AU703697B2

AU703697B2 - DNA encoding a human 5-HT1F receptor and uses thereof

Info

Publication number: AU703697B2
Application number: AU56212/96A
Authority: AU
Inventors: Theresa Branchek; Paul R Hartig; Richard L Weinshank
Original assignee: Synaptic Pharmaceutical Corp
Current assignee: Lundbeck Research USA Inc
Priority date: 1992-01-08
Filing date: 1996-06-26
Publication date: 1999-04-01
Anticipated expiration: 2013-01-08
Also published as: AU5621296A

Description

DNA ENCODING A HUMAN 5-HT 17 RECEPTOR AND USES THEREOF Background of the Invention Throughout this application various publications are referenced by partial citations within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.

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 25 anatomical localization of serotonin and serotonergic neurons in both the peripheral and central nervous 0 systems supports its role in such diverse physiologic and behavioral functions as pain perception, sleep, aggression, sexual activity, hormone secretion, :30 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 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-HTI, 5-HT 2 5-HT 3 and, putatively, 5-HT 4 (Peroutka, 1991). Furthermore, 5-HT 1 sites have been subclassified as: 5-HTIA, 5-HT 1 B, 5-HT 1 c 5-HTID (Hamon et 20 al., 1990) and 5-HTiE (Leonhardt et al., 1989). Although a detailed characterization of the 5-HT1F binding site is lacking, extensive pharmacologic, biochemical and functional properties have clearly shown that the other four subtypes of 5-HT. sites are receptors according to classical criteria.

During the last few years, the field of molecular biology has 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 (Fargin et al., 1988), 5-HTIc (Julius et al., 1988), HTID (Branchek et al., 1990) and 5-HT 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 3 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 has not been previously explored.

This study is a pharmacological characterization of a serotonergic receptor clone with a binding profile different from that of any serotonergic receptor to date.

In keeping with the nomenclature presently accepted for serotonin receptors, this novel site will be termed a HT1F receptor based upon the fact that it possesses high affinity for the endogenous neurotransmitter, S...o **e *ooo* o 4 Summary of the Invention This invention provides an isolated nucleic acid molecule encoding a human 5-HTF receptor (Seq. I.D. No. 1).

This invention also provides an isolated protein which is a human 5-HTIF receptor (Seq. I.D. Nos. 2, 7).

This invention provides a vector comprising an isolated nucleic acid molecule encoding a human 5-HTIF receptor.

This invention also provides vectors such as plasmids comprising a DNA molecule encoding a human 5-HT 1 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 I 2 located relative to the DNA encoding the 5-HTIp receptor 20 as to permit expression thereof.

This invention provides a mammalian cell comprising a DNA molecule encoding a human 5-HT 1 i receptor.

a 00 25 This invention provides a method for determining whether a ligand not known to be capable of binding to a human HTI. receptor can bind to a human 5-HTpI receptor which comprises contacting a mammalian cell comprising an isolated DNA molecule encoding a human 5-HTiF receptor with the ligand under conditions permitting binding of ligands known to bind to a 5-HTpI receptor, detecting the presence of any of the ligand bound to a human 5-HT1F receptor, and thereby determining whether the ligand binds to a human 5-HTIF receptor.

This invention also provides a method for determining whether a ligand not known to be capable of binding to the human 5-HT1F 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 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-HTIF 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-HTIF receptor on the surface of a cell which comprises contacting a mammalian cell Scomprising an isolated DNA molecule encoding a human HTIF receptor with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and 20 bind to, a human 5-HT 1 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-HTIF receptor on the 0 25 surface of a cell which comprises contacting the mammalian cell comprising an isolated DNA molecule encoding and expressing a human 5-HTIF 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-HTIF 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-HTiF receptor.

This invention also provides a method of detecting expression of the 5-HT1F receptor on the surface of a cell by detecting the presence of mRNA coding for a 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-HT1F receptor under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the 5-HTF 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 20 5-HTlF receptor so as to prevent translation of the mRNA molecule.

This invention provides an antibody directed to a human 1 receptor.

go) This invention provides a transgenic nonhuman mammal expressing DNA encoding a human 5-HTIF receptor. This invention also provides a transgenic nonhuman mammal expressing DNA encoding a human 5-HTIF receptor so mutated as to be incapable of normal receptor activity, and not expressing native 5-HTiF receptor. This invention further provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a human 5-HT1F receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a receptor and which hybridizes to mRNA encoding a receptor thereby reducing its translation.

This invention provides a method of determining the physiological effects of expressing varying levels of human 5-HTiF receptors which comprises producing a transgenic nonhuman animal whose levels of human receptor expression are varied by use of an inducible -promoter which regulates human 5-HTIF receptor expression.

This invention also provides a method of determining the physiological effects of expressing varying levels of human 5-HTr, receptors which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of human 5-HTIF receptor.

This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a specific human 5-HT1F 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.

20 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-HT1F receptor and labelled with a detectable marker; e. detecting labelled O 25 bands which have hybridized to the DNA encoding a human 1 F 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-HTIF receptor which comprises inducing cells to express 5-HTIF 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-HTIF receptor which comprises inserting nucleic acid encoding 5-HTF 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.

9 "This invention also provides a transgenic nonhuman mammal 20 expressing DNA encoding a receptor.

9* This invention further provides a transgenic nonhuman mammal expressing DNA encoding a receptor so mutated as to be incapable ot nornal receptor activity, and not 00 25 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 9 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.

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Brief Description of the Ficares Figure 1. (Figures IA-1F) Nucleotide and deduced amino acid sequence of gene 5-HTy, (Seq. I.D. Nos. 1, 2, and 7).

Numbers above the nucleotide sequence indicate nucleotide position. DNA sequence was determined by the chain termination method of Sanger, et al., on denatured double-stranded plasmid templates using the enzyme Sequenase. Deduced amino acid sequence (single letter code) of a long open reading frame is shown.

Figure 2. (Figures 2A-2D) Comparison of the human 5-HT1r receptor primary structures with other serotonin .15 receptors (Seq. I.D. Nos.: 5-HTA 3; 5-HTIe 4; 5-HT 1

D

5; 5-HT1DB 6; 5-HTIF 7; 5-HT2 8).

Amino acid sequences (single letter code) are aligned to optimize homology. The putative transmembrane spanning 20 domains are indicated by stars and identified by Roman numerals (TM I-VII).

Figure 3. 5-HT concentration-effect curves are Srepresented in the absence and in the presence of methiothepin (1.0 MM). Data were normalized to 100% relative to forskolin-stimulated values in the absence of agonist to derive values of Ema x and ES0. The antagonist Kb was estimated by method of Furchgott Kb (Dose of antagonist)/((E 50 in the presence of antagonist/ control E 50 Figure 4. Human tissue distribution of RNA coding for HTIF receptor gene. Total RNA was converted to singlestranded cDNA by random-priming with reverse transcriptase. cDNAs were amplified by PCR using 5-HT 1 y specific PCR primers. PCR products were run on a agarose gel, blotted onto nylon membranes and hybridized 11 to internal gene-specific oligonucleotides and washed under high stringency. Positive controls represent genespecific recombinant plasmids; dH20 served as a negative control. PCR amplification and Southern blotting of RNA samples not treated with reverse transcriptase were negative.

Figure 5 5-HTIF receptor mRNA in the guinea pig brain coronal sections. A. An antisense oligonucleotide probe (4,5 loop) was used. An identical pattern was observed with the 5' untranslated probe (not illustrated).

Hybridization densities are high in layer V of cerebral cortex and in CAl-CA3 of the hippocampus

B.

Control contralateral hemisphere of an adjacent section to that in A. No hybridization was seen using a sense probe of identical specific activity. C. Section hybridized with the antisense probe. The dorsal raphe o. (DR) is densely labeled. D. At high magnification, hybridization (antisense probe) is detected in layer V of 20 sensorimotor cortex. Arrowheads indicate heavily labeled pyramidal cells. E. As in D, through the dorsal raphe.

Arrowheads indicate large, heavily labeled neurons.

Magnification in panels D and E X270.

0* 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 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,,

HT

2 5-HT 3 and 5-HT 4 (Peroutka, 1991). The 5-HT 2 receptor subfamily contains the human 5-HT 2 receptor.

Although no other human members of this family have been 20 described, the rat 5-HT 2 receptor (Pritchett, et al.

1988; Julius, et al. Proc. Natl. Acad. Sci. USA 87:928- 932, 1990) and the rat 5HTIc receptor (Julius, et al.

1988) constitute a rat 5-HT receptor subfamily. The

HT

1 subfamily has been subdivided further as: 5-HT,, 25 WT;I, 5-HTc, 5-HT.: (Hamon et al., 1990) and S(Leonhardt et al., 1989). The 5-HT1A subfamily contains the human 5-HTIA receptor, also known as G-21 (Fargin, et al. 1988) The 5-HTD receptor subfamily contains two members, the 5-HTz.: receptor (also termed 5-HT1D,) and the 5-HT1D-2 receptor (also termed 5-HTIDB). The subfamily contains the human 5-HT1F receptor (also termed clone hll6a). 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-HT1F receptor subfamily so described include the 5-HT 1 receptor and any other receptors which have a greater than 72% homology to the DNA and amino acid sequence shown in Figure 1 (Seq. I.D. Nos. 1, 2, and 7) according to the definition of "subfamily". This invention relates to the discovery of the first member of the human receptor subfamily.

