of protonation.

The AC strand is approximately 80 percent protonated at pH 6.6 and 20

percent protonated at pH 7.8. Thus, changes of only 0.5 of a pH unit create

conditions under which the AC hairpin is either largely destabilized or highly

stable. A corresponding pKa shift of this magnitude for protonation of ring

nitrogens could be easily mediated by the presence of a nearby charged

group, such as local interaction of a charged protein containing regions of

acidic or phosphorylated residues. In vivo, the presence of a nearby positive

charge may be the driving force to induce the formation of a cruciform structure

at this specific region of the enkephalin enhancer. Thus, proton transfer and

stabilization of the AC hairpin, at the expense of duplex formation, may be a

biological switch for the formation of a cruciform structure.

Whether this transition has subsequent effects on the binding of additional

trans-acting factors or polymerase subunits that control transcription from the

proenkephalin promoter remains to be determined. There is, however, an

intriguing piece of information that suggests the functional significance of the

ability to form stable cruciform structures. Extensive site-specific mutational

analysis of the human enkephalin enhancer region (Comb et al. 1988) has

identified two mutations within ENKCRE-1 and ENKCRE-2 that stimulate

approximately twofold basal levels of transcription from the promoter, as well

as retaining a high degree of inducibility by CAMP. For one of these mutations,

specific patterns of protein binding have been characterized and appear to be

unaffected. Interestingly, both mutations serve to make perfect base pairs out

of the mismatched pairs. Thus, mutations that potentially stabilize the cruciform

structure, while having no apparent effect on specific DNA-protein interactions,

result in increased expression in vivo.

It is also noteworthy that other cAMP-responsive genes contain CREs within or

near sequences capable of forming imperfect palindromes (figure 8) (McMurray

et al. 1991). Furthermore, many of these regulatory regions have the potential

to form cruciform structures, with AC or GT mismatches representing the major

126
FIGURE 7. Schematic diagram of the hydrogen binding pattern of an AT

base pair and an AC mismatch at pH 7.0 and 5.5. The AC

mismatch pair contains one hydrogen bond at pH 7.0 and binds

an additional proton under acidic conditions. Protonation can

occur at the N1 of adenine, giving rise to an additional hydrogen

bond.

SOURCE: McMurray et al. 1991. Hairpin formation within the enhancer region

of the human enkephalin gene. Copyright 1991 by Cynthia T.

McMurray (Rochester, MN)

127
FIGURE 8. Model for cruciform structures that can potentially form at or near

the CREs of several genes. Both linear and potential cruciform

structures are shown for the top strand only. Hatched boxes

represent characterized CREs for each gene. In the cruciform

conformation, the horizontal boxes indicate the position of base

pair mismatches. Henk, human enkephalin gene; VIP, human

vasoactive intestinal peptide gene; CRH, rat corticotropinreleasing hormone gene; PEPCK, rat phosphoenolpyruvate

carboxykinase gene; TH, rat tyrosine hydroxylase gene.

SOURCE: McMurray et al. 1991. Hairpin formation within the enhancer region

of the human enkephalin gene. Copyright 1991 by Cynthia T.

McMurray (Rochester, MN).

128
species of mismatch base pairing. Thus, formation of cruciform structures may

be a general feature of certain classes of CREs. This observation, along with

the unique physical and thermodynamic properties of the enkephalin enhancer

region, suggest a model in which protein-mediated structural changes within the

enhancer region DNA play an active role in regulated expression of the human

proenkephalin gene via the formation of a cruciform structure. The main

challenge for the future will be to determine if such a cruciform structure is

capable of forming in vivo when the enhancer region is placed in the context of

nuclear DNA.

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prepyriform cortex kindling differentially alters the levels of prodynorphin

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ACKNOWLEDGMENTS

The studies described were supported in part by National Institute on Drug

Abuse grant DA-04154 (JD) and by postdoctoral fellowship PF-2997 from the

American Cancer Society (CTM).

AUTHORS

Cynthia T. McMurray, Ph.D.

Assistant Professor/Senior Associate Consultant

Departments of Pharmacology and Biochemistry/Molecular Biology

Mayo Foundation

Rochester, MN 55905

Karen Pollock, M.S.

Senior Research Assistant

James Douglass, Ph.D.

