Methods for identifying a compound that binds to a human 5-HT.sub.1E receptor

This invention provides an isolated nucleic acid molecule encoding a human 5-HT.sub.1E receptor, an isolated protein which is a human 5-HT.sub.1E receptor, vectors comprising an isolated nucleic acid molecule encoding a human 5-HT.sub.1E receptors, mammalian cells comprising such vectors, antibodies directed to the human 5-HT.sub.1E receptor, nucleic acid probes useful for detecting nucleic acid encoding human 5-HT.sub.1E receptors, antisense oligonucleotides complementary to any sequences of a nucleic acid molecule which encodes a human 5-HT.sub.1E at receptor, pharmaceutical compounds related to human 5-HT.sub.1E receptors, and nonhuman transgenic animals which express DNA a normal or a mutant human 5-HT.sub.1E receptor. This invention further provides methods for determining ligand binding, detecting expression, drug screening, and treatment involving the human 5-HT.sub.1E receptor.

BACKGROUND OF THE INVENTION 
Since the purification of a pressor substance in blood serum termed 
serotonin (Rapport et al., 1947) and later identified as 
5-hydroxytryptamine (5-HT)(Rapport, 1949), there has been a plethora of 
reports demonstrating that this indoleamine not only plays a role in the 
functioning of peripheral tissues but, indeed, performs a key role in the 
brain as a neurotransmitter. Certainly, the anatomical localization of 
serotonin and serotonergic neurons in both the peripheral and central 
nervous systems supports its role in such diverse physiologic and 
behavioral functions as pain perception, sleep, aggression, sexual 
activity, hormone secretion, thermoregulation, motor activity, 
cardiovascular function, food intake and renal regulation (For review see 
Green, 1985; Osborne and Hamon, 1988; Sanders-Bush, 1988; Peroutka, 1991). 
Taken together, it appears that serotonin plays an important role in 
homeostasis and in modulating responsiveness to environmental stimuli. 
Accordingly, studies demonstrating that abnormalities in the serotonergic 
system may be associated with disease states has created a drug 
development effort towards agents which may selectively modulate the 
function of serotonin (Glennon, 1990). 
In relation to the characterization of physiologic or biochemical responses 
resulting from the release of serotonin are simultaneous investigations 
examining the receptor sites responsible for the actions elicited by the 
indoleamine transmitter. Following early in vitro pharmacological assays 
describing the existence of two different serotonin receptors, designated 
as D and M, in the guinea pig ileum (Gaddum and Picarelli, 1957), the 
advent of receptor binding technique in the 1970's has brought to light 
during the last decade the diversity of 5-HT receptors existing in both 
the brain and peripheral tissues. Thus, although the concept of D and M 
receptors has not been invalidated, serotonin receptors not fitting either 
category have been identified using radioligand methods. To date using 
this technique, there appears to be four classes of serotonin receptors 
found in the brain: 5-HT.sub.1, 5-HT.sub.2, 5-HT.sub.3 and, 5-HT.sub.4 
(Peroutka, 1991). Furthermore, 5-HT1 sites have been subclassified as: 
5-HT.sub.1A, 5-HT.sub.1B, 5-HT.sub.1C, 5-HT.sub.1D (Hamon et al., 1990) 
and 5-HT.sub.1E (Leonhardt et al., 1989). Although a detailed 
characterization of the 5-HT.sub.1E binding site is lacking, extensive 
pharmacologic, biochemical and functional properties have clearly shown 
that the other four subtypes of 5-HT.sub.1 sites are receptors according 
to classical criteria. Interestingly, the 5-HT.sub.1E binding site was 
first observed in human cortical tissue using [.sup.3 H]5-HT as the 
radioligand probe in the presence of 5-carboxyamidotryptamine and 
mesulergine to mask other members of the 5-HT.sub.1 receptor class. The 
affinity constants of the nine drugs tested indicated a unique 
pharmacological profile. In particular, the low affinity of 5-CT and 
ergotamine seemed to clearly discriminate the pharmacologically defined 
5-HT.sub.1D site from that of this novel serotonergic site. Importantly, 
it was demonstrated that 5-HT.sub.1E sites are saturable and exist in a 
density consistent with other known neurotransmitter receptors. 
Furthermore, this site appeared to interact with a GTP-binding protein. 
Overall, the data provided a framework suggesting that the 5-HT.sub.1E 
binding site may represent a functional receptor. 
During the last few years, the field of molecular biology as provided an 
important facet to receptor research by cloning these proteins and 
allowing more precise characterizations in isolated systems (Hartig et 
al.,1990). This has been accomplished for the 5-HT.sub.1A (Fargin et al., 
1988), 5-HT.sub.1C (Julius et al., 1988), 5-HT.sub.1D (Branchek et al., 
1990) and 5-HT.sub.2 receptors (Pritchett et al., 1988). Thus, there is no 
doubt that these binding sites represent "true" functional receptors. 
Indeed, the pharmacological characterization of serotonin receptors 
involved in various physiological or biochemical functions is a key 
component of drug development for the serotonergic system. As one can 
deduce from the diversity of serotonin binding sites, many targets are 
available for advancement in selective drug design. The coupling of 
molecular biological methods to pharmacological characterization 
particularly for cloned human receptors will open new avenues for 
pharmaceutical development which have not been previously explored. 
SUMMARY OF THE INVENTION 
This invention provides an isolated nucleic acid molecule encoding a human 
5-HT.sub.1E receptor. 
This invention also provides an isolated protein which is a human 
5-HT.sub.1E receptor. 
This invention provides a vector comprising an isolated nucleic acid 
molecule encoding a human 5-HT.sub.1E receptor. 
This invention also provides vectors such as plasmids comprising a DNA 
molecule encoding a human 5-HT.sub.1E receptor, adapted for expression in 
a bacterial cell, a yeast cell, or a mammalian cell which additionally 
comprise the regulatory elements necessary for expression of the DNA in 
the bacterial, yeast, or mammalian cells so located relative to the DNA 
encoding the 5-HT.sub.1E receptor as to permit expression thereof. 
This invention provides a mammalian cell comprising a DNA molecule encoding 
a human 5-HT.sub.1E receptor. 
This invention provides a method for determining whether a ligand not known 
to be capable of binding to a human 5-HT.sub.1E receptor can bind to a 
human 5-HT.sub.1E receptor which comprises contacting a mammalian cell 
comprising an isolated DNA molecule encoding a human 5-HT.sub.1E receptor 
with the ligand under conditions permitting binding of ligands known to 
bind to a 5-HT.sub.1E receptor, detecting the presence of any of the 
ligand bound to a human 5-HT.sub.1E receptor, and thereby determining 
whether the ligand binds to a human 5-HT.sub.1E receptor. 
This invention also provides a method for determining whether a ligand not 
known to be capable of binding to the human 5-HT.sub.1E receptor can 
functionally activate its activity or prevent the action of a ligand which 
does so. This comprises contacting a mammalian cell comprising an isolated 
DNA molecule which encodes a human 5-HT.sub.1E receptor with the ligand 
under conditions permitting the activation or blockade of a functional 
response, detected by means of a bioassay from the mammalian cell such as 
a second messenger response, and thereby determining whether the ligand 
activates or prevents the activation of the human 5-HT.sub.1E receptor 
functional output. 
This invention further provides a method of screening drugs to identify 
drugs which specifically interact with, and bind to, the human 5-HT.sub.1E 
receptor on the surface of a cell which comprises contacting a mammalian 
cell comprising an isolated DNA molecule encoding a human 5-HT.sub.1E 
receptor with a plurality of drugs, determining those drugs which bind to 
the mammalian cell, and thereby identifying drugs which specifically 
interact with, and bind to, a human 5-HT.sub.1E receptor. 
This invention also provides a method of screening drugs to identify drugs 
which interact with, and activate or block the activation of, the human 
5-HT.sub.1E receptor on the surface of a cell which comprises contacting 
the mammalian cell comprising an isolated DNA molecule encoding and 
expressing a human 5-HT.sub.1E receptor with a plurality of drugs, 
determining those drugs which activate or block the activation of the 
receptor in the mammalian cell using a bioassay such as a second messenger 
assays, and thereby identifying drugs which specifically interact with, 
and activate or block the activation of, a human 5-HT.sub.1E receptor. 
This invention provides a nucleic acid probe comprising a nucleic acid 
molecule of at least 15 nucleotides capable of specifically hybridizing 
with a sequence included within the sequence of a nucleic acid molecule 
encoding a human 5-HT.sub.1E receptor. 
This invention also provides a method of detecting expression of the 
5-HT.sub.1E receptor on the surface of a cell by detecting the presence of 
mRNA coding for a 5-HT.sub.1E receptor which comprises obtaining total 
mRNA from the cell and contacting the mRNA so obtained with a nucleic acid 
probe comprising a nucleic acid molecule of at least 15 nucleotides 
capable of specifically hybridizing with a sequence included within the 
sequence of a nucleic acid molecule encoding a human 5-HT.sub.1E receptor 
under hybridizing conditions, detecting the presence of mRNA hybridized to 
the probe, and thereby detecting the expression of the 5-HT.sub.1E 
receptor by the cell. 
This invention provides an antisense oligonucleotide having a sequence 
capable of binding specifically with any sequences of an mRNA molecule 
which encodes a human 5-HT.sub.1E receptor so as to prevent translation of 
the mRNA molecule. 
This invention provides an antibody directed to a human 5-HT.sub.1E 
receptor. 
This invention provides a transgenic nonhuman mammal expressing DNA 
encoding a human 5-HT.sub.1E receptor. This invention also provides a 
transgenic nonhuman mammal expressing DNA encoding a human 5-HT.sub.1E 
receptor so mutated as to be incapable of normal receptor activity, and 
not expressing native 5-HT.sub.1E receptor. This invention further 
provides a transgenic nonhuman mammal whose genome comprises antisense DNA 
complementary to DNA encoding a human 5-HT.sub.1E receptor so placed as to 
be transcribed into antisense mRNA which is complementary to mRNA encoding 
a 5-HT.sub.1E receptor and which hybridizes to mRNA encoding a 5-HT.sub.1E 
receptor thereby reducing its translation. 
This invention provides a method of determining the physiological effects 
of expressing varying levels of human 5-HT.sub.1E receptors which 
comprises producing a transgenic nonhuman animal whose levels of human 
5-HT.sub.1E receptor expression are varied by use of an inducible promoter 
which regulates human 5-HT.sub.1E receptor expression. 
This invention also provides a method of determining the physiological 
effects of expressing varying levels of human 5-HT.sub.1E receptors which 
comprises producing a panel of transgenic nonhuman animals each expressing 
a different amount of human 5-HT.sub.1E receptor. 
This invention provides a method for diagnosing a predisposition to a 
disorder associated with the expression of a specific human 5-HT.sub.1E 
receptor allele which comprises: a. obtaining DNA of subjects suffering 
from the disorder; b. performing a restriction digest of the DNA with a 
panel of restriction enzymes; c. electrophoretically separating the 
resulting DNA fragments on a sizing gel; d. contacting the resulting gel 
with a nucleic acid probe capable of specifically hybridizing to DNA 
encoding a human 5-HT.sub.1E receptor and labelled with a detectable 
marker; e. detecting labelled bands which have hybridized to the DNA 
encoding a human 5-HT.sub.1E receptor labelled with a detectable marker to 
create a unique band pattern specific to the DNA of subjects suffering 
from the disorder; f. preparing DNA obtained for diagnosis by steps a-e; 
and g. comparing the unique band pattern specific to the DNA of subjects 
suffering from the disorder from step e and the DNA obtained for diagnosis 
from step f to determine whether the patterns are the same or different 
and to diagnose thereby predisposition to the disorder if the patterns are 
the same. 
This invention provides a method of preparing the isolated 5-HT.sub.1E 
receptor which comprises inducing cells to express 5-HT.sub.1E receptor, 
recovering the receptor from the resulting cells and purifying the 
receptor so recovered. 
This invention also provides a method of preparing the isolated 5-HT.sub.1E 
receptor which comprises inserting nucleic acid encoding 5-HT.sub.1E 
receptor in a suitable vector, inserting the resulting vector in a 
suitable host cell, recovering the receptor produced by the resulting 
cell, and purifying the receptor so recovered. 
This invention provides an antisense oligonucleotide having a sequence 
capable of binding specifically with any sequences of an mRNA molecule 
which encodes a receptor so as to prevent translation of the mRNA 
molecule. 
This invention also provides a transgenic nonhuman mammal expressing DNA 
encoding a receptor. 
This invention further provides a transgenic nonhuman mammal expressing DNA 
encoding a receptor so mutated as to be incapable of normal receptor 
activity, and not expressing native receptor. 
This invention also provides a method of determining the physiological 
effects of expressing varying levels of a receptor which comprises 
producing a transgenic nonhuman animal whose levels of receptor expression 
are varied by use of an inducible promoter which regulates receptor 
expression. 
This invention also provides a method of determining the physiological 
effects of expressing varying levels of a receptor which comprises 
producing a panel of transgenic nonhuman animals each expressing a 
different amount of the receptor. 
This invention further provides a transgenic nonhuman mammal whose genome 
comprises antisense DNA complementary to DNA encoding a receptor so placed 
as to be transcribed into antisense mRNA which is complementary to mRNA 
encoding the receptor and which hybridizes to mRNA encoding the receptor 
thereby preventing its translation. 
This invention provides a method for determining whether a ligand not known 
to be capable of binding to a receptor can bind to a receptor which 
comprises contacting a mammalian cell comprising an isolated DNA molecule 
encoding the receptor with the ligand under conditions permitting binding 
of ligands known to bind to a receptor, detecting the presence of any of 
the ligand bound to the receptor, and thereby determining whether the 
ligand binds to the receptor.

DETAILED DESCRIPTION OF THE INVENTION 
As used herein, the 5-HT receptor family is defined as the group of 
mammalian proteins that function as receptors for serotonin. A 5-HT 
receptor subfamily is defined as a subset of proteins belonging to the 
5-HT receptor family which are encoded by genes which exhibit homology of 
greater than 72% or higher with each other in their deduced amino acid 
sequences within presumed transmembrane regions (linearly contiguous 
stretches of hydrophobic amino acids, bordered by charged or polar amino 
acids, that are long enough to form secondary protein structures that span 
a lipid bilayer). Four human 5-HT receptor subfamilies can be 
distinguished based on the information presently available: 5-HT.sub.1, 
5-HT.sub.2, 5-HT.sub.3, and 5-HT.sub.4 (Peroutka, 1991). The 5-HT.sub.2 
receptor subfamily contains the human 5-HT.sub.2 receptor. Although no 
other human members of this family have been described, the rat 5-HT.sub.2 
receptor (Pritchett, et al. 1988; Julius, et al. Proc. Natl. Acad. Sci. 
USA 87:928-932, 1990) and the rat 5HT.sub.1C receptor (Julius, et al. 
1988) constitute a rat 5-HT receptor subfamily. The 5-HT.sub.1 subfamily 
has been subdivided further as: 5-HT.sub.1A, 5-HT.sub.1B, 5-HT.sub.1C, 
5-HT.sub.1D (Hamon et al., 1990) and 5-HT.sub.1E (Leonhardt et al., 1989). 
The 5-HT.sub.1A subfamily contains the human 5-HT.sub.1A receptor, also 
known as G-21 (Fargin, et al. 1988) The 5-HT.sub.1D receptor subfamily 
contains two members, the 5-HT.sub.1D-1 receptor (also termed 
5-HT.sub.1D.alpha.) and the 5-HT.sub.1D-2 receptor (also termed 
5-HT.sub.1D.beta.). The 5-HT.sub.1E subfamily contains the human 
5-HT.sub.1E receptor (also termed clone hp75d). Although this definition 
differs from the pharmacological definition used earlier, there is 
significant overlap between the present definition and the pharmacological 
definition. Members of the 5-HT.sub.1E receptor subfamily so described 
include the 5-HT.sub.1E receptor and any other receptors which have a 
greater than 72% homology to the DNA and amino acid sequence shown in FIG. 
1 according to the definition of "subfamily". This invention relates to 
the discovery of the first member of the human 5-HT.sup.1E receptor 
subfamily. 
This invention provides an isolated nucleic acid molecule encoding a human 
5-HT.sub.1E receptor. As used herein, the term "isolated nucleic acid 
molecule" means a nucleic acid molecule that is, a molecule in a form 
which does not occur in nature. Such a receptor is by definition a member 
of the 5-HT.sub.1E receptor subfamily. Therefore, any receptor which meets 
the defining criteria given above is a human 5-HT.sub.1E receptor. One 
means of isolating a human 5-HT.sub.1E receptor is to probe a human 
genomic library with a natural or artificially designed DNA probe, using 
methods well known in the art. DNA probes derived from the human receptor 
gene 5-HT.sub.1E are particularly useful probes for this purpose. DNA and 
cDNA molecules which encode human 5-HT.sub.1E receptors may be used to 
obtain complementary genomic DNA, cDNA or RNA from human, mammalian or 
other animal sources, or to isolate related cDNA or genomic clones by the 
screening of cDNA or genomic libraries, by methods described in more 
detail below. Transcriptional regulatory elements from the 5' untranslated 
region of the isolated clones, and other stability, processing, 
transcription, translation, and tissue specificity-determining regions 
from the 3' and 5' untranslated regions of the isolated genes are thereby 
obtained. Examples of a nucleic acid molecule are an RNA, cDNA, or 
isolated genomic DNA molecule encoding a human 5-HT.sub.1E receptor. Such 
molecules may have coding sequences substantially the same as the coding 
sequence shown in FIG. 1. The DNA molecule of FIG. 1 encodes the sequence 
of the human 5-HT.sub.1E receptor gene. 