This invention provides an isolated nucleic acid molecule encoding a human 5-HTIF receptor (Seq. I.D. No. 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-HTIF receptor subfamily.

Therefore, any receptor which meets the defining criteria given above is a human 5-HT1F receptor. One means of isolating a human 5-HTF 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-HTir are particularly useful probes for this purpose. DNA and 20 cDNA molecules which encode human 5-HTIp 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 qenomic clones by the screening of cDNA or genomic libraries, by methods described in S 25 more detail below. Transcriptional regulatory elements Sfrom 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 RNJA. cDNA, or isolated genomic DNA molecule encoding a human 5-HTF receptor. Such molecules may have coding sequences substantially the same as the coding sequence shown in Figure 1. The DNA molecule of Figure 1 encodes the sequence of the human 5-HT1F receptor gene (Seq. I.D. No. 1).

14 This invention further provides a cDNA molecule of encoding a human 5-HT 1 I receptor having a coding sequence substantially the same as the coding sequence shown in Figure 1 (Seq. I.D. No. This molecule is obtained by the means described above.

This invention also provides an isolated protein which is a human 5-HTIF 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 Figure 1 (Seq. I.D. Nos. 2, 7) which is a human 5-HTIF receptor.

One means for obtaining isolated 5-HTIF 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 Sw ith the expression vectors described below in more detail.

25 This invention provides a vector comprising an isolated Snucleic acid molecule such as DNA, RNA, or cDNA encoding 0 a human 5-HTlF receptor. Examples of vectors are viruses such as bacteriophages (such as phage lambda), cosmids, plasmids (such as pUCl8, available from Pharmacia, Piscataway, NJ), 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 Figure 1 and designated clone hll6a.

This invention also provides vectors comprising a DNA molecule encoding a human 5-HTIF 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-HTIF receptor as to permit expression thereof. DNA having coding sequences substantially the same as the coding sequence shown in Figure 1 may usefully be inserted into the vectors to express human 5-HTI receptors. Regulatory elements 20 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.

P:\OPER\MRO\56212-96.021 27/1/99 -16- 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,F 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-HTF receptor as to permit expression thereof. Some plasmids adapted for expression in a mammalian cell are pSVL (available from Pharmacia, Piscataway, NJ), pcEXV- 3 (Miller J. and Germain J. Exp. Med. 164:1478 (1986)) and pM05 (Branchek, T. et al, Mol. Pharm. 38:604-609 (1990)).

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 Figure 1 and the regulatory elements necessary for expression of S. 15 the DNA in the mammalian cell which is designated pMO5-hll6a and deposited under ATCC Accession No. 75175. Those skilled in the art will readily appreciate that numerous plasmids adapted for .0 expression in a mammalian cell which comprise DNA of encoding human 5-HTF receptors and the regulatory elements necessary to 20 express such DNA in the mammalian cell may be constructed utilizing existing plasmids and adapted as appropriate to o 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 25 general, and by other methods well known in the art.

The deposit discussed supra, and the other deposits discussed herein, were made for the purposes of Australian Patent No.

667510, in respect of which this application is a divisional application under the provisions of Section 39 of the Patents Act 1990. The deposits 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, Maryland 20852.

I

This invention provides a mammalian cell comprising a DNA molecule encoding a human 5-HT1F 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-HTIF 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-HTIF 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- cells, Y1 cells, etc. A particular example of an Ltk' cell is a cell designated and deposited under ATCC Accession No. CRL 10957 and comprises the plasmid designated pMO5-hll6a. Another example is the murine fibroblast cell line designated N- 5-HTIF and deposited under ATCC Accession No. CRL 10956.

Expression plasmids such as that described supra may be used to transfect mammalian cells by methods well known 20 in the art such as calcium phosphate precipitation, or DNA encoding these 5-HTlF receptors may be otherwise introduced into mammalian cells, by microinjection, to obtain mammalian cells which comprise DNA, cDNA or a plasmid, encoding either human 5-HTlF receptor.

This invention provides a method for determining whether a ligand not known to be capable of binding to a human HT, receptor can bind to a human 5-HT 1 F receptor which comprises contacting a mammalian cell comprising a DNA molecule encoding a human 5-HT 1 F 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 1 F receptor, detecting the presence of any of the ligand bound to the receptor, and thereby determining whether the ligand binds to the 5-HT1F receptor. This invention also provides a method for determining whether a ligand not known to be capable of binding to the human 5-HT1F 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 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., receptor functional output. The DNA in the cell may have a coding sequence substantially the same as the coding sequence shown in Figure 1 preferably, the mammalian cell is nonneuronal in origin. An example of a nonneuronal mammalian cell is an Ltk- cell, in particular the Ltk- cell designated L-5-HTIF. Another example of a non-neuronal mammalian cell to be used for functional assays is a murine fibroblast cell line, specifically the NIH3T3 cell designated N-5-HTp. The 20 preferred method for determining whether a ligand is capable of binding to the human 5-HT 1 i receptor comprises contacting a transfected nonneuronal mammalian cell (i.e.

a cell that does not naturally express any type of or G-protein coupled receptor, thus will only express 25 such a receptor if it is transfected into the cell) 9V expressing a 5-HT. receptor on its surface, or contacting a membrane preparat:on derived from such a transfected cell, with the ligand under conditions which are known to prevail, and thus to be associated with, jn vivo binding of the ligands to a 5-HTIF receptor, detecting the presence of any of the ligand being tested bound to the receptor on the surface of the cell, and thereby determining whether the ligand binds to, activates or prevents the activation of the 5-HTIF 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 preexisting 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-HT1F 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 20 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-HTl. receptor. The transfection system is also useful for determining the 25 affinity and efficacy of known drugs at the human I receptor sites.

This invention also provides a method of screening drugs to identify drugs which specifically interact with, and bind to, the human 5-HT1r receptor on the surface of a cell which comprises contacting a mammalian cell comprising a DNA molecule encoding a human 5-HTIF 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-HT1F 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-HTIF receptor on the surface of a cell which comprises contacting the mammalian cell comprising an isolated DNA molecule encoding and expressing a human 5-HTIF 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-HT1F receptor. The DNA in the cell may have a coding sequence substantially the same as the coding sequence shown in Figure 1 (Seq. I.D. No. 1).

Preferably, the mammalian cell is nonneuronal in origin.

An example of a nonneuronal mammalian cell is an Ltk" cell, in particular the Ltk- cell designated I Another example of a non-neuronal mammalian cell to be used for functional assays is a murine fibroblast cell line, specifically the NIH3T3 cell designated N-5-HT 1 Drug candidates are identified by choosing chemical compounds which bind with high affinity to the expressed 5-HTlF receptor protein in transfected cells, using radioligand binding methods well known in the art, examples of which are shown in the binding assays 25 described herein. Drug candidates are also screened for Q selectivity by identifying compounds which bind with high affinity to one particular 5-HT 1 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-HT1F 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-HT1F receptor, for example with a coding sequence included within the sequence shown in Figure 1. As used herein, the phrase "specifically 25 hybridizing" means the ability of a nucleic acid molecule 00 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 1 F receptors is useful as a diagnostic test for any disease process in which levels of expression of the corresponding 5-HTLF receptor is altered. DNA probe molecules are produced by insertion of a DNA molecule which encodes human 5-HT1F 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 Figure 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-HTIF receptor of are useful as probes for these genes, for their associated mRNA, or 20 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 25 the mRNA produced by the 5-HTIF gene by in situ Shybridization. An example of such an oligonucleotide is: -TCTCACCACTCTCCAAAAGGACTTGGCCATTCACCTCCTCCTTTG-3' (Seq.

I.D. No. 9).

This invention also provides a method of detecting expression of a 5-HT 1 r receptor on the surface of a cell by detecting the presence of mRNA coding for a 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-HT1F receptor under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the HTIF 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.

20 This invention provides an antisense oligonucleotide S. having a sequence capable of binding specifically with any sequences of an mRNA molecule which encodes a human receptor so as to prevent translation of the mRNA molecule. The antisense oligonucleotide may have a 25 sequence capable of binding specifically .with 'any Ssequences of the cDNA molecule whose sequence is shown in Figure 1. As used herein, the phrase "binding specifically" means tne 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 1

F

receptor by passing through a cell membrane and binding specifically with mRNA encoding a human 5-HT 1 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 celltype 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 Figure 1 may be used as the oligonucleotides of the pharmaceutical composition.