Scientist

Vollum Institute-L474

Oregon Health Sciences University

3181 Southwest Sam Jackson Park Road

Portland, OR 97201

131
The Prohormone and Proprotein

Processing Enzymes PC1 and PC2:

Structure, Selective Cleavage of

Mouse POMC and Human Renin at

Pairs of Basic Residues, Cellular

Expression, Tissue Distribution,

and mRNA Regulation

Nabil G. Seidah, Robert Day, Suzanne Benjannet,

Normand Rondeau, Alain Boudreault, Timothy Reudelhuber,

Martin K.-H. Schafer, Stanley J. Watson, and Michel Chrétien

INTRODUCTION

Limited proteolysis of precursors at specific pairs of basic residues and/or

at single basic amino acids is a widespread mechanism by which the cell

expresses a repertoire of biologically active proteins and peptides (Lazure

et al. 1983; Mains et al. 1990). Until recently, the tissue-specific and

developmentally regulated proteinases responsible for such conversions

were not identified at the molecular level. The cloning and cellular expression

of the yeast Kex2 gene product demonstrated that this enzyme belongs to

the Ca2+-dependent subtilisin family of serine proteinases (Julius et al. 1984;

Fuller et al. 1989a) and that it exhibits exquisite selectivity of cleavage at pairs

of basic residues in several yeast (Fuller et al. 1988) and mammalian (Bathurst

et al. 1987; Thomas et al. 1988; Foster et al. 1991) precursors. Furthermore,

the ability of a yeast enzyme to cleave mammalian precursors both in vitro

and in vivo reinforced the hypothesis that Kex2 represents a prototype for a

mammalian subtilisin-like proteinase(s) physiologically responsible for the

cleavage of proproteins at selected pairs of basic residues. The search for

the homologous mammalian convertases led to the recent identification and

molecular cloning of three distinct proteins. These were called furin (Roebroek

et al. 1986; Fuller et al. 1989b; Van den Ouweland et al. 1990), PC1 (Seidah et

al. 1990, 1991a; Smeekens et al. 1991, who also called this enzyme PC3), and

PC2 (Smeekens and Steiner 1990; Seidah et al. 1990).

132
CHROMOSOMAL ASSIGNMENT

The chromosomal localization of the genes coding for the convertases PC1,

PC2, and furin has been achieved by in situ hybridization of both human and

mouse metaphase spreads (Seidah et al. 1991a, 1991b). The data showed

that in both species these three genes are not synthenic. Thus PC1, PC2,

and furin were located on human chromosomes 5q15-21, 20p11.1-11.2, and

15q25-26 and on mouse chromosomes 13 [C1-C3 band], 2 [F3-H2 region],

and 7 [D1-E2 region], respectively. Genetic linkage analysis of each gene

in the mouse demonstrated that PC1, PC2, and furin loci map close to the

G protein Nras-2, homeobox Pax-1, and fes/fps oncogene Fes (Copeland

et al., in press).

STRUCTURAL ANALYSIS

The comparative architectural features of the subtilisin-like proprotein

convertases reported so far and those of the subtilisins are presented in

figure 1. It is apparent that PC1 , PC2, and Kex2 contain disulphide bridges

only within their catalytic domain, whereas furin also exhibits a Cys-rich

segment before the putative transmembrane domain (TMD) and two other Cys

residues within the TMD and the cytosolic C-terminal segment, respectively.

The glycosylation pattern in the mammalian members shows that they each

contain three N-glycosylation sites, and unlike Kex2, none appears within the

putative prosegment. This figure also shows in the unshaded area a segment

(called P-domain) that has been reported to be important for the folding of the

proteinase and the elaboration of the full enzymatic activity of Kex2 (Fuller et

al. 1991). Finally, although the structures of PC1 and PC2 do not predict the

presence of a transmembrane domain, their C-terminal segments code for an

amphipathic structure that could interact with membranes in a pH-dependent

manner, in a similar fashion to the properties of the soluble carboxypeptidase

E (Fricker et al. 1990).

Figure 2 depicts the conservation of sequences around the active sites Asp*,

His*, and Ser* as well as the catalytically important Asn* residues of some

subtilisins and of all the members of the mammalian subtilisin family reported

so far. Aside from the conserved residues in the family (boxed in), certain

residues (inverted triangles) are found only in the mammalian subtilisin family,

and within these, three of them (Arg, Ser, and Ala, shown in bold) are found

only in the Kex2-like convertases and not in the recent structure reported for

the human tripeptidyl peptidase II (hTPP II) (Tomkinson and Jonsson 1991).