This invention further provides a cDNA molecule of encoding a human 
5-HT.sub.1E receptor having a coding sequence substantially the same as 
the coding sequence shown in FIG. 1. This molecule is obtained by the 
means described above. 
This invention also provides an isolated protein which is a human 
5-HT.sub.1E receptor. As used herein, the term "isolated protein means a 
protein molecule free of other cellular components. An example of such 
protein is an isolated protein having substantially the same amino acid 
sequence as the amino acid sequence shown in FIG. 1 which is a human 
5-HT.sub.1E receptor. One means for obtaining isolated 5-HT.sub.1E 
receptor is to express DNA encoding the receptor in a suitable host, such 
as a bacterial, yeast, or mammalian cell, using methods well known in the 
art, and recovering the receptor protein after it has been expressed in 
such a host, again using methods well known in the art. The receptor may 
also be isolated from cells which express it, in particular from cells 
which have been transfected with the expression vectors described below in 
more detail. 
This invention provides a vector comprising an isolated nucleic acid 
molecule such as DNA, RNA, or cDNA encoding a human 5-HT.sub.1E receptor. 
Examples of vectors are viruses such as bacteriophages (such as phage 
lambda), cosmids, plasmids (such as pUC18, available from Pharmacia, 
Piscataway, N.J.), and other recombination vectors. Nucleic acid molecules 
are inserted into vector genomes by methods well known in the art. For 
example, insert and vector DNA can both be exposed to a restriction enzyme 
to create complementary ends on both molecules which base pair with each 
other and are then ligated together with a ligase. Alternatively, linkers 
can be ligated to the insert DNA which correspond to a restriction site in 
the vector DNA, which is then digested with the restriction enzyme which 
cuts at that site. Other means are also available. A specific example of 
such plasmids is a plasmid comprising cDNA having a coding sequence 
substantially the same as the coding sequence shown in FIG. 1 and 
designated clone hp75d (Seq. I.D. No. 1). 
This invention also provides vectors comprising a DNA molecule encoding a 
human 5-HT.sub.1E receptor, adapted for expression in a bacterial cell, a 
yeast cell, or a mammalian cell which additionally comprise the regulatory 
elements necessary for expression of the DNA in the bacterial, yeast, or 
mammalian cells so located relative to the DNA encoding a human 
5-HT.sub.1E receptor as to permit expression thereof. DNA having coding 
sequences substantially the same as the coding sequence shown in FIG. 1 
may usefully be inserted into the vectors to express human 5-HT.sub.1E 
receptors. Regulatory elements required for expression include promoter 
sequences to bind RNA polymerase and transcription initiation sequences 
for ribosome binding. For example, a bacterial expression vector includes 
a promoter such as the lac promoter and for transcription initiation the 
Shine-Dalgarno sequence and the start codon AUG (Maniatis, et al., 
Molecular Cloning, Cold Spring Harbor Laboratory, 1982). Similarly, a 
eukaryotic expression vector includes a heterologous or homologous 
promoter for RNA polymerase II, a downstream polyadenylation signal, the 
start codon AUG, and a termination codon for detachment of the ribosome. 
Such vectors may be obtained commercially or assembled from the sequences 
described by methods well known in the art, for example the methods 
described above for constructing vectors in general. Expression vectors 
are useful to produce cells that express the receptor. Certain uses for 
such cells are described in more detail below. 
This invention further provides a plasmid adapted for expression in a 
bacterial, yeast, or, in particular, a mammalian cell which comprises a 
DNA molecule encoding a human 5-HT.sub.1E receptor and the regulatory 
elements necessary for expression of the DNA in the bacterial, yeast, or 
mammalian cell so located relative to the DNA encoding a human 5-HT.sub.1E 
receptor as to permit expression thereof. Some plasmids adapted for 
expression in a mammalian cell are pSVL (available from Pharmacia, 
Piscataway, N.J.) and pcEXV-3 (Miller J. and Germain R. N., J. Exp. Med. 
164:1478 (1986)). A specific example of such plasmid is a plasmid adapted 
for expression in a mammalian cell comprising cDNA having coding sequences 
substantially the same as the coding sequence shown in FIG. 1 and the 
regulatory elements necessary for expression of the DNA in the mammalian 
cell which is designated pcEXV-hp75d and deposited under ATCC Accession 
No. 75138. Those skilled in the art will readily appreciate that numerous 
plasmids adapted for expression in a mammalian cell which comprise DNA of 
encoding human 5-HT.sub.1E receptors and the regulatory elements necessary 
to express such DNA in the mammalian cell may be constructed utilizing 
existing plasmids and adapted as appropriate to contain the regulatory 
elements necessary to express the DNA in the mammalian cell. The plasmids 
may be constructed by the methods described above for expression vectors 
and vectors in general, and by other methods well known in the art. 
The deposit discussed supra, and the other deposits discussed herein, were 
made pursuant to, and in satisfaction of, the Budapest Treaty on the 
International Recognition of the Deposit of Microorganisms for the Purpose 
of Patent Procedure with the American Type Culture Collection (ATCC), 
12301 Parklawn Drive, Rockville, Md. 20852. 
This invention provides a mammalian cell comprising a DNA molecule encoding 
a human 5-HT.sub.1E receptor, such as a mammalian cell comprising a 
plasmid adapted for expression in a mammalian cell, which comprises a DNA 
molecule encoding a human 5-HT.sub.1E receptor, the protein encoded 
thereby is expressed on the cell surface, and the regulatory elements 
necessary for expression of the DNA in the mammalian cell so located 
relative to the DNA encoding a human 5-HT.sub.1E receptor as to permit 
expression thereof. Numerous mammalian cells may be used as hosts, 
including, for example, the mouse fibroblast cell NIH3T3, CHO cells, HeLa 
cells, Ltk.sup.- cells, Y1 cells, etc. A particular example of an 
Ltk.sup.- cell is a cell designated 5-HT.sub.1E -7 and deposited under 
ATCC Accession No. CRL 10913 and comprises the plasmid designated 
pcEXV-hp75d. Another example is the murine adrenal Y1 cell line designated 
Y-5-HT.sub.1E and deposited under ATCC Accession No. CRL 10914. Expression 
plasmids such as that described supra may be used to transfect mammalian 
cells by methods well known in the art such as calcium phosphate 
precipitation, or DNA encoding these 5-HT.sub.1E receptors may be 
otherwise introduced into mammalian cells, e.g., by microinjection, to 
obtain mammalian cells which comprise DNA, e.g., cDNA or a plasmid, 
encoding either human 5-HT.sub.1E receptor. 
This invention provides a method for determining whether a ligand not known 
to be capable of binding to a human 5-HT.sub.1E receptor can bind to a 
human 5-HT.sub.1E receptor which comprises contacting a mammalian cell 
comprising a DNA molecule encoding a human 5-HT.sub.1E receptor, the 
protein encoded thereby is expressed on the cell surface, with the ligand 
under conditions permitting binding of ligands known to bind to the 
5-HT.sub.1E receptor, detecting the presence of any of the ligand bound to 
the 5-HT.sub.1E receptor, and thereby determining whether the ligand binds 
to the 5-HT.sub.1E receptor. This invention also provides a method for 
determining whether a ligand not known to be capable of binding to the 
human 5-HT.sub.1E receptor can functionally activate its activity or 
prevent the action of a ligand which does so. This comprises contacting a 
mammalian cell comprising an isolated DNA molecule which encodes a human 
5-HT.sub.1E receptor with the ligand under conditions permitting the 
activation or blockade of a functional response, detected by means of a 
bioassay from the mammalian cell such as a second messenger response, and 
thereby determining whether the ligand activates or prevents the 
activation of the human 5-HT.sub.1E receptor functional output. The DNA in 
the cell may have a coding sequence substantially the same as the coding 
sequence shown in FIG. 1 preferably, the mammalian cell is nonneuronal in 
origin. An example of a nonneuronal mammalian cell is an Ltk.sup.- cell, 
in particular the Ltk.sup.- cell designated 5-HT.sub.1E -7. Another 
example of a non-neuronal mammalian cell to be used for functional assays 
is a Y1 murine adrenal cell, specifically the Y1 cell designated 
Y-5-HT.sub.1E. The preferred method for determining whether a ligand is 
capable of binding to the human 5-HT.sub.1E receptor comprises contacting 
a transfected nonneuronal mammalian cell (i.e. a cell that does not 
naturally express any type of 5-HT or G-protein coupled receptor, thus 
will only express such a receptor if it is transfected into the cell) 
expressing a 5-HT.sub.1E receptor on its surface, or contacting a membrane 
preparation derived from such a transfected cell, with the ligand under 
conditions which are known to prevail, and thus to be associated with, in 
vivo binding of the ligands to a 5-HT.sub.1E receptor, detecting the 
presence of any of the ligand being tested bound to the 5-HT.sub.1E 
receptor on the surface of the cell, and thereby determining whether the 
ligand binds to, activates or prevents the activation of the 5-HT.sub.1E 
receptor. This response system is obtained by transfection of isolated DNA 
into a suitable host cell containing the desired second messenger system 
such as phosphoinositide hydrolysis, adenylate cyclase, guanylate cyclase 
or ion channels. Such a host system is isolated from pre-existing cell 
lines, or can be generated by inserting appropriate components of second 
messenger systems into existing cell lines. Such a transfection system 
provides a complete response system for investigation or assay of the 
activity of human 5-HT.sub.1E receptors with ligands as described above. 
Transfection systems are useful as living cell cultures for competitive 
binding assays between known or candidate drugs and ligands which bind to 
the receptor and which are labeled by radioactive, spectroscopic or other 
reagents. Membrane preparations containing the receptor isolated from 
transfected cells are also useful for these competitive binding assays. 
Functional assays of second messenger systems or their sequelae in 
transfection systems act as assays for binding affinity and efficacy in 
the activation of receptor function. A transfection system constitutes a 
"drug discovery system" useful for the identification of natural or 
synthetic compounds with potential for drug development that can be 
further modified or used directly as therapeutic compounds to activate or 
inhibit the natural functions of the human 5-HT.sub.1E receptor. The 
transfection system is also useful for determining the affinity and 
efficacy of known drugs at the human 5-HT.sub.1E receptor sites. 
This invention also provides a method of screening drugs to identify drugs 
which specifically interact with, and bind to, the human 5-HT.sub.1E 
receptor on the surface of a cell which comprises contacting a mammalian 
cell comprising a DNA molecule encoding a human 5-HT.sub.1E receptor on 
the surface of a cell with a plurality of drugs, determining those drugs 
which bind to the mammalian cell, and thereby identifying drugs which 
specifically interact with, and bind to, the human 5-HT.sub.1E receptor. 
This invention also provides a method of screening drugs to identify drugs 
which interact with, and activate or block the activation of, the human 
5-HT.sub.1E receptor on the surface of a cell which comprises contacting 
the mammalian cell comprising an isolated DNA molecule encoding and 
expressing a human 5-HT.sub.1E receptor with a plurality of drugs, 
determining those drugs which activate or block the activation of the 
receptor in the mammalian cell using a bioassay such as a second messenger 
assays, and thereby identifying drugs which specifically interact with, 
and activate or block the activation of, a human 5-HT.sub.1E receptor. The 
DNA in the cell may have a coding sequence substantially the same as the 
coding sequence shown in FIG. 1. Preferably, the mammalian cell is 
nonneuronal in origin. An example of a nonneuronal mammalian cell is an 
Ltk.sup.- cell, in particular the Ltk.sup.- cell designated 5-HT.sub.1E 
-7. Another example of a non-neuronal mammalian cell to be used for 
functional assays is a Y1 murine adrenal cell, specifically the Y1 cell 
designated Y-5-HT.sub.1E. Drug candidates are identified by choosing 
chemical compounds which bind with high affinity to the expressed 
5-HT.sub.1E receptor protein in transfected cells, using radioligand 
binding methods well known in the art, examples of which are shown in the 
binding assays described herein. Drug candidates are also screened for 
selectivity by identifying compounds which bind with high affinity to one 
particular 5-HT.sub.1E receptor subtype but do not bind with high affinity 
to any other serotonin receptor subtype or to any other known receptor 
site. Because selective, high affinity compounds interact primarily with 
the target 5-HT.sub.1E receptor site after administration to the patient, 
the chances of producing a drug with unwanted side effects are minimized 
by this approach. This invention provides a pharmaceutical composition 
comprising a drug identified by the method described above and a 
pharmaceutically acceptable carrier. As used herein, the term 
"pharmaceutically acceptable carrier" encompasses any of the standard 
pharmaceutical carriers, such as a phosphate buffered saline solution, 
water, and emulsions, such as an oil/water or water/oil emulsion, and 
various types of wetting agents. Once the candidate drug has been shown to 
be adequately bio-available following a particular route of 
administration, for example orally or by injection (adequate therapeutic 
concentrations must be maintained at the site of action for an adequate 
period to gain the desired therapeutic benefit), and has been shown to be 
non-toxic and therapeutically effective in appropriate disease models, the 
drug may be administered to patients by that route of administration 
determined to make the drug bio-available, in an appropriate solid or 
solution formulation, to gain the desired therapeutic benefit. 
This invention provides a nucleic acid probe comprising a nucleic acid 
molecule of at least 15 nucleotides capable of specifically hybridizing 
with a sequence included within the sequence of a nucleic acid molecule 
encoding a human 5-HT.sub.1E receptor, for example with a coding sequence 
included within the sequence shown in FIG. 1. As used herein, the phrase 
"specifically hybridizing" means the ability of a nucleic acid molecule to 
recognize a nucleic acid sequence complementary to its own and to form 
double-helical segments through hydrogen bonding between complementary 
base pairs. Nucleic acid probe technology is well known to those skilled 
in the art who will readily appreciate that such probes may vary greatly 
in length and may be labeled with a detectable label, such as a 
radioisotope or fluorescent dye, to facilitate detection of the probe. 
Detection of nucleic acid encoding human 5-HT.sub.1E receptors is useful 
as a diagnostic test for any disease process in which levels of expression 
of the corresponding 5-HT.sub.1E receptor is altered. DNA probe molecules 
are produced by insertion of a DNA molecule which encodes human 
5-HT.sub.1E receptor or fragments thereof into suitable vectors, such as 
plasmids or bacteriophages, followed by insertion into suitable bacterial 
host cells and replication and harvesting of the DNA probes, all using 
methods well known in the art. For example, the DNA may be extracted from 
a cell lysate using phenol and ethanol, digested with restriction enzymes 
corresponding to the insertion sites of the DNA into the vector (discussed 
above), electrophoresed, and cut out of the resulting gel. An example of 
such DNA molecule is shown in FIG. 1. The probes are useful for `in situ` 
hybridization or in order to locate tissues which express this gene 
family, or for other hybridization assays for the presence of these genes 
or their mRNA in various biological tissues. In addition, synthesized 
oligonucleotides (produced by a DNA synthesizer) complementary to the 
sequence of a DNA molecule which encodes human 5-HT.sub.1E receptor of are 
useful as probes for these genes, for their associated mRNA, or for the 
isolation of related genes by homology screening of genomic or cDNA 
libraries, or by the use of amplification techniques such as the 
Polymerase Chain Reaction. Synthesized oligonucleotides as described may 
also be used to determine the cellular localization of the mRNA produced 
by the, 5-HT.sub.1E gene by in situ hybridization. An example of such an 
oligonucleotide is: GATGGTACACTGGCTGGGGGGTGGGCTGAGTTGACGGTGGCT (Seq. I.D. 
No. 8). 
This invention also provides a method of detecting expression of a 
5-HT.sub.1E receptor on the surface of a cell by detecting the presence of 
mRNA coding for a 5-HT.sub.1E receptor which comprises obtaining total 
mRNA from the cell using methods well known in the art and contacting the 
mRNA so obtained with a nucleic acid probe comprising a nucleic acid 
molecule of at least 15 nucleotides capable of specifically hybridizing 
with a sequence included within the sequence of a nucleic acid molecule 
encoding a human 5-HT.sub.1E receptor under hybridizing conditions, 
detecting the presence of mRNA hybridized to the probe, and thereby 
detecting the expression of the 5-HT.sub.1E receptor by the cell. 
Hybridization of probes to target nucleic acid molecules such as mRNA 
molecules employs techniques well known in the art. In one possible means 
of performing this method, nucleic acids are extracted by precipitation 
from lysed cells and the mRNA is isolated from the extract using a column 
which binds the poly-A tails of the mRNA molecules. The mRNA is then 
exposed to radioactively labelled probe on a nitrocellulose membrane, and 
the probe hybridizes to and thereby labels complementary mRNA sequences. 
Binding may be detected by autoradiography or scintillation counting. 
However, other methods for performing these steps are well known to those 
skilled in the art, and the discussion above is merely an example. 
This invention provides an antisense oligonucleotide having a sequence 
capable of binding specifically with any sequences of an mRNA molecule 
which encodes a human 5-HT.sub.1E receptor so as to prevent translation of 
the mRNA molecule. The antisense oligonucleotide may have a sequence 
capable of binding specifically with any sequences of the cDNA molecule 
whose sequence is shown in FIG. 1. As used herein, the phrase "binding 
specifically" means the ability of a nucleic acid sequence to recognize a 
nucleic acid sequence complementary to its own and to form double-helical 
segments through hydrogen bonding between complementary base pairs. A 
particular example of an antisense oligonucleotide is an antisense 
oligonucleotide comprising chemical analogues of nucleotides. 