20 This invention also provides a method of treating abnormalities which are alleviated by reduction of expression of a 5-HT1F receptor which comprises administering to a subject an amount of the pharmaceutical composition described above effective to 25 reduce expression of the 5-HTIT receptor by the subject.

bThis invention further provides a method of treating an abnormal condition related to 5-HTrI receptor activity which comprises administering to a subject an amount of the pharmaceutical composition described above effective to reduce expression of the 5-HTIF 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

I-

oligonucleotides, or other antisense chemical structures are designed to bind to mRNA encoding the 5-HT 1 I receptor and inhibit translation of mRNA and are useful as drugs to inhibit expression of 5-HTIF receptor genes in patients. This invention provides a means to therapeutically alter levels of expression of human 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 Figure 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 20 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 by designing small, hydrophobic SAOD chemical structures) or by virtue of specific transport systems in the cell which recognize 25 and transport the SAOD into--the cell. -In addition, the SSAOD 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 Figure 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 Cohen, Trends in Pharm. Sci. 10, 435 (1989); H.M. Weintraub, Sci. Am. January (1990) p. In addition, coupling of ribozymes to antisense oligonucleotides is a promising strategy for inactivating target mRNA Sarver et al., Science 247, 1222 (1990)) S: 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 20 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-HTlF receptors.

*o This invention provides an antibody directed to the human 5-HT1r receptor, for example a monoclonal antibody directed to an epitope of a human 5-HTyF 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-HTir receptor included in the amino acid sequence shown in Figure 1 (Seq. I.D. Nos. 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 Figure 1 will bind to a surface epitope of a human 5-HT1F receptor, as described. Antibodies directed to human 5-HT1F 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 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 Figure 1.

As a still further alternative, DNA, such as a cDNA or a .0 fragment thereof, may be cloned and expressed and the 20 resulting polypeptide recovered and used as an immunogen.

SThese antibodies are useful to detect the presence of human 5-HT 1 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 S 25 from animals or humans.

This invention provides a pharmaceutical composition which comprises an amount of an antibody directed to the human 5-HTIF receptor effective to block binding of naturally occurring ligands to the 5-HTlF receptor, and a pharmaceutically acceptable carrier. A monoclonal antibody directed to an epitope of a human 5-HT1F 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 1 F receptor included in the amino acid sequence shown in Figure 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-HTIF 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 receptor and thereby alleviate abnormalities resulting from overexpression of a human 5-HTIF 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-HTIF 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

HT

1 i receptor and thereby alleviate the abnormal condition. Some examples of abnormal conditions are S 20 dementia, Parkinson's disease, feeding disorders, pathological anxiety, schizophrenia, and a migraine headache.

This invention provides a method of detecting the 25 presence of a 5-HT,: receptor on the surface of a cell Qwhich comprises contacting the cell with an antibody directed to the human 5-HTIF 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 1 r 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 1 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-HTIF receptor. This invention also provides a transgenic nonhuman mammal expressing DNA encoding a human 5-HTIF receptor so mutated as to be incapable of normal receptor activity, and not expressing native 5-HT 1 F receptor. This invention also provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a human 5-HTpI receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a receptor and which hybridizes to mRNA encoding a 5-HTiF 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 20 DNA or cDNA molecules having a coding sequence substantially the same as the coding sequence shown in Figure 1 (Seq. I.D. No. An example of a transgenic S. animal is a transgenic mouse. Examples of tissue specificity-determining regions are the metallothionein 25 promotor (Low, Lechan, Hammer, R.E. et al.

Science 231:1002-1004 (1986)) and the L7 promotor (Oberdick, Smeyne, Mann, Jackson, S. and SMorgan, J.I. Science 248:223-226 (1990)).

Animal model systems which elucidate the physiological and behavioral roles of human 5-HT1F receptors are produced by creating transgenic animals in which the expression of a 5-HT 1 F receptor is either increased or decreased, or the amino acid sequence of the expressed

HT

1 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-HTiF 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 1 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 S. 20 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 25 as an example, is as follows: Female mice are mated, 0 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 1 receptor is purified from a vector (such as plasmid hl16a 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 0 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 20 these 5-HT 1 F 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

HT

17 receptors by inducing or inhibiting expression of the native or trans-gene and thus increasing or decreasing expression of normal or mutant 5-HTF, receptors in the living animal. Thus, a model system is produced in which the biological activity of drugs directed against these i 5-HTr receptors are evaluated before such drugs become available. The transgenic animals which over or under produce the 5-HT 1 F receptor indicate by their physiological state whether over or under production of the 5-HTIF 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-HTPI 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-HT1F receptor is achieved therapeutically either by producing agonist or antagonist drugs directed against these 5-HTlF receptors or by any method which increases or decreases the expression of these 5-HTiF.receptors in man.

oS 20 This invention provides a method of determining the physiological effects of expressing varying levels of human 5-HT1F receptors which comprises producing a transgenic nonhuman animal whose levels of human receptor expression are varied by use of an inducible promoter which regulates human 5-HTIj receptor expression.

This invention also provides a method of determining the physiological effects of expressing varying levels of human 5-HTi, receptors which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of human 5-HTIF receptor. Such animals may be produced by introducing different amounts of DNA encoding a human 5-HT 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 1 F receptor comprising administering the substance to a transgenic nonhuman mammal expressing at least one artificially introduced DNA molecule encoding a human 5-HTIF 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 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 Figure 1.

This invention provides a pharmaceutical composition comprising an amount of the substance described suvra effective to alleviate the abnormalities resulting from overexpression of 5-HTI receptor and a pharmaceutically acceptable carrier.

20 This invention further provides a method for treating the abnormalities resulting from overexpression of a human HTIP 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 1 r receptor.

This invention provides a method for identifying a substance capable of alleviating the abnormalities resulting from underexpression of a human 5-HTiF receptor comprising administering the substance to the transgenic nonhuman mammal described above which expresses only nonfunctional human 5-HTF 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 receptor.

34 This invention also provides a pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of receptor and a pharmaceutically acceptable carrier.

This invention further provides a method for treating the abnormalities resulting from underexpression of a human 1 P 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-HTiF receptor.

This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a specific human 5-HTpF receptor allele which comprises: a) obtaining DNA of subjects suffering S. from the disorder; b) performing a restriction digest of the DNA with a panel of restriction enzymes; c.electrophoretically separating the resulting DNA 20 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-HTiF receptor and labelled with a detectable marker; e) detecting labelled bands which have hybridized to the DNA encoding a human 5-HTiy receptor labelled with a detectable marker to 00create 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-HTIF receptor allele.

This invention provides a method of preparing the isolated 5-HTiF receptor which comprises inducing cells to express 5-HTIF receptor, recovering the receptor from the resulting cells, and purifying the receptor so recovered. An example of an isolated 5-HTIF receptor is an isolated protein having substantially the same amino acid sequence as the amino acid sequence shown in Figure 1 (Seq. I.D. Nos. 2, For example, cells can be induced to express receptors by exposure to substances such as hormone s. 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 20 isolated S-HTIF receptor which comprises inserting nucleic acid encoding 5-HT1F 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 or an isolat-ed 5-JiT± re-ceptor -is an -isol-&ted pr-t-ein -ha-v-i-nq 00 substantially the same amino acid sequence as the amino acid sequence shown in Figure 1. This method for preparing 5-HTlF receptor uses recombinant DNA technology 9methods well known in the art. For example, isolated nucleic acid encoding 5-KT,F 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-HTIF receptor is isolated fr om the culture mediu m 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 (Seq. I.D. No. 9).

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 S 20 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 Sthe 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.

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 20 a 5-HT 1 F 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 25 and experimental tools provided by this discovery will be Suseful 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 HTIF receptor. A new human gene for the receptor identified herein as 5-HT1F has been identified and characterized, and a series of related cDNA and genomic clones have been isolated. In addition, the human 5-HT1F receptor has been expressed in Ltk- cells and NIH3T3 P:\OPER\MRO\56212-96.021 27/1/99 -38cells by transfecting the cells with the plasmid pM05-hll6a.

The pharmacological binding properties of the protein encoded have been determined, and these binding properties classify this protein as a serotonin 5-HTiF receptor. Mammalian cell lines expressing this human 5-HT1 receptor at the cell surface have been constructed, thus establishing the first well-defined, cultured cell lines with which to study this 5-HT,, 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.

15 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

EXPERIMENTAL DETAILS Materials and Methods Polvmerase Chain Reaction (PCR): The third (III) and fifth transmembrane domains of the following receptors were aligned and used to synthesize a pair of "degenerate" primers: 5-HTIA (Seq. 1.D. No. 5-HT1c (Seq. I.D. No. 5-HT 2 (Seq. I.D. No. 8) and the HTIDo/o (Seq. I.D. Nos. 5 and 6, respectively) receptors (patent pending). These primers hybridize to opposite strands of target sequences to allow amplification of the region between the corresponding transmembrane domains.

That primer which was designed to anneal to transmembrane domain III is designated 3.17 and consists of a mixture of 192 different 31-mers with two inosine nucleotides; S. the primer which annealed to transmembrane domain V is S- designated 5.5 and consists of a mixture of 288 different 20 27-mers with five inosine nucleotides. EcoRI linkers were included at the 5' end of primer 3.17, to facilitate :".the subcloning of the amplified cDNA in pBluescript (Stratagene) vectors. 5 Ag of poly RNA from rat brain was reverse transcribed by avian myeloblastosis 25 virus reverse transcriptase (AMV) including 3 aM each of 3.17 and 5.5 primers. The resulting single-stranded cDNA was used in a PCR reaction under the following conditions: 94*C for 1 minute, 50 0 C for 2 minutes and 720C for 3 minutes for 40 cycles. Following PCR, 90 Al 30 of the reaction was phenol:chloroform extracted and precipitated; 10 M1 was visualized on a gel using ethidium bromide staining. After precipitation the sample was treated with T4 DNA polymerase and digested with EcoRl prior to separation on a 1% agarose gel. The DNA fragment was isolated from the gel, kinased and cloned into pBluescript. Recombinant clones were analyzed by sequencing.