Finally, only in PC2 is an Asp* found instead of the usual Asn* residue. This

interesting single point mutation of PC2 (but not PC1 or furin) may have an

important bearing on the catalytic efficiency (kcat/Km) of this proteinase, since

133
FIGURE 1. Schematic diagram depicting the various domains of the Kex2-like

enzymes and their comparison to the primitive subtilisin BPN

enzyme. The number of amino acids (# a.a.) in each primary

translation product is shown on the right. The diagram also points

out the positions of the active sites Asp*(D), His*(H), and Ser*(S)

in each enzyme.

it has been reported that this Asn* residue forms a hydrogen bond with the

developing substrate carbonyl oxygen anion in the transition state complex

(Robertus et al. 1972). This interaction was shown to be important, but not

essential, for the stabilization of this oxyanion hole in subtilisins (Bryan et al.

1986). From these data and the sequence similarity of the catalytic domains in

these various enzymes, it becomes apparent that hTPP II is much closer to

subtilisin BPN’ than to any other member of the Kex2-like proteinases. This

suggests a possible classification of hTPP II as a mammalian degradative

enzyme, rather than a Kex2-like specific proprotein convertase, in agreement

with its observed broad substrate specificity (Bålöw et al. 1986).

134
FIGURE 2. Conservation of the sequences around the Asp*, His*, and Ser* of the active sites found in the subtilisins

(Moehle et al. 1987), Kex2 (Mizuno et al. 1988), furin (Van den Ouweland et al. 1990), mPC1 (Seidah et

al. 1991a; Smeekens et al. 1991), mPC2 (Seidah et al. 1990), and hTPP II (Tomkinson and Jonsson

1991). Boxed amino acids, inverted triangles, and bold residues represent invariant residues, amino

acids found only in the eukaryotic members of the family, and conserved amino acids found only in the

Kex2-like members of the eukaryotic subfamily, respectively.
CLEAVAGE SPECIFICITY OF PC1 AND PC2

To investigate the cleavage specificity of PC1 and PC2 the authors used two

different precursors as substrate: mouse proopiomelanocortin (POMC) and

human renin. Vaccinia virus recombinants of each enzyme were used to

coexpress these proteinases with the prosubstrates in several cell lines.

Following their purification, the intracellular and secreted peptide products

were unambiguously characterized by microsequence.

Vaccinia virus recombinants of the mouse prohormone convertases PC1

and PC2 were coexpressed together with mouse POMC in the constitutively

secreting cells, BSC-40, and in the endocrine-derived cell lines PC12 and

AtT-20, which exhibit regulated secretion. Figure 3 summarizes the cleavage

selectivity of PC1 and PC2 as deduced from the monitoring of the POMC

processing. The data demonstrated the distinct cleavage specificities of

PC1 and PC2, since in the cell lines analyzed it was found that (1) PC1

cleaves POMC into adrenocorticotropic hormone (ACTH) and ß-lipotropin;

(2) PC2 cleaves POMC into biologically active ß-endorphin, an N-terminally

extended ACTH containing the joining peptide, and into either -melanocytestimulating hormone (MSH) or des-acetyl -MSH; and (3) PC2 cleaves POMC

at the five pairs of basic residues analyzed, whereas PC1 cleaves preferentially

two of them, suggesting that PC2 has a broader spectrum of activity than

PC1. These data are consistent with the proposed hypothesis concerning the

physiological role of PC1 and PC2 as distinct proprotein convertases acting

alone or together to produce a set of tissue-specific maturation products both in

the brain and in peripheral tissues (Seidah et al. 1990, 1991a). Interestingly,

since POMC processing can also occur in cells devoid of a regulated pathway

of secretion, these data also show that POMC processing by either PC1 or

PC2 is not dependent on the presence of secretory granules.

Recent data from our laboratory showed that neither PC1 nor PC2 can

process human prorenin in a Chinese Hamster Ovary constitutively secreting

cell line (data not shown). To test the hypothesis that, unlike POMC, human

prorenin processing might require the presence of secretory granules, a

somatomammotroph GH4-C1 cell line, which expresses a stable transfectant

of human prorenin, was infected with PC1 or PC2 vaccinia virus recombinants.