This invention also provides a pharmaceutical composition comprising an 
amount of the oligonucleotide described above effective to reduce 
expression of a human 5-HT.sub.1E receptor by passing through a cell 
membrane and binding specifically with mRNA encoding a human 5-HT.sub.1E 
receptor in the cell so as to prevent its translation and a 
pharmaceutically acceptable hydrophobic carrier capable of passing through 
a cell membrane. The oligonucleotide may be coupled to a substance which 
inactivates mRNA, such as a ribozyme. The pharmaceutically acceptable 
hydrophobic carrier capable of passing through cell membranes may also 
comprise a structure which binds to a receptor specific for a selected 
cell type and is thereby taken up by cells of the selected cell type. The 
structure may be part of a protein known to bind a cell-type specific 
receptor, for example an insulin molecule, which would target pancreatic 
cells. DNA molecules having coding sequences substantially the same as the 
coding sequence shown in FIG. 1 may be used as the oligonucleotides of the 
pharmaceutical composition. 
This invention also provides a method of treating abnormalities which are 
alleviated by reduction of expression of a 5-HT.sub.1E receptor which 
comprises administering to a subject an amount of the pharmaceutical 
composition described above effective to reduce expression of the 
5-HT.sub.1E receptor by the subject. This invention further provides a 
method of treating an abnormal condition related to 5-HT.sub.1E receptor 
activity which comprises administering to a subject an amount of the 
pharmaceutical composition described above effective to reduce expression 
of the 5-HT.sub.1E receptor by the subject. Several examples of such 
abnormal conditions are dementia, Parkinson's disease, feeding disorders, 
pathological anxiety, schizophrenia, or a migraine headache. 
Antisense oligonucleotide drugs inhibit translation of mRNA encoding these 
receptors. Synthetic oligonucleotides, or other antisense chemical 
structures are designed to bind to mRNA encoding the 5-HT.sub.1E receptor 
and inhibit translation of mRNA and are useful as drugs to inhibit 
expression of 5-HT.sub.1E receptor genes in patients. This invention 
provides a means to therapeutically alter levels of expression of human 
5-HT.sub.1E receptors by the use of a synthetic antisense oligonucleotide 
drug (SAOD) which inhibits translation of mRNA encoding these receptors. 
Synthetic oligonucleotides, or other antisense chemical structures 
designed to recognize and selectively bind to mRNA, are constructed to be 
complementary to portions of the nucleotide sequences shown in FIG. 1 of 
DNA, RNA or of chemically modified, artificial nucleic acids. The SAOD is 
designed to be stable in the blood stream for administration to patients 
by injection, or in laboratory cell culture conditions, for administration 
to cells removed from the patient. The SAOD is designed to be capable of 
passing through cell membranes in order to enter the cytoplasm of the cell 
by virtue of physical and chemical properties of the SAOD which render it 
capable of passing through cell membranes (e.g. by designing small, 
hydrophobic SAOD chemical structures) or by virtue of specific transport 
systems in the cell which recognize and transport the SAOD into the cell. 
In addition, the SAOD can be designed for administration only to certain 
selected cell populations by targeting the SAOD to be recognized by 
specific cellular uptake mechanisms which binds and takes up the SAOD only 
within certain selected cell populations. For example, the SAOD may be 
designed to bind to a receptor found only in a certain cell type, as 
discussed above. The SAOD is also designed to recognize and selectively 
bind to the target mRNA sequence, which may correspond to a sequence 
contained within the sequence shown in FIG. 1 by virtue of complementary 
base pairing to the mRNA. Finally, the SAOD is designed to inactivate the 
target mRNA sequence by any of three mechanisms: 1) by binding to the 
target mRNA and thus inducing degradation of the mRNA by intrinsic 
cellular mechanisms such as RNAse I digestion, 2) by inhibiting 
translation of the mRNA target by interfering with the binding of 
translation-regulating factors or of ribosomes, or 3) by inclusion of 
other chemical structures, such as ribozyme sequences or reactive chemical 
groups, which either degrade or chemically modify the target mRNA. 
Synthetic antisense oligonucleotide drugs have been shown to be capable of 
the properties described above when directed against mRNA targets (J. S. 
Cohen, Trends in Pharm. Sci. 10, 435 (1989); H. M. Weintraub, Sci. Am. 
January (1990) p. 40). In addition, coupling of ribozymes to antisense 
oligonucleotides is a promising strategy for inactivating target mRNA (N. 
Sarver et al., Science 247, 1222 (1990)). An SAOD serves as an effective 
therapeutic agent if it is designed to be administered to a patient by 
injection, or if the patient's target cells are removed, treated with the 
SAOD in the laboratory, and replaced in the patient. In this manner, an 
SAOD serves as a therapy to reduce receptor expression in particular 
target cells of a patient, in any clinical condition which may benefit 
from reduced expression of 5-HT.sub.1E receptors. 
This invention provides an antibody directed to the human 5-HT.sub.1E 
receptor, for example a monoclonal antibody directed to an epitope of a 
human 5-HT.sub.1E receptor present on the surface of a cell and having an 
amino acid sequence substantially the same as an amino acid sequence for a 
cell surface epitope of the human 5-HT.sub.1E receptor included in the 
amino acid sequence shown in FIG. 1 (Seq. I.D. No. 2). Amino acid 
sequences may be analyzed by methods well known in the art to determine 
whether they produce hydrophobic or hydrophilic regions in the proteins 
which they build. In the case of cell membrane proteins, hydrophobic 
regions are well known to form the part of the protein that is inserted 
into the lipid bilayer which forms the cell membrane, while hydrophilic 
regions are located on the cell surface, in an aqueous environment. 
Therefore antibodies to the hydrophilic amino acid sequences shown in FIG. 
1 will bind to a surface epitope of a human 5-HT.sub.1E receptor, as 
described. Antibodies directed to human 5-HT.sub.1E receptors may be 
serum-derived or monoclonal and are prepared using methods well known in 
the art. For example, monoclonal antibodies are prepared using hybridoma 
technology by fusing antibody producing B cells from immunized animals 
with myeloma cells and selecting the resulting hybridoma cell line 
producing the desired antibody. Cells such as NIH3T3 cells or Ltk.sup.- 
cells may be used as immunogens to raise such an antibody. Alternatively, 
synthetic peptides may be prepared using commercially available machines 
and the amino acid sequence shown in FIG. 1 (Seq. I.D. No. 2). As a still 
further alternative, DNA, such as a cDNA or a fragment thereof, may be 
cloned and expressed and the resulting polypeptide recovered and used as 
an immunogen. These antibodies are useful to detect the presence of human 
5-HT.sub.1E receptors encoded by the isolated DNA, or to inhibit the 
function of the receptors in living animals, in humans, or in biological 
tissues or fluids isolated from animals or humans. 
This invention provides a pharmaceutical composition which comprises an 
amount of an antibody directed to the human 5-HT.sub.1E receptor effective 
to block binding of naturally occurring ligands to the 5-HT.sub.1E 
receptor, and a pharmaceutically acceptable carrier. A monoclonal antibody 
directed to an epitope of a human 5-HT.sub.1E receptor present on the 
surface of a cell and having an amino acid sequence substantially the same 
as an amino acid sequence for a cell surface epitope of the human 
5-HT.sub.1E receptor included in the amino acid sequence shown in FIG. 1 
is useful for this purpose. 
This invention also provides a method of treating abnormalities which are 
alleviated by reduction of expression of a human 5-HT.sub.1E receptor 
which comprises administering to a subject an amount of the pharmaceutical 
composition described above effective to block binding of naturally 
occurring ligands to the 5-HT.sub.1E receptor and thereby alleviate 
abnormalities resulting from overexpression of a human 5-HT.sub.1E 
receptor. Binding of the antibody to the receptor prevents the receptor 
from functioning, thereby neutralizing the effects of overexpression. The 
monoclonal antibodies described above are both useful for this purpose. 
This invention additionally provides a method of treating an abnormal 
condition related to an excess of 5-HT.sub.1E receptor activity which 
comprises administering to a subject an amount of the pharmaceutical 
composition described above effective to block binding of naturally 
occurring ligands to the 5-HT.sub.1E receptor and thereby alleviate the 
abnormal condition. Some examples of abnormal conditions are dementia, 
Parkinson's disease, feeding disorders, pathological anxiety, 
schizophrenia, and a migraine headache. 
This invention provides a method of detecting the presence of a 5-HT.sub.1E 
receptor on the surface of a cell which comprises contacting the cell with 
an antibody directed to the human 5-HT.sub.1E receptor, under conditions 
permitting binding of the antibody to the receptor, detecting the presence 
of the antibody bound to the cell, and thereby the presence of the human 
5-HT.sub.1E receptor on the surface of the cell. Such a method is useful 
for determining whether a given cell is defective in expression of 
5-HT.sub.1E receptors on the surface of the cell. Bound antibodies are 
detected by methods well known in the art, for example by binding 
fluorescent markers to the antibodies and examining the cell sample under 
a fluorescence microscope to detect fluorescence on a cell indicative of 
antibody binding. The monoclonal antibodies described above are useful for 
this purpose. 
This invention provides a transgenic nonhuman mammal expressing DNA 
encoding a human 5-HT.sub.1E receptor. This invention also provides a 
transgenic nonhuman mammal expressing DNA encoding a human 5-HT.sub.1E 
receptor so mutated as to be incapable of normal receptor activity, and 
not expressing native 5-HT.sub.1E receptor. This invention also provides a 
transgenic nonhuman mammal whose genome comprises antisense DNA 
complementary to DNA encoding a human 5-HT.sub.1E receptor so placed as to 
be transcribed into antisense mRNA which is complementary to mRNA encoding 
a 5-HT.sub.1E receptor and which hybridizes to mRMA encoding a 5-HT.sub.1E 
receptor thereby reducing its translation. The DNA may additionally 
comprise an inducible promoter or additionally comprise tissue specific 
regulatory elements, so that expression can be induced, or restricted to 
specific cell types. Examples of DNA are DNA or cDNA molecules having a 
coding sequence substantially the same as the coding sequence shown in 
FIG. 1. An example of a transgenic animal is a transgenic mouse. Examples 
of tissue specificity-determining regions are the metallothionein promotor 
(Low, M. J., Lechan, R. M., Hammer, R. E. et al. Science 231:1002-1004 
(1986)) and the L7 promotor (Oberdick, J., Smeyne, R. J., Mann, J. R., 
Jackson, S. and Morgan, J. I. Science 248:223-226 (1990)). 
Animal model systems which elucidate the physiological and behavioral roles 
of human 5-HT.sub.1E receptors are produced by creating transgenic animals 
in which the expression of a 5-HT.sub.1E receptor is either increased or 
decreased, or the amino acid sequence of the expressed 5-HT.sub.1E 
receptor protein is altered, by a variety of techniques. Examples of these 
techniques include: 1) Insertion of normal or mutant versions of DNA 
encoding a human 5-HT.sub.1E receptor or homologous animal versions of 
these genes, by microinjection, retroviral infection or other means well 
known to those skilled in the art, into appropriate fertilized embryos in 
order to produce a transgenic animal (Hogan B. et al. Manipulating the 
Mouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory (1986)). 
2) Homologous recombination (Capecchi M. R. Science 244:1288-1292 (1989); 
Zimmer, A. and Gruss, P. Nature 338:150-153 (1989)) of mutant or normal, 
human or animal versions of these genes with the native gene locus in 
transgenic animals to alter the regulation of expression or the structure 
of these 5-HT.sub.1E receptors. The technique of homologous recombination 
is well known in the art. It replaces the native gene with the inserted 
gene and so is useful for producing an animal that cannot express native 
receptor but does express, for example, an inserted mutant receptor, which 
has replaced the native receptor in the animal's genome by recombination, 
resulting in underexpression of the receptor. Microinjection adds genes to 
the genome, but does not remove them, and so is useful for producing an 
animal which expresses its own and added receptors, resulting in 
overexpression of the receptor. One means available for producing a 
transgenic animal, with a mouse as an example, is as follows: Female mice 
are mated, and the resulting fertilized eggs are dissected out of their 
oviducts. The eggs are stored in an appropriate medium such as M2 medium 
(Hogan B. et al. Manipulating the Mouse Embryo, A Laboratory Manual, Cold 
Spring Harbor Laboratory (1986)). DNA or cDNA encoding a human 5-HT.sub.1E 
receptor is purified from a vector (such as plasmid pCEXV-hp75d described 
above) by methods well known in the art. Inducible promoters may be fused 
with the coding region of the DNA to provide an experimental means to 
regulate expression of the trans-gene. Alternatively or in addition, 
tissue specific regulatory elements may be fused with the coding region to 
permit tissue-specific expression of the trans-gene. The DNA, in an 
appropriately buffered solution, is put into a microinjection needle 
(which may be made from capillary tubing using a pipet puller) and the egg 
to be injected is put in a depression slide. The needle is inserted into 
the pronucleus of the egg, and the DNA solution is injected. The injected 
egg is then transferred into the oviduct of a pseudopregnant mouse (a 
mouse stimulated by the appropriate hormones to maintain pregnancy but 
which is not actually pregnant), where it proceeds to the uterus, 
implants, and develops to term. As noted above, microinjection is not the 
only method for inserting DNA into the egg cell, and is used here only for 
exemplary purposes. 
Since the normal action of receptor-specific drugs is to activate or to 
inhibit the receptor, the transgenic animal model systems described above 
are useful for testing the biological activity of drugs directed against 
these 5-HT.sub.1E receptors even before such drugs become available. These 
animal model systems are useful for predicting or evaluating possible 
therapeutic applications of drugs which activate or inhibit these 
5-HT.sub.1E receptors by inducing or inhibiting expression of the native 
or trans-gene and thus increasing or decreasing expression of normal or 
mutant 5-HT.sub.1E receptors in the living animal. Thus, a model system is 
produced in which the biological activity of drugs directed against these 
5-HT.sub.1E receptors are evaluated before such drugs become available. 
The transgenic animals which over or under produce the 5-HT.sub.1E 
receptor indicate by their physiological state whether over or under 
production of the 5-HT.sub.1E receptor is therapeutically useful. It is 
therefore useful to evaluate drug action based on the transgenic model 
system. One use is based on the fact that it is well known in the art that 
a drug such as an antidepressant acts by blocking neurotransmitter uptake, 
and thereby increases the amount of neurotransmitter in the synaptic 
cleft. The physiological result of this action is to stimulate the 
production of less receptor by the affected cells, leading eventually to 
underexpression. Therefore, an animal which underexpresses receptor is 
useful as a test system to investigate whether the actions of such drugs 
which result in under expression are in fact therapeutic. Another use is 
that if overexpression is found to lead to abnormalities, then a drug 
which down-regulates or acts as an antagonist to 5-HT.sub.1E receptor is 
indicated as worth developing, and if a promising therapeutic application 
is uncovered by these animal model systems, activation or inhibition of 
the 5-HT.sub.1E receptor is achieved therapeutically either by producing 
agonist or antagonist drugs directed against these 5-HT.sub.1E receptors 
or by any method which increases or decreases the expression of these 
5-HT.sub.1E receptors in man. 
This invention provides a method of determining the physiological effects 
of expressing varying levels of human 5-HT.sub.1E receptors which 
comprises producing a transgenic nonhuman animal whose levels of human 
5-HT.sub.1E receptor expression are varied by use of an inducible promoter 
which regulates human 5-HT.sub.1E receptor expression. This invention also 
provides a method of determining the physiological effects of expressing 
varying levels of human 5-HT.sub.1E receptors which comprises producing a 
panel of transgenic nonhuman animals each expressing a different amount of 
human 5-HT.sub.1E receptor. Such animals may be produced by introducing 
different amounts of DNA encoding a human 5-HT.sub.1E receptor into the 
oocytes from which the transgenic animals are developed. 
This invention also provides a method for identifying a substance capable 
of alleviating abnormalities resulting from overexpression of a human 
5-HT.sub.1E receptor comprising administering the substance to a 
transgenic nonhuman mammal expressing at least one artificially introduced 
DNA molecule encoding a human 5-HT.sub.1E receptor and determining whether 
the substance alleviates the physical and behavioral abnormalities 
displayed by the transgenic nonhuman mammal as a result of overexpression 
of a human 5-HT.sub.1E receptor. As used herein, the term "substance" 
means a compound or composition which may be natural, synthetic, or a 
product derived from screening. Examples of DNA molecules are DNA or cDNA 
molecules having a coding sequence substantially the same as the coding 
sequence shown in FIG. 1. 
This invention provides a pharmaceutical composition comprising an amount 
of the substance described supra effective to alleviate the abnormalities 
resulting from overexpression of 5-HT.sub.1E receptor and a 
pharmaceutically acceptable carrier. 
This invention further provides a method for treating the abnormalities 
resulting from overexpression of a human 5-HT.sub.1E receptor which 
comprises administering to a subject an amount of the pharmaceutical 
composition described above effective to alleviate the abnormalities 
resulting from overexpression of a human 5-HT.sub.1E receptor. 
This invention provides a method for identifying a substance capable of 
alleviating the abnormalities resulting from underexpression of a human 
5-HT.sub.1E receptor comprising administering the substance to the 
transgenic nonhuman mammal described above which expresses only 
nonfunctional human 5-HT.sub.1E receptor and determining whether the 
substance alleviates the physical and behavioral abnormalities displayed 
by the transgenic nonhuman mammal as a result of underexpression of a 
human 5-HT.sub.1E receptor. 
This invention also provides a pharmaceutical composition comprising an 
amount of a substance effective to alleviate abnormalities resulting from 
underexpression of 5-HT.sub.1E receptor and a pharmaceutically acceptable 
carrier. 