Cloning and Sequencing: A human lymphocyte genomic library (Stratagene) was screened using the rat S51 fragment (obtained by PCR) as a probe. The probe was labeled with 32 P by the method of random priming (Feinberg et al., 1983). Hybridization was performed at 50 0 C in a solution containing 50% formamide, 10% dextran sulfate, SSC (IX SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), IX Denhardt's (0.02% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), and 200 gg/ml of sonicated salmon sperm DNA. The filters were washed at 50 0 C in 0.1X SSC containing 0.1% sodium dodecyl sulfate (SDS) and exposed at -70C 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, Nucleotide sequence analysis was done by the Sanger dideoxy nucleotide chain- 20 termination method (Sanger 1977) on denatured doublestranded plasmid templates using Sequenase (U.S.

Biochemical Corp., Cleveland, Ohio).

SExpression: The entire coding region of clone hll6a was 25 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-HTIF receptor gene) and the plasmid pGCcos3neo (containing the aminoglycoside transferase gene) into Ltk" cells or NIH3T3 cells using calcium phosphate (reagents obtained from Specialty Media, Lavellette, NJ).

The cells were grown in a controlled environment (370 C, CO0) as monolayers in Dulbecco's modified Eagle medium (Gibco, Grand Island, containing 25 mM glucose and supplemented with 10% bovine calf serum, 100 U/ml penicillin G and 100 mg/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 3 H)serotonin.

Membrane Preparation: Membranes were prepared from transfected Ltk- cells which were grown to 100% confluency. The cells were washed twice with phosphatebuffered saline, scraped from the culture dishes into ml of ice-cold phosphate-buffered saline, and centrifuged at 200 x g for 5 min at 40. The pellet was resuspended in 2.5 ml of ice-cold Tris buffer (20 mM Tris -HC1, pH 7.4 at 230, 5 mM EDTA) and homogenized by a Wheaton tissue grinder. The lysate was subsequently centrifuged at 200 x g for 5 min at 40 to pellet large fragments which were discarded. The supernatant was collected and centrifuged at 40,000 x g for 20 min at The pellet resulting from this centrifugation was washed once in ice-cold Tris wash buffer and finally resuspended in a S: final buffer containing 50 mM Tris-HCl and 0.5 mM EDTA, 2 pH 7.4 at 23°. Membrane preparations were kept on ice 20 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.

S 25 Radiolioand Binding: 3 }5HT binding was performed using slight modifications of the 5-HTID assay conditions reported by Herrick-Davis and Titeler (1988) with the omission of masking ligands. Radioligand binding studies were achieved at 370 C in a total volume of 250 .l of buffer (50 mM Tris, 10 mM MgCl 2 0.2 mM EDTA, 10 MM pargyline, 0.1 ascorbate, pH 7.4 at 370 C) in 96 well microtiter plates. Saturation studies were conducted using 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 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 MM 5-HT. Binding was initiated by the addition of 50 pl membrane homogenates (10-20 Mg). The reaction was terminated by rapid filtration through presoaked polyethyleneimine) filters using 48R Cell Brandel Harvester (Gaithersburg, MD). Subsequently, filters were washed for 5 sec with ice cold buffer (50 mM Tris HCL, pH 7.4 at 40 dried and placed into vials containing 2.5 ml of Readi-Safe (Beckman, Fullerton, CA) and radioactivity was measured using a Beckman LS 5000TA liquid scintillation counter.

The efficiency of counting of 3 H]5HT averaged between 45-50%. Binding data was analyzed by computer-assisted nonlinear regression analysis (Accufit and Accucomp, Lundon Software, Chagrin Falls, OH). ICso values were converted to K values using the Cheng-Prusoff equation (1973). All experiments were performed in triplicate.

Measurement of cAMP Formation Transfected NIH3T3 cells (estimated Bmax from one point competition studies 488 fmol/mg of protein) were 25 incubated in DMEM. 5mM theophylline, 10mM Hepes o Hydroxyethyl]-l-piperazineethanesulfonic acid), 10 M pargyline, for 20 minutes at 37 0 C, 5% C02. Drug doseeffect curves were then conducted by adding 6 different final concentrations of drug, followed immediately by the addition of forskolin (10 uM). Subsequently, the cells were incubated for an additional 10 minutes at 37 0 C, C02. The media was aspirated and the.reaction terminated by the addition of 100 mM HC1. The plates were stored at 4"C for 15 minutes and centrifuged for 5 minutes (500 x g at 4°C to pellet cellular debris. Aliquots of the supernatant fraction were then stored at -200C prior to assessment of cAMP formation by radioimmunoassay (cAMP 43 Radio immunoas say kit, Advanced Magnetics, Cambridge,

MA).

Tise Localization studies. Human tissues (NDRI) were homogenized and total RNA extracted (Sambrook et al. 1989). cDNA was prepared f rom 5 osg of total RNA with random hexanucleotide primers (500 pmoles) using Superscript reverse transCriPtase (BRL) in PCR reaction buffer (Cetus Corp.) containing 1mM dNTPs, at 42 0 C. for 1 hr. An aliquot of the first strand cDNA was diluted in a 50 ml PCR reaction mixture (200 £LM dNTPs final concentration) containing 1.25 U of Taq polymerase and 1 M4M of primers from the sense strand 3 and from the antisense strand (5 1TGTTGATGCCTCAGATAGACTT 3 The PCR products were run o n a 1.5% aqarose gel and transferred to charged nylon membrane (ZetaProbe, Bio-Rad). Filters were hybridized and washed under high stringency.

*:in situ Hybridization. In situ hybridization was 20 performed as described previously (McCabe et al., 1989) using male Hartley guinea pigs (300-350g.). A fragment of the guinea pig 5-HTIF receptor gene was cloned by homology and sequenced. 4.5-base oliqoprobes synthesized to the loop and 5' untranslated regions were 3' end-labeled with 3 5 S-dATP to a specific activity of 4_XicO The.

00 ~n ucl1e ot i de s eque n c es we re 3' and S. STAGCAGTTCCTCTGAGGTCAACTTTTCATCAAAGAGTTTAG. 31.

Sense probes, melting terperature, and RNase pretreatment were used as controls. Sections were exposed to Kodak X- OMAT AR film for 1 week or coated with Kodak NTB-2 emulsion/2% qlycerol(1:l) -for 2 weeks. similar experiments were also done on human tissue.

Drcs 3 K)5-HT (specific activity 28 Ci/minole) was obtained f rom New England Nuclear, Boston, MA. All other chemicals were obtained from commercial sources and 44 were of the highest grade known purity available.

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Results Cloning of a Novel Gene Encoding a 5HT1F Receptor Polyadenylated (poly RNA prepared from rat brain was reverse transcribed and the resulting cDNAs were subjected to amplification by PCR with the use of a set of "degenerate" primers. The synthesis of these primers were based on sequences corresponding to the third and fifth transmembrane segments of the current set of available serotonin receptors. The primers were designed to amplify only serotonin specific sequences. This was accomplished, particularly with the transmembrane domain V primer, which was designed to anneal at its 3' end only to the sequence "AFY(F)IP". We have determined by sequence analysis that the presence of an alanine (A) rather than a serine in the position immediately amino-terminal to the sequence "FY(F)IP" is an amino acid which can distinguish the closely related adrenergic and 00 0 20 dopaminergic receptor families from the serotonergic receptor family. After 30 amplification cycles, agarose gel electrophoresis revealed a clear pattern of cDNA species of approximately 250 base pairs. Individual ,cDNAs were cloned directly into pBluescript and subjected 0* 25 to sequence analysis. One clone, designated S51, was observed to encode a novel serotonin receptor. We then Sscreened a human genomic placental library with the PCR fragment S51. Isolation of the full-length coding region was obtained from a genomic clone designated hll6a.

Nucleotide Sequence and Deduced Amino Acid Sequence of hll6a DNA sequence information obtained from clone hll6a is shown in Figure 1. An open reading frame extending from an ATG start codon at position 1 to a stop codon at position 1098 can encode a protein 366 amino acids in length, having a relative molecular mass (Mr) of 41,660.

A comparison of this protein sequence with previously characterized neurotransmitter receptors indicates that hll6a 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 hll6a. A comparison of the transmembrane homology of hll6a to the other cloned serotonin receptors is shown if Figure 2 exhibits the following order of identity: 5 -HTIDa 5-HT1A 5-HTic and 5-HT 2 eceptor Expression in Transfected Mammalian Cells Saturation analysis of membranes prepared from stably transfected Ltk- cells demonstrated that the receptor 25 expressed was saturable and of high affinity. Scatchard Splot analysis by non-linear regression revealed a Kd of 9.2 t 0.99 nM (mean n=4) and a Bmax 4.4 t 0.36 picomoles mg of protein (mean The percent specific binding determined at the measured Kd value for 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 3 H]5-HT (ICo 0 243 115, nH 0.71 0.08, Imax 55.6 3.2% mean Additional data demonstrating that this coupling to a G-protein is functionally relevant is provided below.