As shown in figure 4, the results of analysis of the secreted renin activity

demonstrated that only PC1 can activate human prorenin in this cell line and

that neither the endogenous furin nor the exogenous PC2 can activate this

zymogen. Pulse chase analysis of the prorenin maturation product confirmed

that PC1 cleaved human prorenin at the expected LysArg-zymogen activation

site, thereby releasing the N-terminal 45 amino acids prosegment (not shown).

These results point out that not all precursors can be processed efficiently in the

136
FIGURE 3. Major end products of POMC processing by PC1 and PC2. The

arrows represent the cleavage sites following pairs of basic

residues. The dark triangles tally the cleavage sites by each

enzyme. The numbers represent the start and end positions of

the processed peptides based on the mouse POMC sequence.

The N- and O-glycosylation (CHO) sites as well as the pairs of

basic residues are emphasized.

SOURCE: Benjannet et al. 1991

absence of secretory granules and that each case has to be carefully analyzed

before general conclusions can be reached. Furthermore, of the three enzymes

studied, only PC1 is capable of efficient processing of human prorenin. Neither

PC2 nor furin (as shown by work described in this chapter and in Hatsuzawa et

al. 1991) can be shown to activate this zymogen.

BACULOVIRUS EXPRESSION OF PC1 AND PC2

To study the physicochemical and kinetic properties of the convertases

PC1 and PC2 in vitro, we sought to obtain large quantities of each enzyme

using the baculovirus expression system. Recombinant viruses encoding

mouse PC1 and PC2 were obtained using the pJV Nhe I transfer vector

and by a procedure similar to that previously published (Vialard et al. 1990).

Expression of each enzyme was monitored by polyacrylamide electrophoresis

137
FIGURE 4. Human prorenin activation by PC1. A stable transfectant of GH4

cells expressing human prorenin (N.I.=noninfected cells) was

infected with either 5 pfu of the recombinant vaccinia virus

VV:PC1, VV:PC2 or the wild type vaccinia virus VV:WT

(Benjannet et al. 1991). The secreted renin activity was

measured before (active renin) and after (total renin) trypsin

treatment (15 μg for 1 hour at room temperature) by an indirect

assay using human angiotensinogen as a substrate for active

renin and a radioimmunoassay directed against angiotensin I.

The amount of prorenin was calculated from the difference

between the renin activity measured after and before trypsin

treatment.

on SDS/PAGE. Surprisingly, the enzymes produced were not secreted from

the Sf9 cells but, rather, remained intracellular. The proteins were found to

be produced in quantities exceeding 1 mg/L of culture, were associated with

membrane components within the cell, and were recovered by centrifugation

of the cell lysates in the particulate fraction. On SDS/PAGE, PC1 and PC2

migrate with an apparent molecular weight of 87,000 and 74,000 daltons

(not shown). To investigate the enzyme activity of each proteinase, we

selected to affinity label the membrane fraction containing either enzyme

with a pentapeptide chloromethyl ketone 125I-[D-Tyr]GluPheLysArg-COCH2CI,

of which the GluPheLysArg sequence represents the ACTH/ß-lipotropic

hormone junction (Cromlish et al. 1986), which was shown to be cleaved by

either PC1 or PC2 (Benjannet et al. 1991). As shown in figure 5, both PC1

138
FIGURE 5. Labeling of the cellular extract of baculovirus-expressed PC1 and

PC2 with the affinity label [

125I-D-Tyr]GluPheLysArg-COCH2CI.

The labeled proteins migrate exactly at the position of the

Coomassie-colored PC1 (87 kD) and PC2 (67 kD), suggesting

that these proteins are at least partially active proteinases. This

labeling works both at acidic (pH 5.5) and neutral (pH 7.0) pH

conditions, and no labeling is seen if the enzyme preparation is

first incubated with 1 mM diisopropyl fluorophosphate.