This invention further provides a method for treating the abnormalities 
resulting from underexpression of a human 5-HT.sub.1E receptor which 
comprises administering to a subject an amount of the pharmaceutical 
composition described above effective to alleviate the abnormalities 
resulting from underexpression of a human 5-HT.sub.1E receptor. 
This invention provides a method for diagnosing a predisposition to a 
disorder associated with the expression of a specific human 5-HT.sub.1E 
receptor allele which comprises: a) obtaining DNA of subjects suffering 
from the disorder; b) performing a restriction digest of the DNA with a 
panel of restriction enzymes; c.electrophoretically separating the 
resulting DNA fragments on a sizing gel; d) contacting the resulting gel 
with a nucleic acid probe capable of specifically hybridizing to DNA 
encoding a human 5-HT.sub.1E receptor and labelled with a detectable 
marker: e) detecting labelled bands which have hybridized to the DNA 
encoding a human 5-HT.sub.1E receptor labelled with a detectable marker to 
create a unique band pattern specific to the DNA of subjects suffering 
from the disorder; f) preparing DNA obtained for diagnosis by steps a-e; 
and g) comparing the unique band pattern specific to the DNA of subjects 
suffering from the disorder from step e and the DNA obtained for diagnosis 
from step f to determine whether the patterns are the same or different 
and thereby to diagnose predisposition to the disorder if the patterns are 
the same. This method may also be used to diagnose a disorder associated 
with the expression of a specific human 5-HT.sub.1E receptor allele. 
This invention provides a method of preparing the isolated 5-HT.sub.1E 
receptor which comprises inducing cells to express 5-HT.sub.1E receptor, 
recovering the receptor from the resulting cells, and purifying the 
receptor so recovered. An example of an isolated 5-HT.sub.1E receptor is 
an isolated protein having substantially the same amino acid sequence as 
the amino acid sequence shown in FIG. 1. For example, cells can be induced 
to express receptors by exposure to substances such as hormones. The cells 
can then be homogenized and the receptor isolated from the homogenate 
using an affinity column comprising, for example, serotonin or another 
substance which is known to bind to the receptor. The resulting fractions 
can then be purified by contacting them with an ion exchange column, and 
determining which fraction contains receptor activity or binds 
anti-receptor antibodies. 
This invention provides a method of preparing the isolated 5-HT.sub.1E 
receptor which comprises inserting nucleic acid encoding 5-HT.sub.1E 
receptor in a suitable vector, inserting the resulting vector in a 
suitable host cell, recovering the receptor produced by the resulting 
cell, and purifying the receptor so recovered. An example of an isolated 
5-HT.sub.1E receptor is an isolated protein having substantially the same 
amino acid sequence as the amino acid sequence shown in FIG. 1. This 
method for preparing 5-HT.sub.1E receptor uses recombinant DNA technology 
methods well known in the art. For example, isolated nucleic acid encoding 
5-HT.sub.1E receptor is inserted in a suitable vector, such as an 
expression vector. A suitable host cell, such as a bacterial cell, or a 
eukaryotic cell such as a yeast cell, is transfected with the vector. 
5-HT.sub.1E receptor is isolated from the culture medium by affinity 
purification or by chromatography or by other methods well known in the 
art. 
This invention provides an antisense oligonucleotide having a sequence 
capable of binding specifically with any sequences of an mRNA molecule 
which encodes a receptor so as to prevent translation of the mRNA 
molecule. 
This invention also provides a transgenic nonhuman mammal expressing DNA 
encoding a receptor. 
This invention further provides a transgenic nonhuman mammal expressing DNA 
encoding a receptor so mutated as to be incapable of normal receptor 
activity, and not expressing native receptor. 
This invention provides a method of determining the physiological effects 
of expressing varying levels of a receptor which comprises producing a 
transgenic nonhuman animal whose levels of receptor expression are varied 
by use of an inducible promoter which regulates receptor expression. 
This invention also provides a method of determining the physiological 
effects of expressing varying levels of a receptor which comprises 
producing a panel of transgenic nonhuman animals each expressing a 
different amount of the receptor. 
This invention further provides transgenic nonhuman mammal whose genome 
comprises antisense DNA complementary to DNA encoding a receptor so placed 
as to be transcribed into antisense mRNA which is complementary to mRNA 
encoding the receptor and which hybridizes to mRNA encoding the receptor 
thereby preventing its translation. 
This invention provides a method for determining whether a ligand not known 
to be capable of binding to a receptor can bind to a receptor which 
comprises contacting a mammalian cell comprising an isolated DNA molecule 
encoding the receptor with the ligand under conditions permitting binding 
of ligands known to bind to a receptor, detecting the presence of any of 
the ligand bound to the receptor, and thereby determining whether the 
ligand binds to the receptor. 
Applicants have identified individual receptor subtype proteins and have 
described methods for the identification of pharmacological compounds for 
therapeutic treatments. Pharmacological compounds which are directed 
against specific receptor subtypes provide effective new therapies with 
minimal side effects. 
This invention identifies for the first time a new receptor protein, its 
amino acid sequence, and its human gene. Furthermore, this invention 
describes a previously unrecognized group of receptors within the 
definition of a 5-HT.sub.1E receptor. The information and experimental 
tools provided by this discovery are useful to generate new therapeutic 
agents, and new therapeutic or diagnostic assays for this new receptor 
protein, its associated mRNA molecule or its associated genomic DNA. The 
information and experimental tools provided by this discovery will be 
useful to generate new therapeutic agents, and new therapeutic or 
diagnostic assays for this new receptor protein, its associated mRNA 
molecule, or its associated genomic DNA. 
Specifically, this invention relates to the first isolation of a human cDNA 
and genomic clone encoding a 5-HT.sub.1E receptor. A new human gene for 
the receptor identified herein as 5-HT.sub.1E has been identified and 
characterized, and a series of related cDNA and genomic clones have been 
isolated. In addition, the human 5-HT.sub.1E receptor has been expressed 
in Ltk.sup.- cells and Y1 cells by transfecting the cells with the 
plasmid pcEXV-hp75d. The pharmacological binding properties of the protein 
encoded have been determined, and these binding properties classify this 
protein as a serotonin 5-HT.sub.1E receptor. Mammalian cell lines 
expressing this human 5-HT.sub.1E receptor at the cell surface have been 
constructed, thus establishing the first well-defined, cultured cell lines 
with which to study this 5-HT.sub.1E receptor. 
The invention will be better understood by reference to the Experimental 
Details which follow, but those skilled in the art will readily appreciate 
that the specific experiments detailed are only illustrative of the 
invention as described more fully in the claims which follow thereafter. 
Materials and Methods 
Cloning and Sequencing: 
A human placental genomic library (Stratagene) was screened using 
oligonucleotides derived from the human 5-HT.sub.1D.beta. receptor gene 
(U.S. Ser. No. 520,716), as a probe. Overlapping oligomers complementary 
to the 5-HT.sub.1D.beta. sequence in transmembrane domains III, V and VI 
were labeled with .sup.32 P-dATP and .sup.32 P-dCTP by synthesis with the 
large fragment of DNA Polymerase (Maniatis et al., 1982). Hybridization 
was performed at 40.degree. C. in a solution containing 25% formamide, 10% 
dextran sulfate, 5.times. SSC (1.times. SSC is 0.15 M sodium chloride, 
0.015 M sodium citrate), 1.times. Denhardt's (0.02% polyvinylpyrrolidone, 
0.02% Ficoll, and 0.02% bovine serum albumin), and 200 .mu.g/ml of 
sonicated salmon sperm DNA. The filters were washed at 40.degree. C. in 
0.1.times. SSC containing 0.1% sodium dodecyl sulfate (SDS) and exposed at 
-70.degree. C. to Kodak XAR film in the presence of an intensifying 
screen. Lambda phage hybridizing to the probe were plaque purified and DNA 
was prepared for Southern blot analysis (Southern, 1975; Maniatis et al., 
1982). For subcloning and further Southern blot analysis DNA was inserted 
into pUC18 (Pharmacia, Piscataway, N.J.). Nucleotide sequence analysis was 
done by the Sanger dideoxy nucleotide chain-termination method (Sanger 
1977) on denatured double-stranded plasmid templates using Sequenase (U.S. 
Biochemical Corp., Cleveland, Ohio). 
Expression: 
The entire coding region of clone hp75d was cloned into the eukaryotic 
expression vector pcEXV-3 (Miller, 1986). Stable cell lines were obtained 
by cotransfection with the plasmid pcEXV-3 (containing the 5-HT.sub.1E 
receptor gene) and the plasmid pGCcos3neo (containing the aminoglycoside 
transferase gene) into Ltk.sup.- cells and Y1 cells using calcium 
phosphate (reagents obtained from Specialty Media, Lavellette, N.J.). The 
cells were grown in a controlled environment (37.degree. C., 5% CO.sub.2) 
as monolayers in Dulbecco's modified Eagle medium (Gibco, Grand Island, 
N.Y.) or Flo Medium Specialty Media, Inc., Lavallette, N.J.) containing 25 
mM glucose and supplemented with 10% bovine calf serum, 100 U/ml 
penicillin G and 100 .mu.g/ml streptomycin sulfate. Stable clones were 
then selected for resistance to the antibiotic G-418 and harvested 
membranes were screened for their ability to bind [.sup.3 H] serotonin. 
Detection of 5-HT.sub.1E Receptor mRNA in Brain Using PCR: 
Total RNA from human brain tissue was extracted using the RNasol protocol 
as described by the manufacturer. cDNA was prepared from 5 .mu.g of total 
RNA with random hexanucleotide primers (500 pmoles) using Superscript 
reverse transcriptase (BRL, MD.) in 50 mM Tris-HCL pH8.3 buffer containing 
40 u RNasin, 2.5 mM MgCl.sub.2, 50 mM KCL and 1 mM dNTPs, at 42.degree. C. 
for 1 hr. The reaction was stopped by heating at 95.degree. C. for 5 min. 
and chilled on ice. After terminating the reaction, RNase H (2 u) was 
added and incubated at 37.degree. C. for 20 min. An aliquot (1 .mu.g) of 
the first strand cDNA was diluted (1:5) into a 50 .mu.l PCR reaction 
mixture (200 .mu.M dNTPs final concentration) containing 1.25 U of Taq 
polymerase (Cetus Corp., CA) in the buffer supplied by the manufacturer, 
and 1 .mu.M of 5' primer=5'TACCACGCGGCCAAGAGCCTTTACCA 3' (Seq. I.D. No. 9) 
and 3' primer=5'TGGTGCTAGAGATCTGCTGACGTTC 3' (Seq. I.D. No. 10), 
oligonucleotides derived from the third cytoplasmic loop region. The PCR 
amplification reaction was carried out on a Perkin Elmer Cetus thermal 
cycler by first a 5 min. incubation at 95.degree. C. followed by 30 rounds 
of the following cycle: 2 min. at 94.degree. C., 2 min. at 42.degree. C., 
3 min. at 72.degree. C., with a 3" extension, followed at the end by a 15 
min. incubation at 72.degree. C. In order to control for the amplification 
of DNA (carried over during the RNA extraction), control PCR reactions 
were run in parallel with RNA diluted in the same manner as the cDNA 
samples. If necessary, RNA samples were pretreated with RNase-free DNase 
to eliminate any contaminating DNA. Positive controls were included in all 
experiments, consisting of plasmid containing 5HT1E receptor gene 
sequences. The products of the PCR amplification were separated by 
electrophoresis in 1.5% agarose. Amplified fragments were identified by 
blotting the gel to nitrocellulose and probing with an oligonucleotide 
internal to the 5'- and 3'-primers 
(5'GAGAAGTCAGACACACAGAAAGTCTGTGTAAGTTTTACAACTTGC 3') (Seq. I.D. No. 11) 
and end-labeled with [c-.sup.32 P]ATP using T.sub.4 polynucleotide kinase. 
Hybridization was performed at 40.degree. C. in a solution containing 50% 
formamide, 10% dextran sulfate, 5.times. SSC (1.times. SSC is 0.15M sodium 
chloride, 0.015M sodium citrate), 1.times. Denhardt's (0.02% 
polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), and 
200 .mu.g/ml of sonicated salmon sperm DNA. The filters were washed at 
50.degree. C. in 0.1.times. SSC containing 0.1% sodium dodecyl sulfate and 
exposed at -70.degree. C. to Kodak XAR film in the presence of an 
intensifying screen. 
Membrane Preparation: 
Membranes were prepared from transfected Ltk.sup.- cells which were grown 
to 100% confluency. The cells were washed twice with phosphate-buffered 
saline, scraped from the culture dishes into 5 ml of ice-cold 
phosphate-buffered saline, and centrifuged at 200.times.g for 5 min at 
4.degree. C. The pellet was resuspended in 2.5 ml of ice-cold Tris buffer 
(20 mM Tris-HCl, pH 7.4 at 23.degree. C., 5 mM EDTA) and homogenized by a 
Wheaton tissue grinder. The lysate was subsequently centrifuged at 
200.times.g for 5 min at 4.degree. C. to pellet large fragments which were 
discarded. The supernatant was collected and centrifuged at 40,000.times.g 
for 20 min at 4.degree. C. The pellet resulting from this centrifugation 
was washed once in ice-cold Tris buffer and finally resuspended in a final 
buffer containing 50 mM Tris-HCl and 0.5 mM EDTA, pH 7.4 at 23.degree. C. 
Membrane preparations were kept on ice and utilized within two hours for 
the radioligand binding assays. Protein concentrations were determined by 
the method of Bradford (1976) using bovine serum albumin as the standard. 
Radioligand Binding: 
[.sup.3 H]5HT binding was performed using slight modifications of the 
5-HT.sub.1E assay conditions reported by Leonhardt et al. (1989) with the 
omission of masking ligands. Radioligand binding studies were achieved at 
37.degree. C. in a total volume of 250 .mu.l of buffer (50 mM Tris, 10 mM 
MgCl.sub.2, 0.2 mM EDTA, 10 .mu.M pargyline, 0.1% ascorbate, pH 7.4 at 
37.degree. C.) in 96 well microtiter plates. Saturation studies were 
conducted using [.sup.3 H]5-HT at 12 different concentrations ranging from 
0.5 nM to 100 nM. Displacement studies were performed using 4.5-5.5 nM 
[.sup.3 H]5-HT. The binding profile of drugs in competition experiments 
was accomplished using 10-12 concentrations of compound. Incubation times 
were 30 min for both saturation and displacement studies based upon 
initial investigations which determined equilibrium binding conditions. 
Nonspecific binding was defined in the presence of 10 .mu.M 5-HT. Binding 
was initiated by the addition of 50 .mu.l membrane homogenates (10-20 
.mu.g). The reaction was terminated by rapid filtration through presoaked 
(0.5% polyethyleneimine) filters using 48R Cell Brandel Harvester 
(Gaithersburg, Md.). Subsequently, filters were washed for 5 seconds with 
ice cold buffer (50 mM Tris HCL, pH 7.4 at 4.degree. C.), dried and placed 
into vials containing 2.5. ml of Readi-Safe (Beckman, Fullerton, Calif.) 
and radioactivity was measured using a Beckman LS 5000TA liquid 
scintillation counter. The efficiency of counting of [.sup.3 H]5HT 
averaged between 45-50%. Binding data was analyzed by computer-assisted 
nonlinear regression analysis (Accufit and Accucomp, Lundon Software, 
Chagrin Falls, Ohio). IC.sub.50 values were converted to Ki values using 
the Cheng-Prusoff equation (1973). All experiments were performed in 
triplicate. 
Measurement of cAMP Formation: 
Transfected Y1 cells (expression level=688 fmol/mg of protein) were 
incubated in DMEM, 5 mM theophylline, 10 mM Hepes 
(4-[2-Hydroxyethyl]-1-piperazineethanesulfonic acid), 10 .mu.M pargyline, 
for 20 minutes at 37.degree. C., 5% CO.sub.2. A 5-HT dose-effect curve was 
then conducted by adding 12 different final concentrations of drug ranging 
from 10 .mu.M to 0.1 nM, followed immediately by the addition of forskolin 
(10 .mu.M). Subsequently, the cells were incubated for an additional 10 
minutes at 37.degree. C., 5% CO.sub.2. The media was aspirated and the 
reaction terminated by the addition of 100 mM HCl. The plates were stored 
at 4.degree. C. for 15 minutes and centrifuged for 5 minutes (500.times.g 
at 4.degree. C.) to pellet cellular debris. Aliquots of the supernatant 
fraction were then stored at -20.degree. C. prior to assessment of cAMP 
formation by radioimmunoassay (cAMP Radioimmunoassay kit. Advanced 
Magnetics, Cambridge, Mass.). 
Detection of 5-HT.sub.1E Receptor mRNA in Brain Using In Situ Hybridization 
Histochemistry 
Labeling of 5-HT.sub.1E receptor oligo probe with digoxigenin-11-dUTP: The 
5-HT.sub.1E receptor oligo probe (45 mer sequence: 
GATGGTACACTGGCTGGGGGGTGGGCTGAGTTGACGGTGGCT) (Seq. I.D. No. 12) was 
synthesized in Molecular Biology Department of Neurogenetic Corp. The 3' 
end tailing reaction was as follows: To a sterile 1.5 ml Eppendorf tube 
add (1) 8 .mu.l of 5-HT.sub.1E receptor oligo probe (520 ng, denatured at 
95.degree. C., then chilled on ice); (2) 8 .mu.l of 5.times. tailing 
buffer (BRL); (3) 5 .mu.l of digoxigenin-11-dUTP (Boehringer); (4) 4 .mu.l 
of dATP at 1/50 dilution; (5) 7 .mu.l of terminal transferase (TdT, BRL); 
(6) 7 .mu.l of distilled water. The above reaction mixture was incubated 
at 37.degree. C. for 5 min. The tailed oligo probe was purified by ethanol 
precipitation, vacuum dried and reconstituted in 40 .mu.l of distilled 
water. 