Pharmacological analysis of the receptor was accomplished by testing the ability of drugs from different chemical classes to displace [3H]5-HT specific binding (Table 1).

Of the compounds investigated, 5-HT possessed the highest affinity which according to the classification system of Peroutka and Snyder (1979) makes this site a member of the 5-HTI class. Interestingly, 5-CT possessed low affinity and, thus, discriminates this receptor from that of the 5-HTID receptor as well as other members of this class. The one exception appears to be the recently cloned 5-HT 1 E receptor which also has low affinity for CT Serial No. 803,626, filed December 2, 1991, copending). Various ergoline compounds also bound with high affinity including methylergonovine and :methysergide. Excluding l-napthylpiperazine (Ki 54), piperazine derivatives had low affinity. Interestingly, the rauwolfia alkaloids, rauwolscine and yohimbine, which are alpha-2 adrenergic antagonists had fair affinity for this serotonergic receptor. Furthermore, miscellaneous serotonergic agents that possess high affinity for various receptors within the serotonin family including ketanserin (5-HT2), 8-OH-DPAT (5-HTlA), DOI HT2), spiperone (5-HTA/5-HT2) pindolol and zacopride (5-HT3 had very poor affinity. Taken 0. together, the pharmacological profile of the 5-HT1F receptor is unique and contrasts to that of other known serotonin receptors. Interestingly, the agonist rank order of potency (but not antagonist profile) matches one described for large motorneurons in the spinal cord evaluated electrophysiologically (Connel et al., 1989).

Accordingly, the probability of developing selective drugs for this receptor subtype is increased. The functional 5-HT response (1 uM) was completely blocked by the nonselective antagonist methiothepin (10 gM). This antagonism was surmountable (Fig. indicating probable competitive antagonism. The dose shift produced by 48 methiothepin yielded an apparent Kb of 438 t 14 nM, consistent with the Ki for this compound (Table No direct effect of methiothepin was observed. No other compound tested in this study was an antagonist. In addition, no evidence for coupling of this receptor to P1 turnover was detected at a dose of 10 jsM

S

S. S *5

S

S S S S S S

S

-Table 1. Ki (nM) values of various drugs for the inhibition of 3 H)5-HT specific binding to clonlal 5-HT1F cell membranes. Binding assays were performed With nM of 3 H)5-HT and 10-12 different concentrations of each inhibitory drug. Ki values were calculated from the

IC

5 0 values using the Cheng-Prusoff equation. Each value is the mean t S.E.M. of 2-4 independent determinations.

COMPOUND

L~~

.0 5-HT 10.3 Sumatriptan 23.0 ±11.0 Ergonovirle 31.0 Methylergoflovine 31.0 ±11.0 Methysergide 34.0 ±4.9 5-Methoxy-N,N-DMT 37.5 1-Napthylpiperazile 54.0 ±3.8 Yohimbine 92.0 t 11.0 Ergotamine 171 28 a-Methyl-5-HT 184 20NAN 190 203 t 13 Dihydroergotamile 276 49 Metergolirle 341 t 71 2-Methyl-5-HT 413 5.6 Methiothepii 652 t 41 5 -CT -717 71- TFMPP 1,002 t 5-MT 1,166 t 197 SCH 23390 1,492 t 165 I 1,495 t 893 DP-5-CT 1,613 t 817 DOI 1,739 84 S-OHDPAT1,772 38 1,805 t 220 MCPP 2,020 t 36 Tryptamine 2409 t 103 Quipazine Ritanseril Propaflolol 4,668 t 814 3 ,521 86 I 8,706 t97 Spiperofle >10,000 Zacopr ide >10,000 Piradolol >10,000 Mesulergile >10, 000 Harmalime >10,000 Melatoflin >10,000j a a a a.

a a C a a a a a a a *0 a.

a.

I

cAMP Assay Additional supporting evidence that the 5-HTIF receptor is functionally coupled to a G-protein was obtained by testing the ability of 5-HT as well as other representative serotonergic drugs to inhibit forskolin stimulated cAMP production in NIH3T3 cells transfected with the 5-HT1F receptor. The endogenous indoleamine, HT, produced a concentration-related decrease in forskolin-stimulated cAMP production with an EC50 of 7.1 1.3 nM (n The maximum inhibition of cAMP production by 5-HT was 67 5.4 Additionally, the serotonergic compounds l-napthylpiperazine and lysergol inhibited forskolin-stimulated cAMP production with i: 15 values of 4.5 0.2 nM and 8.8 4.3 nM (n 2), respectively.

Receptor Localization Studies .e 20 Expression of the 5-HTLF transcripts was analyzed from PCR-northern blots and in situ hybridization studies. By PCR, we detected 5-HTIF receptor mRNA in the human brain, uterus (endometrium and myometrium) and mesentery (Fig.

but not in kidney, liver, spleen, heart, pancreas, or testes. In in situ hybridization experiments, we observed 5-HTlF transcripts in lamina V of frontal cortex (Fig 5A) in large pyramidal cells (Fig. 5D). Moderate labeling was also detected over layer VI non-pyramidal neurons. In both layer V and layer VI, the labeling was most evident in dorsal sensorimotor neocortex, and in cingulate and retrosplenal cortices (Fig. 5C) The pyramidal cells in the piriform cortex were heavily labeled as were large neurons in the raphe nuclei (Fig.

Hippocampal pyramidal cells in CA1-CA3 were moderately labeled, as were the granule cells in the dentate gyrus, and some neurons in the nucleus of the solitary tract. Little labeling was found in the thalamus and hypothalamus. significant labelling was also found in the large iotoneurons of the ventral horn of the spinal cord. The localization in the human was found to be in good concordance with that observed in the guinea pig.

Discussion The deduced amino acid sequence of hll6a 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-HT1D a receptor (Fig. 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 60%) to the 0 and 5-HTD.o receptor subfamily. Closely related members of a "subfamily" of serotonin receptors (i.e.

"subtypes") generally share a common transmitter and also S" 15 have similar pharmacological profiles and physiological roles (for example, 5-HT 2 and 5-HTIc or 5-HTID a and HTa) 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; Si.e. 5-HT 2 and 5-HTDa) generally show much lower transmembrane homoloqy (approximately Such 0 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 subfamily, it is likely to be in a subfamily distinct from all the other serotonin receptors. Interestingly, the transmembrane homology between the 5HT1E (Levy et al., 1992; McAllister et al. 1992; Zgombick et al., 1992) and (Amlaiky et al., 1992; Adham et al., in press) receptors is 72%. It is therefore possible that these receptors may be "subtypes", rather than members of distinct "subfamilies".

The present pharmacological evidence substantiates the existence of a novel serotonin receptor in the human brain and peripheral tissues. Comparison of the binding affinities for various drugs observed in native membranes for other known serotonergic receptors (see Hoyer, 1989) to that of the 5-HTF receptor demonstrates that the pharmacological profile does not fit any known receptor to date. The cloning of the 5-HT 1 F site will now allow more extensive investigations into the nature of this unique serotonergic receptor.

The structure-activity relationships observed in the present study suggest that there are important requirements for high affinity binding to the 5 receptor. Substitution or removal of the 5-hydroxy group on serotonin significantly decreases the affinity for the receptor (egs., tryptamine, 5-methoxytryptamine and carboxyamidotryptamine). Additionally, a-methylation and 2-methylation of 5-HT lowers its affinity by 20 and 20 fold, respectively, for the 5-HTlF 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).

yInterestingly, 5-methoxy-N,N-dimethyltryptamine which possesses both a 5-hydroxy substitution as well as a NJNdimethylation has an affinity much higher than the other 5-substituted tryptamine derivatives. Basic structural requirements of the ergoline derivatives demonstrate that N-methylation of the indole ring does not decrease affinity as does bulky substitutions. Furthermore, piperazine derivatives are not bound at high affinity.

Notably, the application of the human 5-HTIF receptor clone to pharmaceutical research can lead to new drug design and development. In this regard, it is important to point out that the affinities of sumatriptan, methylergonovine and methysergide for this receptor suggest that this site may be involved in the control of migraine headaches. Certainly, these compounds have had success in the clinic for the treatment of this debilitating disorder (Sleight et al., 1990). Notably, however, it has been thought that the action of these compounds is mediated at 5-HT1D receptors for sumatriptan and 5-HT2 receptors for methysergide. Interestingly, methylergonovine may be an active metabolite of methysergide which can be responsible for some of the therapeutic antimigraine effects of methysergide. This novel site with affinity for these agents would now suggest that there is one serotonergic receptor which may be responsible for both the pathogenesis and, accordingly, the pharmacological treatment. Importantly, 15 the agents escribed for migraine are not selective for any one p. ticular serotonin receptor and, thus, the physiological significance of drugs acting at one specific site remains controversial (Humphrey P.P.A. et al., 1990). The notion that the 5-HTIF receptor is involved in migraine may be supported by evidence demonstrating that metergoline which has high affinity for the 5-HT 1 D receptor does not block the effects of sumatriptan in the dog saphenous vein (Sumner and tHumphrey, 1990) inferring that this vascular model may contain the novel 5-HTI, site. Furthermore, this data can support the idea that sumatriptan acts at 5-HT 1 p receptors as an anti-migraine drug. Localization of transcripts for the 5-HTIF receptor in the spinal trigeminal nucleus by in situ hybridization strongly supports this contention (Buzzi et al.,1990,1991; Moskowitz et al., 1992). The potential of the 5-HTIF receptor as a novel target for migraine where selective drugs may be developed is an exciting possibility which needs to be explored.