139
and PC2 could be specifically labeled with this pentapeptide, and the labeled

proteins migrate with a similar molecular weight as the unlabeled proteins

stained by Coomassie Blue. However, the specific radioactivity associated

with the labeled proteins were found to be much inferior to that obtained with a

similar amount of active trypsin or plasma kallikrein (N.G. Seidah, unpublished

data). This suggested that either the proteins were synthesized mostly as

inactive enzymes in the Sf9 cells or that the presumed prosegment (see figure

1) was not cleaved in this system. The NH2-terminal sequence analysis of each

protein eluted from the SDS/PAGE gel was found to be KRQFVNEWAAEIPGG

and ERPVFTNHFLVELHK for mPC1 and mPC2, respectively (figure 6). Aside

from confirming the predicted signal peptidase cleavage site in each proteinase

(Seidah et al. 1991a), these data revealed that even though both intracellular

enzymes retained their N-terminal prosegment (figure 6), they retain partial

binding activity toward the pentapeptide chloromethyl ketone, which selectively

labels the His* at the active site of each enzyme (Cromlish et al. 1986).

Efforts are now under way to understand the reason for the absence of

zymogen activation of PC1 and PC2 in the Sf9 cells, which, under similar

conditions, express the active form of the Kex2 proteinase with the concomitant

removal of the prosegment (figure 5) (Germain et al. 1992). A possible

explanation is that, unlike Kex2 and the subtilisins, PC1 and PC2 might not

be capable of autoactivation but, rather, require the presence of another

enzyme to start the zymogen activation process.

PC1 AND PC2 IN THE RAT PITUITARY AND BRAIN

In situ hybridization studies in the mouse central nervous tissues and in

peripheral organs demonstrated that PC1 and PC2 transcripts are mostly

found in endocrine and neuroendocrine tissues and cells (Seidah et al.

1990, 1991a). In contrast, furin was found to be widely distributed both in

endocrine and in nonendocrine tissues and cells (Schalken et al. 1987).

Figure 7 depicts the comparative in situ hybridization of PC1 and PC2 in

the rat pituitary. PC1 transcripts are found in all endocrine cells of the

intermediate lobe and in a majority of pituitary anterior lobe endocrine cells.

PC2 mRNA is highly abundant in the intermediate lobe, with much smaller

amounts found in the anterior lobe. A similar conclusion can also be drawn

from Northern blots (figure 7), which show the relative amounts of PC1 and

PC2 in both the anterior and the neurointermediate lobes of the pituitary. In

the rat, two transcripts are found for both enzymes at about 3 and 5 kb (figure

7), as previously reported in mouse PC1 and PC2 (Seidah et al. 1990, 1991a;

Smeekens et al. 1991) and human PC2 (Smeekens and Steiner 1990).

The difference between these two mRNAs is not yet fully understood, but

preliminary data indicate that the longer forms (5 kb) do not contain an

extended coding region for each enzyme (N.G. Seidah, unpublished data).

140
FIGURE 6. Alignment of the N-terminal segments of the mammalian Kex2-like enzymes. The inverted dark

triangles point to the predicted positions of the signal peptidase cleavage sites, based on von Heijne

(1986) criteria and confirmed by microsequence analysis of the baculovirus-expressed forms of mPC1

and mPC2 (underlined residues). The computer alignment of the prosegments delineates two regions

(shown in bold) where the presumed multibasic cleavage sites exactly align in all cases. By analogy to

the position of the prosegment cleavage site in Kex2 (Wilcox and Fuller 1991), the arrows point to the

probable equivalent site in the other members of the family.
FIGURE 7. (Left) Autoradiography of the rat pituitary distribution of PC1 and

PC2 by in situ hybridization. (Right) Northern gel analysis of PC1

and PC2 mRNA transcripts found in total RNA of anterior (AP;

10 μg) and neurointermediate (NIL; 4 μg) pituitary lobes. X-ray

exposures limes are 4 and 16 hours for PC2 and PC1,

respectively. Markers are in kilobases.

Figure 8 depicts a representative photomicrograph of the distribution of PC1

and PC2 mRNA in a coronal section of the rat brain by in situ hybridization.

It is clear that these enzymes exhibit distinct distribution patterns in the central

nervous system. In general, PC1 has a more restricted distribution, whereas

PC2 is widespread. PC1 mRNA is most highly abundant in the hypothalamic

nuclei such as the supraoptic, paraventricular, and suprachiasmatic nuclei.

142
FIGURE 8. Autoradiography of the rat brain distribution of PC1 and

PC2 by in situ hybridization. CTX=cortex; HC=hippocampus;

PVN=paraventricular nucleus; SON=supraoptic nucleus;

SCN=suprachiasmatic nucleus; Hb=habenula; TH=thalamus;

STR=striatum; Pir=piriform cortex.