Tissue Preparation: 
Guinea pig brains were dissected and frozen, first in methylbutane, then on 
dry ice. Brain sections were cut at 11 .mu.m on a cryostat, thaw-mounted 
on gelatin-coated slides, and stored at -80.degree. C. On the day of the 
experiment, the brain sections were quickly brought to room temperature 
using a cool air stream, and fixed in 3% paraformaldehyde made up in 0.1 M 
PBS containing 0.02% diethylpyrocarbonate(DEPC). After rinsed in 0.1M PBS 
and 2.times. SSC, the tissue sections were dehydrated in graded ethanol 
and air dried. 
Prehybridization: 
All the tissue sections were pre-incubated with hybridization buffer in a 
humid chamber at 25.degree. C. for 1 hr. Twenty ml of the hybridization 
buffer contained (1). 10 ml of 100% formamide; (2) 4 ml of 20.times.SSC; 
(3) 0.4 ml of 50.times. Denhardts; (4) 1.0 ml of 10 mg/ml salmon sperm 
DNA; (5) 0.5 ml of 10 mg/ml yeast tRNA; (6) 4 ml of Dextran sulfate. 
Hybridization: 
The digoxigenin labeled 5-HT.sub.1E oligo probe (40 .mu.l) was diluted in 1 
ml of hybridization buffer. Each tissue section was then covered with 0.1 
ml of the hybridization buffer with probe and incubated at 40.degree. C. 
for 82 hr. After incubation all the sections were washed in 2.times. SSC 
(25.degree. C., 1 hr), in 1.times. SSC (25.degree. C., 1 hr), finally in 
0.5.times. SSC, first 37.degree. C. 30 min, then 25.degree. C. 30 min. 
Immunological Detection: 
Following the post-hybridization washes slides were rinsed in Buffer #1 
(100 mM Tris-HCl; 150 mM NaCl; pH 7.5) for 1 min, then incubated with 2% 
normal sheep serum plus 0.3% triton X-100 in Buffer #1 for 30 min. After 
this preparation the sections were incubated with anti-digoxigenin 
conjugated with alkaline phosphatase at dilution 1:500 in Buffer #1 
containing 1% normal sheep serum and 0.3% triton X-100 at RT for 1.5 hr, 
then at 4.degree. C. overnight. The next day the tissue sections were 
rinsed in Buffer #1 for 10 min, then in Buffer #2 (100 mM Tris-HCl; 100 mM 
NaCl; 50 mM MgCl.sub.2 ; pH9.5) for 10 min. The chromagen solution was 
prepared immediately before the colorimetric reaction: i.e. to 10 ml of 
Buffer #2, (1) 45 .mu.l of 4-nitro blue tetrazolium chloride (NBT, 75 
mg/ml in 70% dimethylformamide); (2) 35 .mu.l of 
5-bromo-4-chloro-3-indolyl-phosphate (x-phosphate, 50 mg/ml in 100% 
dimethylformamide); and (3) 2.5 mg of levamisole were added. In order to 
carry out the colorimetric reaction sections were incubated with the 
chromogen solution in a humid, light-tight box overnight. The chromogen 
reaction was halted by rinsing the slides in Buffer #3 (10 mM Tris-HCl; 1 
mM EDTA; pH 8.0). The sections were then rinsed in PBS, covered with 
Aquamount, and examined under a light microscope. 
Drugs: 
[.sup.3 H]5-HT (specific activity=28 Ci/mmole) was obtained from New 
England Nuclear, Boston, Mass. All other chemicals were obtained from 
commercial sources and were of the highest grade purity available. 
Result 
Isolation of a Genomic Clone Encoding a 5HT.sub.1E Receptor 
A human genomic placental library was screened with oligonucleotide probes 
derived from transmembrane domains III, V and VI of the 5-HT.sub.1D.beta. 
receptor gene. The hybridization of these probes with the library was 
performed at low stringency and the result was the appearance of several 
hundred positive signals. Subsequently, approximately 350 of these clones 
were purified and categorized into various groups, based upon which of the 
three probes were responsible for the hybridization signal associated with 
a given clone. One group of clones exhibited hybridization signals with 
both transmembrane domain probes III and V. A number of these clones were 
subject to Southern blot analysis and determined to be identical or 
overlapping clones. A representative of this group, hp75d, was further 
characterized by nucleic acid sequence analysis and encoded what appeared 
to be a new serotonin receptor based upon its deduced amino acid sequence. 
Nucleotide Sequence and Deduced Amino Acid Sequence of hp75d 
DNA sequence information obtained from clone hp75d is shown in FIG. 1. An 
open reading frame extending from an ATG start codon at position 1 to a 
stop codon at position 1095 can encode a protein 365 amino acids in 
length, having a relative molecular mass (M.sub.f) of 41,633. A comparison 
of this protein sequence with previously characterized neurotransmitter 
receptors indicates that hp75d encodes a receptor which is a new member of 
a family of molecules which span the lipid bilayer seven times and couple 
to guanine nucleotide regulatory proteins (the G protein-coupled receptor 
family). A variety of structural features which are invariant in this 
family were present including the aspartic acid residues of transmembrane 
regions II and III, the DRY sequence at the end of transmembrane region 
III, and the conserved proline residues of transmembrane regions IV, V, VI 
and VII (Hartig et al. and references therein), were present in clone 
hp75d. A comparison of the transmembrane homology of hp75d to the other 
cloned serotonin receptors is shown in FIG. 2 and exhibits the following 
order of identity: 5-HT.sub.1D.alpha. (65%), 5-HT.sub.1D.beta. (64%), 
5-HT.sub.1A (52%), 5-HT.sub.1C (40%) and 5-HT.sub.2 (39%). 
Receptor Expression in Transfected Mammalian Cells 
Saturation analysis of membranes prepared from stably transfected Ltk.sup.- 
cells demonstrated that the receptor expressed was saturable and of high 
affinity. Scatchard plot analysis by non-linear regression revealed a Kd 
of 10.3.+-.1.2 nM (mean.+-.S.E.M., n=7) and a B max consistent with a high 
level of expression, 10.9.+-.2.6 picomoles/mg of protein (mean.+-.S.E.M., 
n=7). The percent specific binding determined at the measured Kd value for 
[.sup.3 H]5-HT was greater than 85% of total binding. Furthermore, 
evidence that the receptor is coupled to a G-protein was demonstrated by 
the ability of Gpp(NH)p, a non-hydrolyzable analog of GTP, to inhibit the 
specific binding of [.sup.3 H]5-HT (IC.sub.50 =1885.+-.556, n.sub.N 
=0.87.+-.0.04, I.sub.max =26.4.+-.5.6%). 
Pharmacological analysis of the receptor was accomplished by testing the 
ability of drugs from different chemical classes to displace [.sup.3 
H]5-HT specific binding (Table 1). Of the compounds investigated, 5-HT and 
5-hydroxylated tryptamine derivatives possessed the highest affinity which 
according to the classification system of Peroutka Table 1 Ki (nM) values 
of various drugs for the inhibition of [.sup.3 H]5-HT specific binding to 
clonal 5-HT.sub.1E cell membranes. Binding assays were performed with 
4.5-5.5 nM of [.sup.3 H]5-HT and 10-12 different concentrations of each 
inhibitory drug. Ki values were calculated from the IC.sub.50 values using 
the Cheng-Prusoff equation. Each value is the mean.+-.S.E.M. of 3-4 
independent determinations. 
TABLE 1 
______________________________________ 
Ki (nM) values of various drugs for the inhibition of [.sup.3 H]5-HT 
specific binding to clonal 5-HT.sub.1E cell membranes. Binding assays 
were 
performed with 4.5-5.5 nM of [.sup.3 H]5-HT and 10-12 different 
concentrations of each inhibitory drug. Ki values were calculated from 
the 
IC.sub.50 values using the Cheng-Prusoff equation. Each value is the 
mean .+-. S.E.M. of 3-4 independent determinations. 
COMPOUND Ki (nM) 
______________________________________ 
5-NT 10.9 .+-. 1.0 
Lysergol 42.8 .+-. 5.3 
Ergonovine 87.7 .+-. 7.6 
Methylergonovine 89.4 .+-. 4.2 
a-Methyl-5-NT 121 .+-. 13 
Methiothepin 194 .+-. 4 
1-Napthylpiperazine 207 .+-. 69 
Methysergide 228 .+-. 16 
Oxymetazoline 419 .+-. 49 
5-Methoxy-N,N-DMT 528 .+-. 32 
Ergotamine 599 .+-. 39 
2-Methyl-5-NT 817 .+-. 203 
1270 .+-. 233 
Sumatriptan 2520 .+-. 135 
Tryptamine 2559 .+-. 827 
DOI 2970 .+-. 592 
5-Methoxytryptamine 3151 .+-. 1041 
8-OM-DPAT 3333 .+-. 310 
3434 .+-. 102 
Spiperone 5051 .+-. 689 
TFMPP 6293 .+-. 259 
5-CT 7875 .+-. 284 
Ketanserin &gt;10,000 
&gt;10,000 
&gt;10,000 
LY-165163 (PAPP) &gt;10,000 
DP-5-CT &gt;10,000 
______________________________________ 
and Snyder (1979) makes this site a member of the 5-HT.sub.1 class. 
Interestingly, 5-CT possessed low affinity and, thus, discriminates this 
receptor from that of the 5-HT.sub.1D receptor as well as other members of 
this class. Various ergoline compounds also bound with high affinity 
including agents which have potent hallucinogenic activity. Excluding 
methiothepin and 1-napthylpiperazine (Ki values=194 and 207 nM, 
respectively), piperazine derivatives had low affinity and displayed Ki 
values greater than 700 nM. Furthermore, rauwolfia alkaloids and 
serotonergic agents that possess high affinity for various subtypes of 
receptor within the serotonin family including ketanserin (5-HT.sub.2), 
8-OH-DPAT (5-HT.sub.1A), DOI (5-HT.sub.1C /5-HT.sub.2), spiperone 
(5-HT.sub.1A /5-HT.sub.2), pindolol (5-HT.sub.1A /5-HT.sub.1B) and 
zacopride (5-HT.sub.3) had very poor affinity. In all cases, the Hill 
Coefficients did not differ significantly from unity. Taken together, the 
pharmacological profile of the 5-HT.sub.1E receptor is unique and 
contrasts to that of other known serotonin receptors. Accordingly, the 
probability of developing selective drugs for this receptor subtype is 
increased. Additional supporting evidence that the 5-HT.sub.1E receptor is 
functionally coupled to a G-protein was obtained by testing the ability of 
5-HT to inhibit forskolin-stimulated cAMP production in Y1 cells 
transfected with the 5-HT.sub.1E receptor. FIG. 4 demonstrates that the 
endogenous indoleamine, 5-HT, produced a concentration-related decrease in 
forskolin-stimulated cAMP production with an EC.sub.50 of 23.1 nM. The 
maximum inhibition of cAMP production was 32%. 
The tissue distribution of 5HT.sub.1E receptor mRNA was detected using PCR 
technology on cDNA from tissue-derived total RNA. The mRNA localization in 
human brain tissues demonstrates the presence of 5HT.sub.1E in: frontal 
cortex, cerebellar cortex, temporal cortex, choroid plexus, hippocampus, 
brain stem and cortex. We have not identified an area of the brain which 
does not contain 5HT.sub.1E. We conclude that the 5HT.sub.1E mRNA is 
abundant in human brain. These findings suggest that the 5HT.sub.1E 
receptor is not restricted to any one region of the brain and possibly 
cell type, but is expressed in numerous neuronal cell groups in many 
distinct regions of the human brain, as has been described for 5HT.sub.1C. 
The presence of 5HT.sub.1E receptor mRNA in various regions of the human 
brain suggest that 5HT.sub.1E may modulate a number of the central actions 
attributed to serotonin. The abundance of 5HT.sub.1E receptors (mRNA) in 
the hippocampus may affect mood, behavior and hallucinogenesis. A greater 
understanding of possible physiological roles of this receptor subtype may 
be realized by the development of more specific 5HT.sub.1E receptor drugs 
as well as physiological manipulations of 5HT.sub.1E mRNAs/receptors. 
The cellular localization of 5-HT.sub.1E receptor mRNA was detected with 
digoxigenin-11-dUTP labeled oligo probes employing in situ 
hybridization(ISHH) technology. Digoxigenin-11-dUTP labeled oxytocin(OT) 
oligo probes were used as a positive control for the experiment since the 
distribution of OT neurons in the central nervous system is well known. OT 
cells were intensely stained in the Guinea Pig's hypothalamus. Tissue 
sections pre-treated with RNase A were used as a negative control. Guinea 
pig brain sections were examined under a light microscope. 5-HT.sub.1E 
receptor mRNA was found in cells located in the frontal cortex, piriform 
cortex, hippocampus (CA1, CA2 and CA3), lateral septal nucleus, triangular 
septal nucleus, septofimbrial nucleus and the basal ganglia 
(caudate-putamen and globus pallidus). It was detected in the amygdaloid 
complex, the bed nucleus stria terminalis and the hypothalamic area 
including anterior hypothalamus, periventricular nucleus, paraventricular 
nucleus (magnocellular and parvocellular populations), supraoptic nucleus 
which seemed to include both vasopressin and oxytocin cell populations, 
and the lateral hypothalamus. The 1E receptor mRNA was also detected in 
the thalamic area including anteroventral thalamic, anterodorsal thalamic, 
mediodorsal thalamic, ventrolateral thalamic, reticular thalamic 
paracentral thalamic, paratenial thalamic nuclei and the nucleus stria 
medullaris. Control sections pre-treated with RNase A did not exhibit any 
staining pattern. 
Experimental Discussion 
The deduced amino acid sequence of hp75d was analyzed to uncover 
relationships between it and the other cloned serotonin receptor 
sequences. Although the homology within the membrane spanning domains was 
greatest with the 5-HT.sub.1D.alpha. receptor (FIG. 2), the nature of 
this newly cloned receptor could not be clearly predicted. The rational 
for this ambiguity is the interpretation of the transmembrane domain 
homology (approximately 65%) to the 5-HT.sub.1D.alpha. and 
5-HT.sub.1D.beta. receptor subfamily. Closely related members of a 
"subfamily" of serotonin receptors (i.e. "subtypes") generally share a 
common transmitter and also have similar pharmacological profiles and 
physiological roles (for example, 5-HT.sub.2 and 5-HT.sub.1C or 
5-HT.sub.1D.alpha. and 5-HT.sub.1D.beta.). Such "subtypes" display an 
amino acid identity of approximately 75-80% in their transmembrane 
domains. Serotonin receptors which are not members of the same 
"subfamily", but are members of the serotonin "family" (in which the 
receptors use the same neurotransmitter; i.e. 5-HT.sub.2 and 
5-HT.sub.1D.alpha.) generally show much lower transmembrane homology 
(approximately 45%). Such transmembrane amino acid homologies can, 
therefore, give insight into the relationship between receptors and be 
used as predictors of receptor pharmacology. According to this type of 
analysis, although the newly cloned receptor appears to be more related to 
the 5-HT.sub.1D subfamily, it is likely to be in a subfamily distinct from 
all the other serotonin receptors. 
The present pharmacological evidence substantiates the existence of a novel 
serotonin receptor in the human brain as first suggested by Leonhardt et 
al. (1989). Comparison of the pharmacological profile observed in native 
cortical membranes to that revealed for the cloned 5-HT.sub.1E receptor 
yielded a correlation coefficient of 0.987 (FIG. 3). Indeed, there were 
some differences in measured affinity constants but the relative values of 
selected drugs performed in both studies was similar. In this regard, a 
close examination of the Scatchard plot analysis performed in the study by 
Leonhardt et al. (1989) reveals that the reported Kd value may have been 
underestimated since the radioligand concentrations did not exceed 10 nM. 
In order to have accurately determined the dissociation constant, the 
radioligand concentration should have been extended to at least 50 nM or 
10 times the estimated Kd (Yamamura et al., 1985). The initial study, 
however, was limited in tissue supply and lacked a comprehensive 
pharmacological characterization. The cloning of the 5-HT.sub.1E site will 
now allow more extensive investigations into the nature of this unique 
receptor. 
The structure-activity relationships observed in the present study suggest 
that there are important requirements for high affinity binding to the 
5-HT.sub.1E receptor. Substitution or removal of the 5-hydroxy group on 
serotonin decreases the affinity 300 fold for the receptor (egs., 
tryptamine, 5-methoxytryptamine and 5-carboxyamidotryptamine). 
Additionally, 2-methylation and .alpha.-methylation of 5-HT essentially 
abolishes its affinity for the 5-HT.sub.1E site. In contrast to these 
substitutions, N,N-dimethylation of the aliphatic side chain of the indole 
ring increases the affinity approximately 20 fold (unpublished 
observations). Basic structural requirements of the ergoline derivatives 
demonstrate that N-methylation of the indole ring decreases affinity as 
does bulky substitutions. Furthermore, piperazine derivatives are not 
bound at high affinity. 