Further insight into potential therapeutic significance of the 5-HT1F receptor has been obtained through localization studies using PCR and in situ hybridization.

Localization of transcripts for this receptor indicates a relatively selective tissue distribution. Of tissues reported here, the 5-HT1F receptor was only detected in a few including the brain, uterus, and mesentery. The possible role of this receptor in uterine or vascular function is intriguing. Future studies defining the specific cell type(s) in these tissues which express the receptor may provide insight into its function in the periphery. Possibilities for therapeutic benefit include dysmenorrhea and labor induction uterus) and hypertension (vascular components of mesentery) and obesity (adipose components). In the brain, the expression of the receptor had a limited distribution compared to that of :15 other serotonin receptors In the neocortex, labelling of layer V pyramidal neurons may indicate a functional role for the 5-HT 17 receptor protein in the integration of sensorimotor (somatodendritic; frontal cortex) or afferent information associated with limbic functions S. 20 (somatodendritic; cingulate/retrosplenial cortex), or in spinal cord processes (axonal). Intense labeling was detected in the large motoneurons of the ventral horn of the spinal cord. Strong labeling was also detected in Shippocampal pyramidal cells, in several thalamic nuclei, and in the dorsal raphe. The detection of transcripts for this gene in the dorsal raphe nucleus indicates a possible role as an autoreceptor. Autoreceptor function opens the possibility that the 5-HT1F receptor could be involved in any or all of the known actions of serotonin including therapeutic potential in anxiety, depression, sleep disorders including jet lag, appetite control, sexual dysfunction, gastrointestinal motility including irritable bowel disease, and cardiovascular regulation.

In addition, localization to the large motoneurons indicates a possible role in spasticity and other disorders of movement.

Another consideration for therapeutic application of this site may be related to the treatment of feeding disorders such as obesity, bulimea 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 wellcharacterized 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). The fact that the 5-HTi, receptor has been cloned from both peripheral and central sites, and has been localized by both PCR and by in situ hybridization, suggests from an i* 15 anatomical standpoint that it can be found in strategic "o locations where feeding may be altered. 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).

SOverall, the 5-HTF receptor can be an important site stimulated by nonselectively blocking serotonin uptake as is accomplished with certain antidepressants. In regard Sto this, serotonin uptake blockers are effective in treating neuropsychiatric disorders such as depression and obsessive-compulsive illness (Asberg et al., 1986; Sleight et al., 1990; 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-HTIF 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. The localization of the 5-HT1F receptor in the raphe nuclei, and therefore its potential role as an autoreceptor, further supports the role for this receptor subtype in depression.

In summary, the pharmacological profile of the cloned human 5-HT 1 F 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. Ultimately, indepth investigations into the localization of this receptor in brain and peripheral 15 tissue will target new sites that may lead to functional roles of the serotonergic receptor. Indeed, the potential .i therapeutic applications may extend to neuropsychiatric disorders including depression, anxiety, schizophrenia, dementia and obsessive-compulsive illness as well as obesity and migraine.

Additionally, the localization of the 5-HTlF receptor in the spinal cord suggests possible roles for this subtype in analgesia as well as spasticity. The clear evidence of involvement of this receptor in the ventral horn further supports the possible role in motor control.

Interestingly, the agonist profile of the 5-HTiF receptor matches that reported for large motoneurons of the spinal cord measured electrophysiologically (Connel et al., 1989). In addition, the presence of the 5-HTir receptor in the mesentery, at ma)or resistance bed of the vascular tree, indicated a role in the control of blood pressure.

A detailed accounting of the localization and therapeutic potential is presented in Table II.

59- Table II. Summary of the localization of mRNA for the receptor in the guinea pig and human CNS. Experiments were performed as described (methods). Each experiment was replicated 2times. potential therapeutic roles anticipated base on these data are indicated.

a a. a a.

a a a a a *aa.

a.

a a a a a a a a a.

a.

a a a.

*aa.a.

a LOCALIZATION OF HUMAN 5HT 1 F mORA' a.

a.

a a .a a a.

AREAS PROJECTIONS THERAPEUTIC

RELEVANCE

FRONTAL CORTEX Main projections to Potential application striatum, dorsal for the development thalamus, and of treatments for superior colliculus. schizophrenia and mood disorders.

CAUDATE Primary projections Potential treatment NUCLEUS to globus pallidus, of any basal ganglia substantia nigra. disorder, including Parkinson's disease, Huntington's chorea, or tardive dyskinesia.

HIPPOCAMPAL Pyramidal neurons Primary locus for FORMATION project mainly treatment of memory within the disorders, e.g.

hippocampus, and Alzheimer's disease also to the septum. or for cognitive enhancement in people with learning disabilities. Also possible treatment for temporal lobe epilepsy.

AMYGDALA Cells in amygdala Wide range of have widespread potential projections to applications. These cortex, hippocampus, include treatment of basal ganglia, autonomic hypothalamus, and dysfunctions such as brainstem autonomic cardiac arrhythmias centers. and non-adaptive response to environmental stressors. Also potential treatment of mood disorders, such as bipolar syndrome.

Table 2 continued -61- AREAS PROJECTIONS THERAPEUTIC

RELEVANCE

HYPOGLOSSAL Main projections to Treatment of verbal NUCLEUS somatic skeletal apraxia.

musculature of the tongue.

DORSAL Principal May have some EFFERENT projections are to application to the NUCLEUS OF THE the parasympathetic treatment of stress- VAGUS ganglia and related ulcers and abdominal viscera. iritable bowel disease.

NUCLEUS OF THE Main projections are Varied potential SOLITARY TRACT to thalamus, applications, with amygdala, regulation of cardiorostroventral vascular function the medulla, and the Al most prominent, e.g.

noradrenergic cell an anti-hypertensive.

group of the dorsal medulla.

GRACILE Provides innervation Potential NUCLEUS of lumbosacral applications for the spinal cord. treatment of dermatitis, or pain associated with itching.

CUNEATE Provides innervation Potential NUCLEUS of cervical spinal applications for the cord. treatment of dermatitis, or pain associated with itching.

a a. a a a 4aa a #4 a a a a a a.

Table 2 continued -62- AREA" PROJECTIONS

TERAPEUTC

SPIN~AL Main projections are Potential treatment TRIGEMINAL to the contralateral of migraine NUCLEUS ventrobasal headaches, and other thalamus, the pain syndromes such posterior thalamic as trigeminal the zona neuralgia.

incerta, the superior colliculus, and the motor nuclei of trigeminal.

OLIVARY Primary projectionls Treatment of ataxia COMPLEX are to the associated with cerebellum. olivopontocerebellar atrophy, or tremors accompanying some neurodegene rat ive diseases RETICUXLAR Pro)ect ions to the Involvement in FORKjATION ihtra-lamiflar and cardiac pressor and dorsomedial n. of depressor responses thalamus, the suggests a role in hypothalamus, blood pressure supramammillary and regulation and lateral mammillary possibly a treatment nuclei, iheseptun, for hypertension.

the diagonal band, Also possible spinal cord, application for the cerebellun. treatment of urinary braist--7. autonomic retention disorders, nuclei, and in the management of pain.

0* 0* @0 0 0 *0 0 00 0 .9 0 0 04 0000 I 0 0 0 000 0 00 0 0 S 0 0004 0**0 00

SR

00 *000 9* 0 5.

000000 0

MEDIAL

VESTI BULAR

NUCLEUS

Pro~ect ions to oculomotor- complex and cerv'ica! spinal cord rotor ne urons.

sickness.

Table 2 continued -63- AREAS ~~PROJECTIONSTHRPUI .1 RELEVANCE CEREBELLAR Projections only to Potential treatment PLTRKINJE CELLS deep cerebellar of movement nuclei, disorders, particularly those involving planned movements, or those invloving abnormalities of gait or stance.

SPINAL CORD Ascending dorsal Primary site for VENTRAL HORN horn projections to treatment of pain, thalamus, brainstem and for possible reticular formation anesthetic and central gray. applications. Also Ventral horn possible therapies pro~ect ions to for spasticity and skeletal and/or movement disorders.

smooth muscle.

00 0 SO 000 0 0 60 0 0 00 0 4 0 005 Table 2 continued -64- LOCZLIZ3TION OF GUINEA PIG SIT 1 IFsi AREAS PROJECTIONS IRELEVANCE TO HUMAN

ITHER.APIES

ANTERIOR Widespread Treatment of OLFACTORY projections to olfactory disorders NU CLEI and brain olfactory (dysosmias) PIRIFORM centers, to limbic associated with-many CORTEX system, neurological hypothalamus, syndromes.

striatum. ri LAYER V of Cells of layer V Enhancement of memory NEOCORTEX project primarily for motor tasks, to other cortical particularly in areas, and to certain amnestic basal ganglia. syndromes, e.g.