143
The habenula and the hippocampus represent extrahypothalamic areas rich in

PC1. Although present throughout the hippocampus, PC1 is expressed with

high abundance in the dentate gyrus. PC1 is also more abundant in the deeper

cortical layers compared with the superficial ones. On the other hand, PC2 is

widely expressed in many brain areas, the most striking example of which is the

thalamus, whose numerous subnuclei all appear to express intermediate to high

levels of PC2. The thalamic distribution of PC1 is much more restricted. PC2 is

also moderately abundant in both the superficial and deeper cortical layers as

well as in the striatum. In the hippocampus, PC2 is found mainly in the CA1,

CA2, and CA3 regions and, to a much lesser extent, in the dentate gyrus.

These unique localization patterns of PC1 and PC2 indicate the differential

roles of these convertases in the brain and may have some significance in

terms of understanding tissue-specific posttranslational processing.

DOPAMINERGIC REGULATION OF PC1 AND PC2 mRNAs IN PITUITARY

PARS INTERMEDIA

PC1 and PC2 are expressed in all pituitary intermediate lobe cells (figure 7).

The rat intermediate lobe melanotrophs, a highly homogeneous cell population,

are highly abundant in POMC and are under inhibitory dopaminergic control.

Figure 9 shows a representative experiment demonstrating the up-regulation

of PC2 in the neurointermediate lobes of rats chronically treated with the

dopaminergic antagonist haloperidol. Also shown is the down-regulation

effect of the dopaminergic agonist bromocryptine on PC2 mRNA in the

neurointermediate lobes of chronically treated rats. Parallel effects were

observed for PC1 mRNA. In the present study, POMC mRNA levels also

increased with haloperidol and decreased with bromocryptine treatments

(data not shown), as previously reported (Chen et al. 1983). Accordingly, in

intermediate lobe melanotrophs, the authors’ data demonstrate the coregulation

of the gene expression of POMC with that of PC1 and PC2.

CONCLUSION

The data presented in this chapter show that PC1 and PC2 are distinct

mammalian subtilisin-like proteinases that originate from two different genes

and that are expressed in neuronal and endocrine tissues in a discrete fashion.

Even though both enzymes cleave POMC at specific pairs of basic residues,

only PC1 can activate human prorenin in cells containing secretory granules.

In the brain, PC2 is generally more abundant and more widely expressed

than PC1. In the pituitary, PC2 mRNA is more abundant than PC1 in the

intermediate lobe, whereas the reverse is true in the anterior lobe. Similar

to POMC, in the intermediate lobe of the pituitary both enzymes are under

144
FIGURE 9. Northern gel analysis of total RNA (2 μg per lane) of

neurointermediate lobes of rats chronically treated with the

dopaminergic antagonist haloperidol (CNT=vehicle; HAL 1=5.0

mg/kg; HAL 2=10 mg/kg) and the dopaminergic agonist

bromoctyptine (CNT=vehicle; BC 1=2.0 mg/kg; BC 2=10 mg/kg).

X-ray exposure times are 4 and 16 hours for PC2 and PC1,

respectively. Markers are in kilobases.

145
negative dopaminergic control. The data from the baculovirus-expressed

enzymes suggest that the zymogen form of PC1 and PC2 is only partially

active and that this form is not secretable. Recent pulse chase experiments

demonstrated that the intracellular activation of PC1 is more efficient than that

of PC2 and that neither enzyme can undergo autocatalytic activation (Benjannet

et al. 1992). Therefore, the zymogen activation of PC1 and PC2 may require

the presence of yet another enzyme, which together with PC1 and PC2 will

fashion in a tissue-specific manner the final molecular products generated from

a given proprotein.

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ACKNOWLEDGMENTS

This chapter was prepared with support from Medical Research Council of

Canada grant PG-2, Mt11268 and from J.A. de Sève Succession.

AUTHORS

Nabil G. Seidah, Ph.D.

Full Professor

Robert Day, Ph.D.

Assistant Professor

Suzanne Benjannet, M.Sc.

Assistant Researcher

Normand Rondeau, M.Sc.

Technician

Alain Boudreault, B.Sc.

Timothy Reudelhuber, Ph.D.