Notably, the application of the human 5-HT.sub.1E receptor clone to 
pharmaceutical research can lead to new drug design and development. The 
localization of this receptor in the cerebral cortex (Leonhardt et al., 
1989) and parts of the basal ganglia such as the putamen and globus 
pallidus (Lowther et al., 1991) suggests a putative link to limbic, 
cognitive and/or motor function (Nieuwenhuys et al., 1988; Kandel and 
Schwartz, 1985) and, thus, may be involved in such abnormal conditions as 
dementia, Parkinson's disease, feeding disorders, anxiety and 
schizophrenia. Notably, the ergot compounds that possess affinity for this 
site have been demonstrated to affect these type of behaviors in humans as 
well as animals (Wilkinson and Dourish, 1991). In relation to this, it 
appears that the 5-HT.sub.1E binding site is also present in rat and 
bovine brain (Leonhardt et al., 1989) as well as guinea-pig, rabbit and 
dog where the data was initially interpreted as evidence for subtypes of 
the 5-HT.sub.1D receptor (Middlemiss, 1990). Nonetheless, it must be taken 
into consideration that this novel site can possibly lead to selective 
drug therapy devoid of side effects. In regard to this, serotonin uptake 
blockers are effective in treating neuropsychiatric disorders such as 
depression and obsessive-compulsive illness (Blier et al., 1987; Asberg et 
al., 1986; Insel et al., 1985). However, these agents have side effects 
and, in fact, the mechanism of action for these compounds are not linked 
to any particular serotonergic receptor. The possibility that agents 
selective for the 5-HT.sub.1E receptor may have clinical utility as 
antidepressants, for example, without the side effects attributed to 
current treatment modalities can have significant implications for drug 
therapy. Furthermore, it should be noted that ergoline derivatives have 
had clinical usefulness as drugs capable of relieving migraines and, thus, 
the involvement of the 5-HT.sub.1E receptor in this disorder deserves 
future attention. Ultimately, in depth investigations into the 
localization of the 5-HT.sub.1E receptor in brain and peripheral tissue 
will target new sites that can lead to specific functional roles for this 
serotonergic receptor. 
Another consideration for therapeutic application of this site may be 
related to the treatment of feeding disorders such as obesity, bulimia 
nervosa and/or anorexia nervosa. The involvement of serotonin and feeding 
behavior has received much attention during the last decade. It is now 
known that many of the identified and well-characterized serotonergic 
receptors are capable of modulating feeding (Blundell and Lawton, 1990). 
Notably, serotonin uptake blockers which have been used to treat feeding 
disorders act nonselectively and as such have side-effect potential 
(Jimerson et al., 1990). Although many different serotonergic receptors 
are involved in feeding, the search for the one site that can be exploited 
for selective drug development has yet to be found. There is no doubt that 
interest exists in finding drugs that interact with the serotonin system 
for the treatment of feeding disorders (Cooper, 1989). 
Thus, the pharmacological profile of the cloned human 5-HT.sub.1E receptor 
is unique and contrasts to other known serotonergic receptors. The utility 
of this site expressed in a cellular system and, thus, isolated for study 
will create excellent opportunities in drug development directed towards a 
novel serotonergic receptor that may have wide-range implications for drug 
therapy. Indeed, the potential therapeutic applications may extend to 
neuropsychiatric disorders including depression, anxiety, schizophrenia, 
dementia and obsessive-compulsive illness as well as obesity and migraine. 
The localization of 5-HT.sub.1E receptor mRNA by in situ hybridization 
makes it possible to predict its physiological and pathological functions. 
5-HT.sub.1E receptor mRNA is detected in the limbic structures, such as 
the hippocampus, septal nuclei, piriform cortex (olfactory system), 
amygdaloid complex and the bed nucleus stria terminalis. The olfactory 
system sends afferent fibers to the hippocampus through the subiculum, and 
to the amygdaloid complex (Kupfermann, 1985). In turn the outputs of the 
hippocampus project to the septal area and hypothalamus while the 
amygdaloid complex projects to the hypothalamus via the stria terminalis 
(Kupfermann, 1985). The involvement of the limbic system in emotional 
behavior (e.g., fear, pleasure, sexual activities) and memory is well 
known (Kupfermann, 1985). Therefore, the finding of 5-HT.sub.1E receptor 
mRNA in these structures indicates a potential role in neuropsychiatric 
disorders such as depression, obsessive-compulsive illness, anxiety, 
schizophrenia and dementia. 
The hypothalamus regulates the body adjustments to the external and 
internal environments. It can control hunger (the ventromedial nucleus and 
the lateral hypothalamus), endocrine functions (e.g. the supraoptic 
nucleus, the paraventricular nucleus and the periventricular nucleus), 
affective (emotional) behavior (the ventromedial and dorsomedial nuclei) 
and the activity of the visceral nervous system (the anterior 
hypothalamus) (Diamond et al., 1985). The discovery of 5-HT.sub.1E 
receptor mRNA in these nuclei indicates physiological and pathological 
roles of this receptor subtype in cardiovascular, gastrointestinal, 
endocrine, neurological and psychiatric systems. 
The thalamus is a relay station where the sensory and motor-related 
pathways passing up the brain stem synapse before proceeding on to the 
cerebral cortex for more elaborate integration and analysis. The 
5-HT.sub.1E receptor mRNA was found in the anterior thalamic nucleus which 
receives the input from the mammillothalamic tract and sends fibers to the 
cingulate gyrus. Thus, this nucleus is an important part of the circuit 
connecting the hypothalamus, the thalamus, and the limbic lobe (Diamond et 
al., 1985). The 5-HT.sub.1E receptor mRNA was also found in the 
mediodorsal thalamic nucleus which is involved in emotional behavior and 
in the ventral lateral thalamic nucleus which connects the basal ganglia 
and premotor area. Furthermore, 5-HT.sub.1E receptor mRNA was found in the 
nucleus reticularis thalami through which pass most of the thalamocortical 
and corticothalamic fibers. The axons of corticothalamic and 
thalamocortical neurons provide collaterals for synapse with reticular 
cells, exerting a facilitatory effect, while the axons of the GABA-rich 
reticularis cells project back on the specific thalamic neurons, 
continuously modulating (by inhibition) the ascending flow of 
thalamocortical impulses (Diamond et al., 1985). The reticularis neurons 
play a significant role in the interactions between the thalamus and the 
frontal cortex. In addition, the 5-HT.sub.1E receptor mRNA was also 
visualized in the basal ganglia including the caudate-putamen and globus 
pallidus both of which control motor activity (Alheid et al., 1990). These 
findings indicate important roles of the 5-HT.sub.1E receptors in 
emotional behavior, sensation (e.g., pain) and motor activity. 
REFERENCES 
Alheid, G. F.; Heimer, L. & Switzer III, R. C., In: The Human Nervous 
System. Ed. George Paxinos, Academic Press, Inc., 1990. 
Asberg, M., Eriksson, B., Matensson, B., Traskman-Bendz, L. and Wagner, A.: 
Therapeutic effects of serotonin uptake inhibitors in depression. J. Clin. 
Psychiat. 47:23-35, 1986. 
Blier, P., DeMontigny, C. and Chaput, Y.: Modifications of the serotonin 
system by antidepressant treatments: Implications for the therapeutic 
response in major depression. J. Clin. Psychpharmacol. 7(6):24s-35s, 1987. 
Blundell, J. E. and Lawton, C. L.: Serotonin receptor sub-types and the 
organization of feeding behaviour: Experimental models. In: Serotonin: 
From cell biology to pharmacology and therapeutics. (eds. Paoletti, R., 
Vanhoutte, P. M., Brunello, N. and Maggi, F. M.) Boston: Kluwer Academic 
Publishers, pp. 213-219, 1990. 
Bradford, M.: A rapid and sensitive method for the quantification of 
microgram quantities of protein utilizing the principle of protein-dye 
binding. Anal. Biochem. 72:248-254, 1976. 
Branchek, T., Weinshank, R. L., Macchi, M. J., Zgombick, J. M. and Hartig, 
P. R.: Cloning and expression of a human 5-HT1D receptor. The Second 
IUPHAR Satellite Meeting on Serotonin, Basel, Switzerland, Jul. 11-13, 
1990, Abstract #2. 
Cheng, Y. C. and Prusoff, W. H.: Relationship between the inhibition 
constant (Ki) and the concentration of inhibitor which causes 50% 
inhibition (IC50) of an enzyme reaction. Biochem. Pharmacol. 22:3099-3108, 
1973. 
Cooper, S. J.: Drugs interacting with 5-HT systems show promise for 
treatment of eating disorders. TIPS 10:56-57, 1989. 
Diamond, M. C.: Scheibel, A. B. and Elson, L. M.: The human brain coloring 
book. Barnes & Noble Books, 1985 
Fargin, A., Raymond, J. R., Lohse, M. J., Kobilka, B. K. Caron, M. G. and 
Lefkowitz, R. J.: The genomic clone G-21 which resembles a 
.beta.-adrenergic receptor sequence encodes the 5-HT1A receptor. Nature 
335:358-360, 1988. 
Gaddum, J. H. and Picarelli, Z. P.: Two kinds of tryptamine receptor. Brit. 
J. Pharmacol. 12:323-328, 1957. 
Glennon, R. A.: Serotonin receptors: Clinical implications. Neurosci. 
Biobehav. Rev. 14:35-47, 1990. 
Green, A. R.: Neuropharmacology of serotonin. Oxford: Oxford University 
Press, 1985. 
Hamon, M., Lanfumey, L., El Mestikawy, S., Boni, C., Miquel, M.-C., 
Bolanos, F., Schechter, L. and Gozlan, H.: The main features of central 
5-HT1 receptors. Neuropsychopharmacol. 3(5/6):349-360, 1990. 
Hartig, P. R., Kao, H.-T., Macchi, M., Adham, N., Zgombick, J., Weinshank, 
R. and Branchek, T.: The molecular biology of serotonin receptors: An 
overview. Neuropsychopharmacol. 3(5/6):335-347, 1990. 
Insel, T. R., Mueller, E. A., Alterman, I., Linnoila, M. and Murphy, D. L.: 
Obsessive-compulsive disorder and serotonin: Is there a connection? Biol. 
Psychiat. 20:1174-1188, 1985. 
Jimerson, D. C., Lesem, M. D., Hegg, A. P. and Brewerton, T. D.: Serotonin 
in human eating disorders. Ann. N.Y. Acad. Sci. 600:532-544, 1990. 
Julius, D., MacDermott, A. B., Axel, R. and Jessell, T. M.: Molecular 
characterization of a functional cDNA encoding the serotonin 1C receptor. 
Science 241:558-564, 1988. 
Kandel, E. R. and Schwartz, J. H.: Principles of neuroscience. New York: 
Elsevier Publishing Co., 1985. 
Kupfermann, I.: Hypothalamus and limbic system. In: Principles of 
neuroscience. Eds: Kandel, E. R. and Schwartz, J. H., New York: Elsevier 
Publishing Co., 1985 
Leonhardt, S., Herrick-Davis, K. and Titeler, M.: Detection of a novel 
serotonin receptor subtype (5-HT1E) in human brain: Interaction with a 
GTP-binding protein. J. Neurochem. 53(2):465-471, 1989. 
Lowther, S., De Paermentier, F., Crompton, M. R., Katona, C. L. E. and 
Horton, R. W.: 5HT.sub.1D and 5HT.sub.1E binding sites in depression: A 
post-mortem study in suicide victims. Serotonin 
1991--5-Hydroxytryptamine--CNS Receptors and Brain Function, Birmingham, 
UK, Jul. 14-17, 1991, p. 175, Abstract #P. 145 
Maniatis, T., Fritsch, E. F., and Sambrook, J. Molecular Cloning: A 
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 
N.Y., 1982. 
Middlemiss, D. N., Suman-Chauhan, N., Smith, S. M., Picton, C., Shaw, D. 
and Bevan, Y.: Subpopulations of 5-HT1D recognition sites in guinea-pig, 
rabbit, dog and human cortex. The Second IUPHAR Satellite Meeting on 
Serotonin, Basel, Switzerland, Jul. 11-13, 1990, Abstract #P-30. 
Miller, J., and R. N. Germain. Efficient cell surface expression of class 
II MHC molecules in the absence of associated invariant chain. J.Exp.Med. 
164:1478-1489, 1986. 
Nieuwenhuys, R., Voogd, J. and van Huijzen, C.: The human nervous system: A 
synopsis and atlas. New York: Springer Verlag, 1988. 
Osborne, N. N. and Hamon, M.: Neuronal serotonin. Chichester: John Wiley 
and Sons, Inc., 1988. 
Peroutka, S. J.: Serotonin receptor subtypes: Basic and clinical aspects. 
New York: Wiley-Liss, Inc., 1991. 
Peroutka, S. J. and Snyder, S. H.: Multiple serotonin receptors, 
differential binding of [.sup.3 H]5-hydroxytryptamine, [.sup.3 H]lysergic 
acid diethylamide and [.sup.3 H]spiroperidol. Mol. Pharmacol. 16:687-699, 
1979. 
Pritchett, D. B., Bach, A. W. J., Wozny, M., Taleb, O., Dal Toso, R., Shih, 
J. and Seeburg, P. H.: Structure and functional expression of cloned rat 
serotonin 5-HT2 receptor. EMBO J. 7:4135-4140, 1988. 
Rapport, M. M., Green, A. A. and Page, I. H.: Purification of the substance 
which is responsible for vasoconstrictor activity of serum. Fed. Proc. 
6:184, 1947. 
Rapport, M. M.: Serum vasoconstrictor (serotonin) V. Presence of creatinine 
in the complex. A proposed structure of the vasoconstrictor principle. J. 
Biol. Chem. 180:961-969, 1949. 
Sanger, S. DNA sequencing with chain-terminating inhibitors. Proc. Natl. 
Acad. Sci. USA 74:5463-5467, 1977. 
Sanders-Bush, E.: The Serotonin Receptors. Clifton, N.J.: Humana Press, 
1988. 
Southern, E. M. Detection of specific sequences among DNA fragments 
separated by gel electrophoresis. J.Mol.Biol. 98:503-505, 1975. 
Wilkinson, L. O. and Dourish, C. T.: Serotonin and animal behavior. In: 
Serotonin receptor subtypes: Basic and clinical aspects. (ed. Peroutka, S. 
J.) New York: Wiley-Liss, Inc., pgs. 147-210, 1991. 