Alzheimer's disease.

CAUDATE- Medium spiny Potential treatment PUTAMEN and neurons project to lof any basal ganglia NUCLEUS globus pallidus, Isodr nldn ACCLJMBENS entopeduncular Parkinson's disease 9 and substantia Huntington's chorea: nigra. or tardive dyskinesia.

AMYGDALA Cells in amyqdal a Wide range of have widespread 1potential -pro'lect xoTIs tc appl i-cat-ion-s. These cortex. i include treatment of hippocampus. oasa: :autonomic ganglia. ;dysfunctions such as hypothalamus. anz 'cardiac arrhythmias brainstem and non-adaptive autonomic centers. response to environmental stressors Also potential treatment of mood disorders, such as bipolar syndrome.

9* 6~.9 Table 2 continued S *5 S

S

a

S

*SSSS.

S

ARMPR03ICTIONS RELEVANCZ TO NUXPJI

THERXPIES

HIPPOCAMPIS Pyramidal neurons Primary locus for project mainly treatment of memory within the disorders, e.g.

hippocampus, and Alzheimer's disease also to the or for cognitive septum. enhancement in people with learning disabilities. Also possible treatment for temporal lobe epilepsy.

DORSAL RAPHE Extensive Treatment of pain projections to syndromes, including cerebral cortex, migraine headache.

frontal striatum, Involvement of raphe limbic structures, in general olfactory arousal/attentional tubercle, central processes makes this gray, hippocampus, a possible target for and spinal cord. treatment of attentiona 1 dysfunctions, such as those observed in Alzheimer's disease, or in developmental disabilities.

Potential application in the treatment of depression.

PON TINE Ma)-or projection -Potential treatment HUC=EI is to the of movement cerebellar cortex. disorders, particularly planned movement, and gait disorders such as.

Friedrich's ataxia.

COLLICULUS synaptic station in ascending auditory pathway.

I

Table 2 continued -66- ARZEA PROJECTIONS RELEVANCE TO UNAN

THERAPIES

ii

I

E

TRIGEMINAL

NUCLEAR

COMPLEX

PONTINE

RETICULAR

FORMATION

A.GIGANTOCEL

LULAR

RETICULAR

NUCLEUS

B.PARAGIGANT

OCELLULAR

RETICULAR

NUCLEUS

C.RAPHE

MAGNUS

MEDIAL

VESTIBULAR

NUCLEUS

CEREBELLAR

PURKINJE

CELLS

Main projections are to the contralateral ventrobasal thalamus, the posterior thalamic the zona incerta, the superior colliculus, and the motor nuclei of trigeminal.

I

Projections to the intra-laminar and dorsomedial n. of thalamus, the hypothalamus, supramammillary and lateral mammillary nuclei, the septum, the diagonal band, spinal cord, cerebellum, brainstem autonomic nuclei Involvement in cardiac pressor and depressor responses suggests a role in blood pressure regulation and possibly a treatment for hypertension.

Also possible application for the treatment of urinary retention disorders, and in the management of pain.

Potential treatment of migraine headaches, and other pain syndromes such as trigeminal neuralgia.

Projections t oculomotor complexi and cervical spinal cord motor neurons.

Treatment of motion sickness.

Projections only Potential treatment to deep cerebellar of movement nuclei, disorders, Sparticularly those 'involving planned movements, or those invloving abnormalities of gait or stance.

Table 2 continued -7 -67- SPINAL CORD Ascending dorsal horn projections to thalamus, bra instem reticular formation and central gray.

Ventral horn projections to skeletal and/or smooth muscle.

Primary site for treatment of pain, and for possible anesthetic applications. Also possible therapies for spasticity and movement disorders.

i i a a a a a a a a -68sef erences Adham, Kao, Schechter, Bard, 32., Olsen, Urquhart, Durkin, Hartig, Weinshank and Branchek, T.A. Cloning of Another Human Serotonin Receptor (5-HTIF): A fifth 5-HT, Receptor Subtype Coupled to the Inhibition of Adenylate Cyclase.

Proc. Natl. Acad. Sci. in press.

Amlaiky, Ramboz, Boschert, Plassat, J-L. and Hen, Isolation of a mouse 1'5HTIE-likel" serotonin receptor expressed predominantly in hippocampus. J. Biol.

Chem. 267:19761-19764, 1992.

Asberg, Erikssofl, Matensson, Traskmafl-Bendz, L. and Wagner, Therapeutic effects of serotonin uptake inhibitors in depression. 3. Clin. Psychiat.

47:23-35, 1986.

Blundell, J.E. and Lawtonl, Serotoflin receptor subtypes and the organisation of feeding behaviour: Experimental models. In: Serotonifl From cell biology to pharmacology and therapeutics. (eds. Paoletti,

R.,

Vanhoutte, Brunello, N. a-nd Ma-gg-i

F.M.)

00 Boston:Kluwer Academiuc Publishers, pp 213-219, 1990.

.*Bradford, A rapid and sensitive method for the quantification of mic-roqram quantities of protein utilizing the principle of protein-dye binding. Anal.

Biochem. 72:248-254. 1976.

Branchek, Weinshank, Macchi, Zgombick, J.M. and Hartig, Cloning and expression of a human 5-HT1D receptor. The Second IUPHAR Satellite Meeting on Serotonin, Basel, Switzerland, July 11-13, 1990, Abstract 1 2.

-69- Buzzi, M.G. and Moskowitz, the antimigraifle drug, suinatriptan (GR43175), selectively blocks neurogenic plasma extravasation from blood vessels in dura mater.

Br. J. PharmaCol. 99:202-206, 1990.

Buzzi, M4oskowitz, Peroutka, S.J. arnd Byun, Further characterization of the putative receptor which mediates blockade of neurogenic plasma extravasation in rat dura mater. Br. J.Pharmacol.

;03:1421-1428, 1991.

Cheng, Y.C. and Prusoff, Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50% inhibition (1C50) of an enzyme reaction. Biochem. Pharmacol. 22:3099-3108, 1973.

Conihel, L.A. and Wallis, depolarizes neonatal rat motorneurones through a receptor unrelated to an identified binding site.

Neuropharmacology 28:625-634, 1989.

Cooper, S.J. Drugs interacting with 5-HT systems show promise f or treatment of eating disorders. TIPS 10:56-57, 00 Fargin, Raymond, Lohse, Kobilka,

B'.K.

Caron, M.G. and Lefkowitz, R.JT.: The genomic clone G-21 which resembles a 8-adrenergic receptor sequence encodes the 5-HT1A receptor. Nature 335:358-360, 1988.

Feinberg, and Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal.Biochemh. 132:6-13, 1983.

Gaddum, 3.H. and Picarelli, Two kinds of tryptamifle receptor. Brit. J. Pharmacol. 12:323-328, 1957.

Glennon, Serotoflin receptors: Clinical implications. Neurosci. Biobehav. Rev. 14:35-47, 1990.

Green, Neuropharmacology of serotonin. Oxford: Oxford University Press, 1985.

Hamoni, Lanfumey, El Mestikawy, Boni, C., Miguel, M. Bolanos, F. Schechter, L. and Goz lan, H. The main features of central 5-HTl receptors.

Neuropsychopharmacol. 3(5/6) :349-360, 1990.

Hartig, Kao, Macchi, Adham, N., Zgombick, Weinshank, R. and Branchek, The molecular biology of serotonin receptors: An overview.

Neuropsychopharmacol. 3(5/6) :335-347, 1990.

~:Herrick-Davis K. and Titeler, Detection and' characterization of the serotonin 5-HTD receptor in rat and human brain. J. Neurochem. 50:1624-1631, 1988.

Hoyer, Biochemical mechanisms of 5-HT receptor- .~effector coupling in peripheral tissues. In: Peripheral actions of 5-HT. (ed. Fozard, Oxford:Oxford University Press, pp 72-99, 1989.

*25 00 Humphrey, Feniuk, Perren, Beresford.

Skingle, M. and Whalley, E.T. Serotonin and migraine. Ann. N.Y. Acad. Sci. 600:587-600, 1990.

Insel, Mueller, Alterman, Linnoila,

M.

and Murphy, Obsessive-compulsive disorder and serotonin: Is there a connection? Biol. Psychiat.

20:1174-1188, 1985.

JimerSon, Lesem, Hegg, A.P. and Brewerton, Serotonin in human eating disorders. Ann. N.Y.

Acad. Sci. 600:532-544, 1990.

-71- Julius, MacDermott, Axel, R. and Jessell, T.M.: Molecular characterization of a functional CDNA encoding the serotonin 1C receptor. Science 241:558-564, 1988.

tLeonhardt, Herrick-Davis, and Titeler, M.: Detection of a novel serot ofin receptor subtype in human brain: Interaction with a GTP-binding protein.