Associate Professor

Michel Chrétien, O.C., M.D.

Scientific Director and Chief Executive Officer

Clinical Research Institute of Montreal

Affiliated to I’Université de Montreal

110 Pine Avenue West

Montreal, Quebec H2W 1R7

CANADA

Martin K.-H. Schafer, M.D.

Assistant Professor

Anatomisches lnstitut

Johannes Gutenberg-Universitat

D-6500 Mainz

GERMANY

149
Stanley J. Watson, M.D., Ph.D.

Professor

Mental Health Research Institute

205 Washtenaw Place

Ann Arbor. MI 48109

150
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93 AIDS AND INTRAVENOUS DRUG USE: FUTURE DIRECTIONS FOR

COMMUNITY-BASED PREVENTION RESEARCH. C.G. Leukefeld, D.S.W.;

R.J. Battjes, D.S.W.; and Z. Amsel, D.Sc., eds.

GPO Stock #017-024-01388-9 $10 NTIS PB #90-148941/AS $39

94 PHARMACOLOGY AND TOXICOLOGY OF AMPHETAMINE AND

RELATED DESIGNER DRUGS. Khursheed Asghar, Ph.D., and Errol De

Souza, Ph.D., eds.

GPO Stock #017-024-01386-2 $11 NTIS PB #90-148958/AS $39

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.

GPO Stock #017-024-01399-4 $21 NTIS PB #90-237660/AS $67

96 DRUGS OF ABUSE: CHEMISTRY, PHARMACOLOGY, IMMUNOLOGY,

AND AIDS. Phuong Thi Kim Pham, Ph.D., and Kenner Rice, Ph.D., eds.

GPO Stock #017-024-01403-6 $8 NTIS PB #90-237678/AS $31

97 NEUROBIOLOGY OF DRUG ABUSE: LEARNING AND MEMORY. Lynda

Erinoff, Ph.D., ed.

GPO Stock #017-024-01404-4 $8 NTIS PB #90-237686/AS $31

98 THE COLLECTION AND INTERPRETATION OF DATA FROM HIDDEN

POPULATIONS. Elizabeth Y. Lambert, M.S., ed.

GPO Stock #017-024-01407-9 $4.75 NTIS PB #90-237694/AS $23

99 RESEARCH FINDINGS ON SMOKING OF ABUSED SUBSTANCES.

C. Nora Chiang, Ph.D., and Richard L. Hawks, Ph.D., eds.

GPO Stock #017-024-01412-5 $5 NTIS PB #91-141119 $23

154
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.

GPO Stock #017-024-01458-3 $8

101 RESIDUAL EFFECTS OF ABUSED DRUGS ON BEHAVIOR. John W.

Spencer, Ph.D., and John J. Boren, Ph.D., eds.

GPO Stock #017-024-01426-7 $6 NTIS PB #91-172858/AS $31

102 ANABOLIC STEROID ABUSE. Geraline C. Lin, Ph.D., and Lynda Erinoff,

Ph.D., eds.

GPO Stock #017-024-01425-7 $8 NTIS PB #91-172866/AS $31

103 DRUGS AND VIOLENCE: CAUSES, CORRELATES, AND

CONSEQUENCES. Mario De La Rosa, Ph.D.; Elizabeth Y. Lambert, M.S.;

and Bernard Gropper, Ph.D., eds.

GPO Stock #017-024-01427-3 $9 NTIS PB #91-172841/AS $31

104 PSYCHOTHERAPY AND COUNSELING IN THE TREATMENT OF

DRUG ABUSE. Lisa Simon Onken, Ph.D., and Jack D. Blaine, M.D., eds.

GPO Stock #017-024-01429-0 $4 NTIS PB #91-172874/AS $23

105 PROBLEMS OF DRUG DEPENDENCE, 1990: PROCEEDINGS OF THE

52ND ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS

OF DRUG DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01435-4 $22

106 IMPROVING DRUG ABUSE TREATMENT. Roy W. Pickens, Ph.D.; Carl

G. Leukefeld, D.S.W.; and Charles R. Schuster, Ph.D., eds.

GPO Stock #017-024-01439-7 $12 NTIS PB #92-105873

Paperback $50 Microfiche $19

107 DRUG ABUSE PREVENTION INTERVENTION RESEARCH:

METHODOLOGICAL ISSUES. Carl G. Leukefeld, D.S.W., and William J.