Yamamura, H. I., Enna, S. J. and Kuhar, M. J.: Neurotransmitter receptor 
binding. New York: Raven Press, 1985. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 12 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2463 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: human plac - #ental genomic 
(B) CLONE: hp75d 
- - (viii) POSITION IN GENOME: 
(C) UNITS: bp 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 736..1830 
- - (ix) FEATURE: 
(A) NAME/KEY: mat.sub.-- - #peptide 
(B) LOCATION: 736..1830 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - ATATACATCA TGGAATACTA TGCAGCCCCC CCCAAGGATG CGTTCCATGT CC - 
#TTTGCAGG 60 
- - GACATGGATG AGTTGCAAAC CATATTCTCA ACAAACTAAC ACAGCCACAG AA - 
#AACCAAAC 120 
- - ACCACATGCT CTCACTCACG AGTGGAGTTG AACAATGAGA ACACATGGCA CA - 
#GGGCCGGG 180 
- - AACATGACAC ACCAGGGCCT GTTGGGGGGT GGAGGGCTAG GGGAGGGATG GC - 
#ATTAGGAG 240 
- - AAGTACCTAA TGTAGATGAT TGGTTGTTGG GTGCAGCAAA CCACCATGGC AC - 
#ATGTATAC 300 
- - CTATGTAGCA AACCTGCAAG TTCTGCACAT GTATCCCAGG ACTTAAAGTA TA - 
#ATTTAAAA 360 
- - AAAAACAGTT TGAAAACTTC CCTGAAGTAA AAAAAGTATC CTTTGAGGAA CA - 
#ATGTAACG 420 
- - ATGAGCTCAA GTTCCACAGG AAAGAGAAAA TTAAAATTTA TAAAGAATTT AT - 
#AAATATCA 480 
- - AACTATTTTC ATGTTTTCCA GGAAAAGTGT GGCTTTCTCA TTCATTAACC AA - 
#TAGCATAA 540 
- - TATTTTCCAG GAACCTTCAC TCAGAAGAAA TGCTGTGGCC CTTCCCTTTA CC - 
#AACAGAAA 600 
- - ATGGAACACA AGAGACCACA TAGCTGAACA AATTATAGCC TCCTTACAAG TG - 
#AGAAACCT 660 
- - TCGAGGCTAC ATAGTTTTCA GCCAAAGGAA AATAACCAAC AGCTTCTCCA CA - 
#GTGTAGAC 720 
- - TGAAACAAGG GAAAC ATG AAC ATC ACA AAC TGT ACC - #ACA GAG GCC AGC 
ATG 771 
Met - #Asn Ile Thr Asn Cys Thr Thr Glu Ala Ser M - #et 
- # 1 5 - # 10 
- - GCT ATA AGA CCC AAG ACC ATC ACT GAG AAG AT - #G CTC ATT TGC ATG ACT 
819 
Ala Ile Arg Pro Lys Thr Ile Thr Glu Lys Me - #t Leu Ile Cys Met Thr 
15 - # 20 - # 25 
- - CTG GTG GTC ATC ACC ACC CTC ACC ACG TTG CT - #G AAC TTG GCT GTG ATC 
867 
Leu Val Val Ile Thr Thr Leu Thr Thr Leu Le - #u Asn Leu Ala Val Ile 
30 - # 35 - # 40 
- - ATG GCT ATT GGC ACC ACC AAG AAG CTC CAC CA - #G CCT GCC AAC TAC CTA 
915 
Met Ala Ile Gly Thr Thr Lys Lys Leu His Gl - #n Pro Ala Asn Tyr Leu 
45 - # 50 - # 55 - # 60 
- - ATC TGT TCT CTG GCC GTG ACG GAC CTC CTG GT - #G GCA GTG CTC GTC ATG 
963 
Ile Cys Ser Leu Ala Val Thr Asp Leu Leu Va - #l Ala Val Leu Val Met 
65 - # 70 - # 75 
- - CCC CTG AGC ATC ATC TAC ATT GTC ATG GAT CG - #C TGG AAG CTT GGG TAC 
1011 
Pro Leu Ser Ile Ile Tyr Ile Val Met Asp Ar - #g Trp Lys Leu Gly Tyr 
80 - # 85 - # 90 
- - TTC CTC TGT GAG GTG TGG CTG AGT GTG GAC AT - #G ACC TGC TGC ACC TGC 
1059 
Phe Leu Cys Glu Val Trp Leu Ser Val Asp Me - #t Thr Cys Cys Thr Cys 
95 - # 100 - # 105 
- - TCC ATC CTC CAC CTC TGT GTC ATT GCC CTG GA - #C AGG TAC TGG GCC ATC 
1107 
Ser Ile Leu His Leu Cys Val Ile Ala Leu As - #p Arg Tyr Trp Ala Ile 
110 - # 115 - # 120 
- - ACC AAT GCT ATT GAA TAC GCC AGG AAG AGG AC - #G GCC AAG AGG GCC GCG 
1155 
Thr Asn Ala Ile Glu Tyr Ala Arg Lys Arg Th - #r Ala Lys Arg Ala Ala 
125 1 - #30 1 - #35 1 - 
#40 
- - CTG ATG ATC CTT ACC GTC TGG ACC ATC TCC AT - #T TTC ATC TCC ATG 
CCC 1203 
Leu Met Ile Leu Thr Val Trp Thr Ile Ser Il - #e Phe Ile Ser Met Pro 
145 - # 150 - # 155 
- - CCT CTG TTC TGG AGA AGC CAC CGC CGC CTA AG - #C CCT CCC CCT AGT CAG 
1251 
Pro Leu Phe Trp Arg Ser His Arg Arg Leu Se - #r Pro Pro Pro Ser Gln 
160 - # 165 - # 170 
- - TGC ACC ATC CAG CAC GAC CAT GTT ATC TAC AC - #C ATT TAC TCC ACG CTG 
1299 
Cys Thr Ile Gln His Asp His Val Ile Tyr Th - #r Ile Tyr Ser Thr Leu 
175 - # 180 - # 185 
- - GGT GCG TTT TAT ATC CCC TTG ACT TTG ATA CT - #G ATT CTC TAT TAC CGG 
1347 
Gly Ala Phe Tyr Ile Pro Leu Thr Leu Ile Le - #u Ile Leu Tyr Tyr Arg 
190 - # 195 - # 200 
- - ATT TAC CAC GCG GCC AAG AGC CTT TAC CAG AA - #A AGG GGA TCA AGT CGG 
1395 
Ile Tyr His Ala Ala Lys Ser Leu Tyr Gln Ly - #s Arg Gly Ser Ser Arg 
205 2 - #10 2 - #15 2 - 
#20 
- - CAC TTA AGC AAC AGA AGC ACA GAT AGC CAG AA - #T TCT TTT GCA AGT 
TGT 1443 
His Leu Ser Asn Arg Ser Thr Asp Ser Gln As - #n Ser Phe Ala Ser Cys 
225 - # 230 - # 235 
- - AAA CTT ACA CAG ACT TTC TGT GTG TCT GAC TT - #C TCC ACC TCA GAC CCT 
1491 
Lys Leu Thr Gln Thr Phe Cys Val Ser Asp Ph - #e Ser Thr Ser Asp Pro 
240 - # 245 - # 250 
- - ACC ACA GAG TTT GAA AAG TTC CAT GCC TCC AT - #C AGG ATC CCC CCC TTC 
1539 
Thr Thr Glu Phe Glu Lys Phe His Ala Ser Il - #e Arg Ile Pro Pro Phe 
255 - # 260 - # 265 
- - GAC AAT GAT CTA GAT CAC CCA GGA GAA CGT CA - #G CAG ATC TCT AGC ACC 
1587 
Asp Asn Asp Leu Asp His Pro Gly Glu Arg Gl - #n Gln Ile Ser Ser Thr 
270 - # 275 - # 280 
- - AGG GAA CGG AAG GCA GCA CGC ATC CTG GGG CT - #G ATT CTG GGT GCA TTC 
1635 
Arg Glu Arg Lys Ala Ala Arg Ile Leu Gly Le - #u Ile Leu Gly Ala Phe 
285 2 - #90 2 - #95 3 - 
#00 
- - ATT TTA TCC TGG CTG CCA TTT TTC ATC AAA GA - #G TTG ATT GTG GGT 
CTG 1683 
Ile Leu Ser Trp Leu Pro Phe Phe Ile Lys Gl - #u Leu Ile Val Gly Leu 
305 - # 310 - # 315 
- - AGC ATC TAC ACC GTG TCC TCG GAA GTG GCC GA - #C TTT CTG ACG TGG CTC 
1731 
Ser Ile Tyr Thr Val Ser Ser Glu Val Ala As - #p Phe Leu Thr Trp Leu 
320 - # 325 - # 330 
- - GGT TAT GTG AAT TCT CTG ATC AAC CCT CTG CT - #C TAT ACG AGT TTT AAT 
1779 
Gly Tyr Val Asn Ser Leu Ile Asn Pro Leu Le - #u Tyr Thr Ser Phe Asn 
335 - # 340 - # 345 
- - GAA GAC TTT AAG CTG GCT TTT AAA AAG CTC AT - #T AGA TGC CGA GAG CAT 
1827 
Glu Asp Phe Lys Leu Ala Phe Lys Lys Leu Il - #e Arg Cys Arg Glu His 
350 - # 355 - # 360 
- - ACT TAGACTGTAA AAAGCTAAAA GGCACGACTT TTTCCAGAGC CTCATGAGT - #G 
1880 
Thr 
365 
- - GATGGGGGTA AGGGGTGCAA CTTATTAATT CTTGAACATA CTTGGTTCAG GA - 
#GAGTTTGT 1940 
- - AAGTATGTGT GGTCTTGTTT CCTTGTTTGT TTGTTTGTTT TGTTCTGTTT TG - 
#TTTGAGGA 2000 
- - TTGTTATTTG GCCTCCTGTT TTCTACCTCT GGTCTTATCT GTGATACATA AT - 
#TTCAAATA 2060 
- - AACATTATCA TACAAAAACA GAAATTTTGC CAGAAGTAAT AATAAGATGA AA - 
#TACTAAAT 2120 
- - ACCTTTTATG GGTTTTTTTT TTTTAGCCAT TTCAGTTACC CTGCAATTAA AG - 
#AATGCCAA 2180 
- - AAATATCTTT ATTTGCAGAA TTTCTTATTA CTTATAAATT AAATACCTGA TA - 
#ATGCCCTC 2240 
- - CATGGCATTA AATCTGAGAT TATGGCTCTA TCTGCGTACA TATTCCAGTG GG - 
#AATTGCAT 2300 
- - GACTACATAA AGAATTAAAA GAAAGTGATG TGCTGTCATC TACGGCTTGC GA - 
#CCTGAGCT 2360 
- - AAAGTCGGGG GCTGTAGCAC TGTGACTACG TAGCCTATCA TTTCAGGTAA AA - 
#ATAGTACA 2420 
- - GCTGGCTTGT CTTGTTAGTT CATGATTAAA TAAACTTCTC TTT - # 
246 - #3 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 365 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Met Asn Ile Thr Asn Cys Thr Thr Glu Ala Se - #r Met Ala Ile Arg Pro 
1 5 - # 10 - # 15 
- - Lys Thr Ile Thr Glu Lys Met Leu Ile Cys Me - #t Thr Leu Val Val Ile 
20 - # 25 - # 30 
- - Thr Thr Leu Thr Thr Leu Leu Asn Leu Ala Va - #l Ile Met Ala Ile Gly 
35 - # 40 - # 45 
- - Thr Thr Lys Lys Leu His Gln Pro Ala Asn Ty - #r Leu Ile Cys Ser Leu 
50 - # 55 - # 60 
- - Ala Val Thr Asp Leu Leu Val Ala Val Leu Va - #l Met Pro Leu Ser Ile 
65 - # 70 - # 75 - # 80 
- - Ile Tyr Ile Val Met Asp Arg Trp Lys Leu Gl - #y Tyr Phe Leu Cys Glu 
85 - # 90 - # 95 
- - Val Trp Leu Ser Val Asp Met Thr Cys Cys Th - #r Cys Ser Ile Leu His 
100 - # 105 - # 110 
- - Leu Cys Val Ile Ala Leu Asp Arg Tyr Trp Al - #a Ile Thr Asn Ala Ile 
115 - # 120 - # 125 
- - Glu Tyr Ala Arg Lys Arg Thr Ala Lys Arg Al - #a Ala Leu Met Ile Leu 
130 - # 135 - # 140 
- - Thr Val Trp Thr Ile Ser Ile Phe Ile Ser Me - #t Pro Pro Leu Phe Trp 
145 1 - #50 1 - #55 1 - 
#60 
- - Arg Ser His Arg Arg Leu Ser Pro Pro Pro Se - #r Gln Cys Thr Ile 
Gln 
165 - # 170 - # 175 
- - His Asp His Val Ile Tyr Thr Ile Tyr Ser Th - #r Leu Gly Ala Phe Tyr 
180 - # 185 - # 190 
- - Ile Pro Leu Thr Leu Ile Leu Ile Leu Tyr Ty - #r Arg Ile Tyr His Ala 
195 - # 200 - # 205 
- - Ala Lys Ser Leu Tyr Gln Lys Arg Gly Ser Se - #r Arg His Leu Ser Asn 
210 - # 215 - # 220 
- - Arg Ser Thr Asp Ser Gln Asn Ser Phe Ala Se - #r Cys Lys Leu Thr Gln 
225 2 - #30 2 - #35 2 - 
#40 
- - Thr Phe Cys Val Ser Asp Phe Ser Thr Ser As - #p Pro Thr Thr Glu 
Phe 
245 - # 250 - # 255 
- - Glu Lys Phe His Ala Ser Ile Arg Ile Pro Pr - #o Phe Asp Asn Asp Leu 
260 - # 265 - # 270 
- - Asp His Pro Gly Glu Arg Gln Gln Ile Ser Se - #r Thr Arg Glu Arg Lys 
275 - # 280 - # 285 
- - Ala Ala Arg Ile Leu Gly Leu Ile Leu Gly Al - #a Phe Ile Leu Ser Trp 
290 - # 295 - # 300 
- - Leu Pro Phe Phe Ile Lys Glu Leu Ile Val Gl - #y Leu Ser Ile Tyr Thr 
305 3 - #10 3 - #15 3 - 
#20 
- - Val Ser Ser Glu Val Ala Asp Phe Leu Thr Tr - #p Leu Gly Tyr Val 
Asn 
325 - # 330 - # 335 
- - Ser Leu Ile Asn Pro Leu Leu Tyr Thr Ser Ph - #e Asn Glu Asp Phe Lys 
340 - # 345 - # 350 
- - Leu Ala Phe Lys Lys Leu Ile Arg Cys Arg Gl - #u His Thr 
355 - # 360 - # 365 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 422 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
- - (ii) MOLECULE TYPE: protein 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - Met Asp Val Leu Ser Pro Gly Gln Gly Asn As - #n Thr Thr Ser Pro Pro 
1 5 - # 10 - # 15 
- - Ala Pro Phe Glu Thr Gly Gly Asn Thr Thr Gl - #y Ile Ser Asp Val Thr 
20 - # 25 - # 30 
- - Val Ser Tyr Gln Val Ile Thr Ser Leu Leu Le - #u Gly Thr Leu Ile Phe 
35 - # 40 - # 45 
- - Cys Ala Val Leu Gly Asn Ala Cys Val Val Al - #a Ala Ile Ala Leu Glu 
50 - # 55 - # 60 
- - Arg Ser Leu Gln Asn Val Ala Asn Tyr Leu Il - #e Gly Ser Leu Ala Val 
65 - #70 - #75 - #80 
- - Thr Asp Leu Met Val Ser Val Leu Val Leu Pr - #o Met Ala Ala Leu Tyr 
85 - # 90 - # 95 
- - Gln Val Leu Asn Lys Trp Thr Leu Gly Gln Va - #l Thr Cys Asp Leu Phe 
100 - # 105 - # 110 
- - Ile Ala Leu Asp Val Leu Cys Cys Thr Ser Se - #r Ile Leu His Leu Cys 
115 - # 120 - # 125 
- - Ala Ile Ala Leu Asp Arg Tyr Trp Ala Ile Th - #r Asp Pro Ile Asp Tyr 
130 - # 135 - # 140 
- - Val Asn Lys Arg Thr Pro Arg Arg Ala Ala Al - #a Leu Ile Ser Leu Thr 
145 1 - #50 1 - #55 1 - 
#60 
- - Trp Leu Ile Gly Phe Leu Ile Ser Ile Pro Pr - #o Met Leu Gly Trp 
Arg 
165 - # 170 - # 175 
- - Thr Pro Glu Asp Arg Ser Asp Pro Asp Ala Cy - #s Thr Ile Ser Lys Asp 
180 - # 185 - # 190 
- - His Gly Tyr Thr Ile Tyr Ser Thr Phe Gly Al - #a Phe Tyr Ile Pro Leu 
195 - # 200 - # 205 
- - Leu Leu Met Leu Val Leu Tyr Gly Arg Ile Ph - #e Arg Ala Ala Arg Phe 
210 - # 215 - # 220 
- - Arg Ile Arg Lys Thr Val Lys Lys Val Glu Ly - #s Thr Gly Ala Asp Thr 
225 2 - #30 2 - #35 2 - 
#40 
- - Arg His Gly Ala Ser Pro Ala Pro Gln Pro Ly - #s Lys Ser Val Asn 
Gly 
245 - # 250 - # 255 
- - Glu Ser Gly Ser Arg Asn Trp Arg Leu Gly Va - #l Glu Ser Lys Ala Gly 
260 - # 265 - # 270 
- - Gly Ala Leu Cys Ala Asn Gly Ala Val Arg Gl - #n Gly Asp Asp Gly Ala 
275 - # 280 - # 285 
- - Ala Leu Glu Val Ile Glu Val His Arg Val Gl - #y Asn Ser Lys Glu His 
290 - # 295 - # 300 
- - Leu Pro Leu Pro Ser Glu Ala Gly Pro Thr Pr - #o Cys Ala Pro Ala Ser 
305 3 - #10 3 - #15 3 - 
#20 
- - Phe Glu Arg Lys Asn Glu Arg Asn Ala Glu Al - #a Lys Arg Lys Met 
Ala 
325 - # 330 - # 335 
- - Leu Ala Arg Glu Arg Lys Thr Val Lys Thr Le - #u Gly Ile Ile Met Gly 
340 - # 345 - # 350 
- - Thr Phe Ile Leu Cys Trp Leu Pro Phe Phe Il - #e Val Ala Leu Val Leu 
355 - # 360 - # 365 
- - Pro Phe Cys Glu Ser Ser Cys His Met Pro Th - #r Leu Leu Gly Ala Ile 
370 - # 375 - # 380 
- - Ile Asn Trp Leu Gly Tyr Ser Asn Ser Leu Le - #u Asn Pro Val Ile Tyr 
385 3 - #90 3 - #95 4 - 
#00 
- - Ala Tyr Phe Asn Lys Asp Phe Gln Asn Ala Ph - #e Lys Lys Ile Ile 
Lys 
405 - # 410 - # 415 
- - Cys Leu Phe Cys Arg Gln 
420 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 460 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
- - (ii) MOLECULE TYPE: protein 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - Met Val Asn Leu Gly Asn Ala Val Arg Ser Le - #u Leu Met His Leu Ile 
1 5 - # 10 - # 15 
- - Gly Leu Leu Val Trp Gln Phe Asp Ile Ser Il - #e Ser Pro Val Ala Ala 
20 - # 25 - # 30 
- - Ile Val Thr Asp Thr Phe Asn Ser Ser Asp Gl - #y Gly Arg Leu Phe Gln 
35 - # 40 - # 45 