Bukoski, Ph.D., eds.

GPO Stock #017-024-01446-0 $9 NTIS PB #92-160985

Paperback $35 Microfiche $17

108 CARDIOVASCULAR TOXICITY OF COCAINE: UNDERLYING

MECHANISMS. Pushpa V. Thadani, Ph.D., ed.

GPO Stock #017-024-01446-0 $7 NTIS PB #92-106608

Paperback $35 Microfiche $17

155
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.

GPO Stock #017-024-01445-1 $4.50 NTIS PB #92-106616

Paperback $26 Microfiche $12.50

110 THE EPIDEMIOLOGY OF COCAINE USE AND ABUSE. Susan Schober,

Ph.D., and Charles Schade, M.D., M.P.H., eds.

GPO Stock #017-024-01456-7 $11 NTIS PB #92-14624-0

Paperback $43 Microfiche $17

111 MOLECULAR APPROACHES TO DRUG ABUSE RESEARCH VOLUME I:

RECEPTOR CLONING, NEUROTRANSMITTER EXPRESSION, AND

MOLECULAR GENETICS. Theresa N.H. Lee, Ph.D., ed.

Not for sale at GPO NTIS PB #92-135743

Paperback $35 Microfiche $17

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.

GPO Stock #017-024-01455-9 $11

113 ECONOMIC COSTS, COST-EFFECTIVENESS, FINANCING, AND

COMMUNITY-BASED DRUG TREATMENT. William S. Cartwright, Ph.D.,

and James M. Kaple, Ph.D., eds.

Not for sale at GPO NTIS PB #92-155795

Paperback $35 Microfiche $17

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.

GPO Stock #017-024-01459-1 $12 NTIS PB #92-146216

Paperback $43 Microfiche $17

115 METHAMPHETAMINE ABUSE: EPIDEMIOLOGIC ISSUES AND

IMPLICATIONS. Marissa A. Miller, D.V.M., M.P.H.. and Nicholas J. Kozel, M.S.,

eds.

GPO Stock #017-024-01460-5 $4

116 DRUG DISCRIMINATION: APPLICATIONS TO DRUG ABUSE

RESEARCH. Richard A. Glennon, Ph.D.; Torbjörn U.C. Järbe, Ph.D.; and

Jerry Frankenheim, Ph.D., eds.

GPO Stock #017-024-01470-2 $13

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117 METHODOLOGICAL ISSUES IN EPIDEMIOLOGICAL, PREVENTION,

AND TREATMENT RESEARCH ON DRUG-EXPOSED WOMEN AND THEIR

CHILDREN. M. Marlyne Kilbey, Ph.D., and Khursheed Asghar, Ph.D., eds.

GPO Stock #017-024-01472-9 $12

118 DRUG ABUSE TREATMENT IN PRISONS AND JAILS. Carl G.

Leukefeld, D.S.W., and Frank M. Tims, Ph.D., eds.

GPO Stock #017-024-01473-7 $16

119 PROBLEMS OF DRUG DEPENDENCE 1991: 53RD ANNUAL

SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01474-5 $22

120 BIOAVAILABILITY OF DRUGS TO THE BRAIN AND THE BLOODBRAIN BARRIER. Jerry Frankenheim, Ph.D., and Roger M. Brown, Ph.D., eds.

121 BUPRENORPHINE: AN ALTERNATIVE TREATMENT FOR OPIOID

DEPENDENCE. Jack D. Blaine, Ph.D., ed.

122 RESEARCH METHODS IN WORKPLACE SETTINGS. Helen Axel, M.A.,

and Dennis J. Crouch, M.B.A., eds.

123 ACUTE COCAINE INTOXICATION: CURRENT METHODS OF

TREATMENT. Heinz Sorer, Ph.D., ed.

124 NEUROBIOLOGICAL APPROACHES TO BRAIN-BEHAVIOR

INTERACTION. Roger M. Brown, Ph.D., and Joseph Frascella, Ph.D., eds.

125 ACTIVATION OF IMMEDIATE EARLY GENES OF ABUSE. Reinhard

Grzanna, Ph.D., and Roger M. Brown, Ph.D., eds.

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DHHS Publication No. (ADM) 92-1945

Alcohol, Drug Abuse, and Mental Health Administration

Printed 1992