- - Phe Pro Asp Gly Val Gln Asn Trp Pro Ala Le - #u Ser Ile Val Val Ile 
50 - # 55 - # 60 
- - Ile Ile Met Thr Ile Gly Gly Asn Ile Leu Va - #l Ile Met Ala Val Ser 
65 - #70 - #75 - #80 
- - Met Glu Lys Lys Leu His Asn Ala Thr Asn Ty - #r Phe Leu Met Ser Leu 
85 - # 90 - # 95 
- - Ala Ile Ala Asp Met Leu Val Gly Leu Leu Va - #l Met Pro Leu Ser Leu 
100 - # 105 - # 110 
- - Leu Ala Ile Leu Tyr Asp Tyr Val Trp Pro Le - #u Pro Arg Tyr Leu Cys 
115 - # 120 - # 125 
- - Pro Val Trp Ile Ser Leu Asp Val Leu Phe Se - #r Thr Ala Ser Ile Met 
130 - # 135 - # 140 
- - His Leu Cys Ala Ile Ser Leu Asp Arg Tyr Va - #l Ala Ile Arg Asn Pro 
145 1 - #50 1 - #55 1 - 
#60 
- - Ile Glu His Ser Arg Phe Asn Ser Arg Thr Ly - #s Ala Ile Met Lys 
Ile 
165 - # 170 - # 175 
- - Ala Ile Val Trp Ala Ile Ser Ile Gly Val Se - #r Val Pro Ile Pro Val 
180 - # 185 - # 190 
- - Ile Gly Leu Arg Asp Glu Ser Lys Val Phe Va - #l Asn Asn Thr Thr Cys 
195 - # 200 - # 205 
- - Val Leu Asn Asp Pro Asn Phe Val Leu Ile Gl - #y Ser Phe Val Ala Phe 
210 - # 215 - # 220 
- - Phe Ile Pro Leu Thr Ile Met Val Ile Thr Ty - #r Phe Leu Thr Ile Tyr 
225 2 - #30 2 - #35 2 - 
#40 
- - Val Leu Arg Arg Gln Thr Leu Met Leu Leu Ar - #g Gly His Thr Glu 
Glu 
245 - # 250 - # 255 
- - Glu Leu Ala Asn Met Ser Leu Asn Phe Leu As - #n Cys Cys Cys Lys Lys 
260 - # 265 - # 270 
- - Asn Gly Gly Glu Glu Glu Asn Ala Pro Asn Pr - #o Asn Pro Asp Gln Lys 
275 - # 280 - # 285 
- - Pro Arg Arg Lys Lys Lys Glu Lys Arg Pro Ar - #g Gly Thr Met Gln Ala 
290 - # 295 - # 300 
- - Ile Asn Asn Glu Lys Lys Ala Ser Lys Val Le - #u Gly Ile Val Phe Phe 
305 3 - #10 3 - #15 3 - 
#20 
- - Val Phe Leu Ile Met Trp Cys Pro Phe Phe Il - #e Thr Asn Ile Leu 
Ser 
325 - # 330 - # 335 
- - Val Leu Cys Gly Lys Ala Cys Asn Gln Lys Le - #u Met Glu Lys Leu Leu 
340 - # 345 - # 350 
- - Asn Val Phe Val Trp Ile Gly Tyr Val Cys Se - #r Gly Ile Asn Pro Leu 
355 - # 360 - # 365 
- - Val Tyr Thr Leu Phe Asn Lys Ile Tyr Arg Ar - #g Ala Phe Ser Lys Tyr 
370 - # 375 - # 380 
- - Leu Arg Cys Asp Tyr Lys Pro Asp Lys Lys Pr - #o Pro Val Arg Gln Ile 
385 3 - #90 3 - #95 4 - 
#00 
- - Pro Arg Val Ala Ala Thr Ala Leu Ser Gly Ar - #g Glu Leu Asn Val 
Asn 
405 - # 410 - # 415 
- - Ile Tyr Arg His Thr Asn Glu Arg Val Ala Ar - #g Lys Ala Asn Asp Pro 
420 - # 425 - # 430 
- - Glu Pro Gly Ile Glu Met Gln Val Glu Asn Le - #u Glu Leu Pro Val Asn 
435 - # 440 - # 445 
- - Pro Ser Asn Val Val Ser Glu Arg Ile Ser Se - #r Val 
450 - # 455 - # 460 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 375 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
- - (ii) MOLECULE TYPE: protein 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - Met Ser Pro Leu Asn Gln Ser Ala Glu Gly Le - #u Pro Gln Glu Ala Ser 
1 5 - # 10 - # 15 
- - Asn Arg Ser Leu Asn Ala Thr Glu Thr Ser Gl - #u Ala Trp Asp Pro Arg 
20 - # 25 - # 30 
- - Thr Leu Gln Ala Leu Lys Ile Ser Leu Pro Va - #l Leu Leu Ser Val Ile 
35 - # 40 - # 45 
- - Thr Leu Ala Thr Val Leu Ser Asn Ala Phe Va - #l Leu Thr Thr Ile Leu 
50 - # 55 - # 60 
- - Leu Thr Arg Lys Leu His Thr Pro Ala Asn Ty - #r Leu Ile Gly Ser Leu 
65 - #70 - #75 - #80 
- - Ala Thr Thr Asp Leu Leu Val Ser Ile Leu Va - #l Met Pro Ile Ser Met 
85 - # 90 - # 95 
- - Ala Tyr Thr Ile Thr His Thr Trp Asn Phe Gl - #y Gln Ile Leu Cys Asp 
100 - # 105 - # 110 
- - Ile Trp Leu Ser Ser Asp Ile Thr Cys Cys Th - #r Ala Ser Ile Leu His 
115 - # 120 - # 125 
- - Leu Cys Val Ile Ala Leu Asp Arg Tyr Trp Al - #a Ile Thr Asp Ala Leu 
130 - # 135 - # 140 
- - Glu Tyr Ser Lys Arg Arg Thr Ala Gly His Al - #a Ala Thr Met Ile Ala 
145 1 - #50 1 - #55 1 - 
#60 
- - Ile Val Trp Ala Ile Ser Ile Cys Ile Ser Il - #e Pro Pro Leu Phe 
Trp 
165 - # 170 - # 175 
- - Arg Gln Glu Lys Ala Gln Glu Glu Met Ser As - #p Cys Leu Val Asn Thr 
180 - # 185 - # 190 
- - Ser Gln Ile Ser Tyr Thr Ile Tyr Ser Thr Cy - #s Gly Ala Phe Tyr Ile 
195 - # 200 - # 205 
- - Pro Ser Val Leu Leu Ile Ile Leu Tyr Gly Ar - #g Ile Tyr Arg Ala Ala 
210 - # 215 - # 220 
- - Arg Asn Arg Ile Leu Asn Pro Pro Ser Leu Se - #r Gly Lys Arg Phe Thr 
225 2 - #30 2 - #35 2 - 
#40 
- - Thr Ala His Leu Ile Thr Gly Ser Ala Gly Se - #r Val Cys Ser Leu 
Asn 
245 - # 250 - # 255 
- - Ser Ser Leu His Glu Gly His Ser His Ser Al - #a Gly Ser Pro Leu Phe 
260 - # 265 - # 270 
- - Phe Asn His Val Lys Ile Lys Leu Ala Asp Se - #r Ala Leu Glu Arg Lys 
275 - # 280 - # 285 
- - Arg Ile Ser Ala Ala Arg Glu Arg Lys Ala Th - #r Lys Ile Leu Gly Ile 
290 - # 295 - # 300 
- - Ile Leu Gly Ala Phe Ile Ile Cys Trp Leu Pr - #o Phe Phe Val Val Ser 
305 3 - #10 3 - #15 3 - 
#20 
- - Leu Val Leu Pro Ile Cys Arg Asp Ser Cys Tr - #p Ile His Pro Gly 
Leu 
325 - # 330 - # 335 
- - Phe Asp Phe Phe Thr Trp Leu Gly Tyr Leu As - #n Ser Leu Ile Asn Pro 
340 - # 345 - # 350 
- - Ile Ile Tyr Thr Val Phe Asn Glu Glu Phe Ar - #g Gln Ala Phe Gln Lys 
355 - # 360 - # 365 
- - Ile Val Pro Phe Arg Lys Ala 
370 - # 375 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 398 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
- - (ii) MOLECULE TYPE: protein 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - Met Glu Glu Pro Gly Ala Gln Cys Ala Pro Pr - #o Ala Pro Ala Gly Ser 
1 5 - # 10 - # 15 
- - Glu Thr Trp Val Pro Gln Ala Asn Leu Ser Se - #r Ala Pro Ser Gln Asn 
20 - # 25 - # 30 
- - Cys Ser Ala Lys Asp Tyr Ile Tyr Gln Asp Se - #r Ile Ser Leu Pro Trp 
35 - # 40 - # 45 
- - Lys Val Leu Leu Val Met Leu Leu Ala Leu Il - #e Thr Leu Ala Thr Thr 
50 - # 55 - # 60 
- - Leu Ser Asn Ala Phe Val Ile Ala Thr Val Ty - #r Arg Thr Arg Lys Leu 
65 - #70 - #75 - #80 
- - His Thr Pro Ala Asn Tyr Leu Ile Ala Ser Le - #u Asp Val Thr Asp Leu 
85 - # 90 - # 95 
- - Leu Val Ser Ile Leu Val Ile Pro Ile Ser Th - #r Met Tyr Thr Val Thr 
100 - # 105 - # 110 
- - Asp Arg Trp Thr Leu Ser Gln Val Val Cys As - #p Phe Trp Leu Ser Ser 
115 - # 120 - # 125 
- - Asp Ile Thr Cys Cys Thr Ala Ser Ile Leu Hi - #s Leu Cys Val Ile Ala 
130 - # 135 - # 140 
- - Leu Asp Arg Tyr Trp Ala Ile Thr Asp Ala Va - #l Glu Tyr Ser Ala Lys 
145 1 - #50 1 - #55 1 - 
#60 
- - Arg Thr Pro Lys Arg Ala Ala Val Met Ile Al - #a Leu Val Trp Val 
Phe 
165 - # 170 - # 175 
- - Ser Ile Ser Ile Ser Leu Pro Pro Phe Phe Tr - #p Arg Gln Ala Lys Ala 
180 - # 185 - # 190 
- - Glu Glu Glu Val Ser Glu Cys Val Val Asn Th - #r Asp His Ile Leu Tyr 
195 - # 200 - # 205 
- - Thr Val Tyr Ser Thr Val Gly Ala Phe Tyr Ph - #e Pro Thr Leu Leu Leu 
210 - # 215 - # 220 
- - Ile Ala Leu Tyr Gly Arg Ile Tyr Val Glu Al - #a Arg Ser Arg Ile Leu 
225 2 - #30 2 - #35 2 - 
#40 
- - Lys Gln Thr Pro Asn Arg Thr Gly Lys Arg Le - #u Thr Arg Ala Gln 
Leu 
245 - # 250 - # 255 
- - Ile Thr Asp Ser Pro Gly Ser Thr Ser Ser Va - #l Thr Ser Ile Asn Ser 
260 - # 265 - # 270 
- - Arg Val Pro Asp Val Pro Ser Glu Ser Gly Se - #r Pro Val Tyr Val Asn 
275 - # 280 - # 285 
- - Gln Val Lys Val Arg Val Ser Asp Ala Leu Le - #u Glu Lys Lys Lys Leu 
290 - # 295 - # 300 
- - Met Ala Ala Arg Glu Arg Lys Ala Thr Lys Th - #r Leu Gly Ile Ile Leu 
305 3 - #10 3 - #15 3 - 
#20 
- - Gly Ala Phe Ile Val Cys Trp Leu Pro Phe Ph - #e Ile Ile Ser Leu 
Val 
325 - # 330 - # 335 
- - Met Pro Ile Cys Lys Asp Ala Cys Trp Phe Hi - #s Leu Ala Ile Phe Asp 
340 - # 345 - # 350 
- - Phe Phe Thr Trp Leu Gly Tyr Leu Asn Ser Le - #u Ile Asn Pro Ile Ile 
355 - # 360 - # 365 
- - Tyr Thr Met Ser Asn Glu Asp Phe Lys Gln Al - #a Phe His Lys Leu Ile 
370 - # 375 - # 380 
- - Arg Leu Ser Ala Gln Val Asp Leu Pro Phe Al - #a Val Gly Pro 
385 3 - #90 3 - #95 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 471 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
- - (ii) MOLECULE TYPE: protein 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - Met Asp Ile Leu Cys Glu Glu Asn Thr Ser Le - #u Ser Ser Thr Thr Asn 
1 5 - # 10 - # 15 
- - Ser Leu Met Gln Leu Asn Asp Asp Thr Arg Le - #u Tyr Ser Asn Asp Phe 
20 - # 25 - # 30 
- - Asn Ser Gly Glu Ala Asn Thr Ser Asp Ala Ph - #e Asn Trp Thr Val Asp 
35 - # 40 - # 45 
- - Ser Glu Asn Arg Thr Asn Leu Ser Cys Glu Gl - #y Cys Leu Ser Pro Ser 
50 - # 55 - # 60 
- - Cys Leu Ser Leu Leu His Leu Gln Glu Lys As - #n Trp Ser Ala Leu Leu 
65 - #70 - #75 - #80 
- - Thr Ala Val Val Ile Ile Leu Thr Ile Ala Gl - #y Asn Ile Leu Val Ile 
85 - # 90 - # 95 
- - Met Ala Val Ser Leu Glu Lys Lys Leu Gln As - #n Ala Thr Asn Tyr Phe 
100 - # 105 - # 110 
- - Leu Met Ser Leu Ala Ile Ala Asp Met Leu Le - #u Gly Phe Leu Val Met 
115 - # 120 - # 125 
- - Pro Val Ser Met Leu Thr Ile Leu Tyr Gly Ty - #r Arg Trp Pro Leu Pro 
130 - # 135 - # 140 
- - Ser Lys Leu Cys Ala Val Trp Ile Tyr Leu As - #p Val Leu Phe Ser Thr 
145 1 - #50 1 - #55 1 - 
#60 
- - Ala Ser Ile Met His Leu Cys Ala Ile Ser Le - #u Asp Arg Tyr Val 
Ala 
165 - # 170 - # 175 
- - Ile Gln Asn Pro Ile His His Ser Arg Phe As - #n Ser Arg Thr Lys Ala 
180 - # 185 - # 190 
- - Phe Leu Lys Ile Ile Ala Val Trp Thr Ile Se - #r Val Gly Ile Ser Met 
195 - # 200 - # 205 
- - Pro Ile Pro Val Phe Gly Leu Gln Asp Asp Se - #r Lys Val Phe Lys Glu 
210 - # 215 - # 220 
- - Gly Ser Cys Leu Leu Ala Asp Asp Asn Phe Va - #l Leu Ile Gly Ser Phe 
225 2 - #30 2 - #35 2 - 
#40 
- - Val Ser Phe Phe Ile Pro Leu Thr Ile Met Va - #l Ile Thr Tyr Phe 
Leu 
245 - # 250 - # 255 
- - Thr Ile Lys Ser Leu Gln Lys Glu Ala Thr Le - #u Cys Val Ser Asp Leu 
260 - # 265 - # 270 
- - Gly Thr Arg Ala Lys Leu Ala Ser Phe Ser Ph - #e Leu Pro Gln Ser Ser 
275 - # 280 - # 285 
- - Leu Ser Ser Glu Lys Leu Phe Gln Arg Ser Il - #e His Arg Glu Pro Gly 
290 - # 295 - # 300 
- - Ser Tyr Thr Gly Arg Arg Thr Met Gln Ser Il - #e Ser Asn Glu Gln Lys 
305 3 - #10 3 - #15 3 - 
#20 
- - Ala Cys Lys Val Leu Gly Ile Val Phe Phe Le - #u Phe Val Val Met 
Trp 
325 - # 330 - # 335 
- - Cys Pro Phe Phe Ile Thr Asn Ile Met Ala Va - #l Ile Cys Lys Glu Ser 
340 - # 345 - # 350 
- - Cys Asn Glu Asp Val Ile Gly Ala Leu Leu As - #n Val Phe Val Trp Ile 
355 - # 360 - # 365 
- - Gly Tyr Leu Ser Ser Ala Val Asn Pro Leu Va - #l Tyr Thr Leu Phe Asn 
370 - # 375 - # 380 
- - Lys Thr Tyr Arg Ser Ala Phe Ser Arg Tyr Il - #e Gln Cys Gln Tyr Lys 
385 3 - #90 3 - #95 4 - 
#00 
- - Glu Asn Lys Lys Pro Leu Gln Leu Ile Leu Va - #l Asn Thr Ile Pro 
Ala 
405 - # 410 - # 415 
- - Leu Ala Tyr Lys Ser Ser Gln Leu Gln Met Gl - #y Gln Lys Lys Asn Ser 
420 - # 425 - # 430 
- - Lys Gln Asp Ala Lys Thr Thr Asp Asn Asp Cy - #s Ser Met Val Ala Leu 
435 - # 440 - # 445 
- - Gly Lys Gln His Ser Glu Glu Ala Ser Lys As - #p Asn Ser Asp Gly Val 
450 - # 455 - # 460 
- - Asn Glu Lys Val Ser Cys Val 
465 4 - #70 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (v) FRAGMENT TYPE: N-terminal 
- - (vi) ORIGINAL SOURCE: 
(A) ORGANISM: oligonucleot - #ide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - GATGGTACAC TGGCTGGGGG GTGGGCTGAG TTGACGGTGG CT - # 
- # 42 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vi) ORIGINAL SOURCE: 
(A) ORGANISM: oligonucleot - #ide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - TACCACGCGG CCAAGAGCCT TTACCA - # - # 
26 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vi) ORIGINAL SOURCE: 
(A) ORGANISM: oligonucleot - #ide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- - TGGTGCTAGA GATCTGCTGA CGTTC - # - # 
25 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 45 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vi) ORIGINAL SOURCE: 
(A) ORGANISM: oligonucleot - #ide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- - GAGAAGTCAG ACACACAGAA AGTCTGTGTA AGTTTTACAA CTTGC - # 
- #45 
- - - - (2) INFORMATION FOR SEQ ID NO:12: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (iii) HYPOTHETICAL: NO 
- - (iv) ANTI-SENSE: NO 
- - (vi) ORIGINAL SOURCE: 
(A) ORGANISM: OLIGONUCLEOT - #IDE 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- - GATGGTACAC TGGCTGGGGG GTGGGCTGAG TTGACGGTGG CT - # 
- # 42 
__________________________________________________________________________