J. Neurochem. 53(2):465-471, 1989.

Levy, F. Gudermann, Birnbaumer, Kaumann, A.

J. Birnbaumer, L. Molecular cloning of a human gene (S31) encoding a novel serotonin receptor mediating inhibition of adenylyl cyclase. FEBS Lett. 296, 201-206, 1992.

Maniatis, Fritsch, and Sambrook, J. Molecular *~Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.

McAllister, G. Charlesworth, A. Snodin, Beer, M.

Noble, A. Middlemiss, D. Iversen, L. .Whiting, P. Molecular cloning of a serotonin receptor from human brain (5-HT1E) A f if th 5HT1-like subtype.

Proc. Natl. Acad. Sci. (USA). 89, 5517-5521, 1992.

00 McCabe, J.T. and Pfaff, in Gene Probes (Academic Press, San Diego) Conn, P.M. pp. 98-126, 1989.

Moskowitz, M.A. Neurogenic versus vascular mechanisms of sumatriptan and ergot alkaloids in migraine. Trends Pharmacol. Sci. 13:307-311, 1992.

Osborne, N.N. and Hanion, M1.: Neuronal serotonin.

Chichester: John Wiley and Sons, Inc., 1988.

Peroutka, Serotonin receptor subtypes: Basic and clinical aspects. New York: Wiley-Liss, Inc., 1991.

-72- Peroutka, S.J. and Snyder, S.H. .Multiple serotonin receptors, differential binding of 3 hydroxytryptahile, 3 H3lysergic acid diethylainide and I 3 H)spiroperidol. Mol. Pharmacol. 16:687-699, 1979.

Pritchett, Bach, Wozfly, Taleb, Dal Toso, Shih, J. and Seeburg, Structure and functional expression of cloned rat serotonin 5-HT2 receptor. EMBO J. 7:4135-4140, 1988.

Rapport, Green, A.A. and Page, Purification of the the substance which is responsible for vasoconstrictor activity of serum. Fed. Proc. 6:184, 1947.

Rapport, Serum vasoconstrictor (serotonin)

V.

Presence of creatinine in the complex. A proposed structure of the vasoconstrictor principle. J. Biol.

Chem. 180:961-969, 1949.

*20 Sambrook, Fritsch, E. Maniatis, in Molec'ular Cloning: A Laboratory Manual; 2nd edition.

(Cold Spring Harbor Laboratory, Cold Spring Harbor,

NY),

pp. 7.19-7.22, 1989.

Sanders-Bush, The Serotonin Receptors. Clifton, New Jersey: Humana Press, 1988.

Sleight, Pierce, Schmidt, Hekmatpanah, C.R. and Peroutka, The clinical utility of serotoflin receptor active agents in reuropsychiatric disease. In: Serotoflin receptor. subtypes: Basic and clinical aspects. (ed. Peroultka, New Inc., pp 211-227, 1990.

Sumner, M.3. and Humnphrey, Sumatriptan (CR43175) inhibits cyclic-AMP accumulation in dog isolated -73saphenous vein. Br. J. Pharmacol. 99:219-220, 1990.

Zgombick, Schechter, Macchi, Hartig, Branchek, and Weinshaik, R.L. The human gene S31 encodes the pharmacological ly-def ined serotonin receptor. Mol. Pharmacol. .42, 180-185, 1992.

-74- SEQUENCE

LISTING

GENERAL INFORMATION: APPLICANT: Weinshank, Richard

L.

Branchek, Theresa Hartig, Paul R.

(ii) TITLE OF INVENTION: DNA ENCODING A HUMAN 5-HTlF RECEPTOR

AND

USES THEREOF (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Cooper Dunham STREET: 30 Rockefeller Plaza CITY: New York STATE: New York COUNTRY:

USA

ZIP: 10112 COMPUTER READABLE

FORM:

MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM:

PC-DOS/MS-DOS

SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT

INFORMATION:

NAME: White, John P REGISTRATION NUMBER: 28,678 REFERENCE/DOCKET NUMBER: 1795/39318 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 212-977-9550 TELEFAX: 212-664-0525 TELEX: 422523 COOP UI INFORMATION FOR SEQ ID NO:1: SEQUENCE

CHARACTERISTICS:

LENGTH: 1730 base pairs TYPE: nucleic acid S STRANDEDNESS: both TOPOLOGY: linear (il) MOLECULE TYPE: DNA (qenomiri (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

FRAGMENT TYPE: N-terminal (vii) IMMEDIATE SOURCE: LIBRARY: human lymphocyte genothic CLONE: hll6a (ix) FEATURE: NAME/KEY: CDS LOCATION: 616..-1713 (ix) FEATURE: NAME/KEY: mat peptide LOCATION: 6167.1713 (xi) SEQUENCE DESCRIPTION: SEQ ID 140:1: TTGCATGCCT GCAGGTCGAC TCTAGAGGAT

CCCCGGOTAC

ATTTTGTCAT

ACATTCCAGC

GTCAGTCAGT

CACACTTTTA

ATTrTCACTGA

AGAAATGTTT

AGOAACTA

ANCATAACAT

CTCTGAACTG

m11AANACAA

GCTTCAAGCC

CTTTGAATAA

AGTACTGAAA

AACTTTTTCT

GTAACAAGCT

TGGAAGTTAC

AAACCTrCAA

ACACTTTTTA

TTTTTTCTCT

TAGGAA.AAGC

GGGCACTGGC

CAGTTGTTAC

OAcATGGACA

AGAOTATCAT

TGGCTTTTTT

TCTGAACCTC

AAAATTATTC

TCCCTTGTTA

CTAAOCAAAA

TCTATCAGCT

GGAGGCCTGC

AAGAGAAAA

TAAAAATTGT

TACTCTTCTC

ATTT =.TAA

TGAAAGGAAG

CAGGTATCCA

CGAGCTCGAA

CTCTTGGTGG

TTGN.ATATAT

GTTATTGAGA

CCAATTCTAT

TGTATTAPAC

ATTPATTTC

TCTATAGAAT

AGAAAAGTTC

TTTTTCAGCT

TTCCTTTGTT,

GCTCTTTGTT

A6ACTCAACTA

TCGGGCCTGC

AATGGCAGAG

CTATATTTTA

TTAAATAAAN

ATTCTGGGTA

TTGAAGCCTT

ATAT'TAATCT

a a.

a a. a a a a .a a a 00 a a AGAAA ATG GAT TTC TTA ANT TCA TCT1 GAT CAA AAC TTG ACC Met Ap Phe Leu Auh Ser Ser Asp Gin Nun Leu Thr 1 5 120 1 8C 240 300 360 420 480 540 600 651 699 74-7 795 843 892 TCA GAG ONA CTG TTA NAC AGA ATO CCA TCC AA ATT CTG GTq TCC CTC Ser Glu Giu Lou Lou Non Nrg met Pro Ser Lys Ile Leu Val Ser Leu 1520 ACT CTC TOT 000 CTG GCA CTG ATG ACA ACA ACT ATC ANC TCC OTT GTG Tbr Lou Swr 0-ly Lou Ala Lou Met Thir.Thr Thr Ile an Ser Leu Val.

35 ATC OCT OCA ATT NTT GTO ACC COG AAG CTG CAC CAT CCA CCC AAT TAT Ile Ala Ala Ile Ii. Val Thr Arg Lys Leu His His Pro Ala Nun Tyr 45 so 55 TTA AT TOT TCC OTT GCA GTC ACN GAT TTT CTT GTG GOT GTC CTG OTG Lou Ile Cym Ser Leu Ala Val Thr Ap Phe Leu Val Ala Val. Leu Val.

A 70 ATG CCC TTC met Pro Phe CAA GTC OTC Gin Val Val AGC ATT GTG Ser Ile Val.

TOT GAC ATT Cys Ap Ile TAT ATT G70 Tyr Il~e Val 85 TOG CTG ACT Trp Leu Ser 100 AGN GAG NOC Arg Glu Ser GTT GAC ATT Val. Asp Ile TOG ATT ATG CG Trp Ile met Giy ACC TGC TOC ACC Thr Cys Cys Thr 105 a. a a a.

a a a a a

TGC

Cys

ATC

II

125

GGC

Giy

CCT

Pro

ATC

Ile

GCT

Ala

TAT

Tyr 205

GCA

Ala

AGC

Ser GAc As An

GG

G1 28 Al

AP

As

GC

AJ

TCC ATC T1 Ser lIe LI 110 ACA CAT GC Thr Amp A.

ATT ATG A lie Met I CCT CTA T Pro Leu P 1 ATC AAG C Ile Lys H 175 TTC TAC A Phe Tyr I 190 AGA GCA C Arg Ala P AAG GAG C Lye Glu C ACT AA Thr Lye CCA TCA p Pro Ser 255 TCT GAA Ser Glu 2'70 T ACA ACA y Thr Arg 5 A TT T TA a Ph. Val T OTC TOT In Val CyS :A TG CTT La Trp Lou 335

'U

uu

-T

la

TT

1 e

TC

he 60

AC

is

ITC

1.e

;CA

1 a

AC

I u rci Sex 24C

ACJ

Tn

TT(

P h

GA

G1

I

AT

3: 1C CAT Cl His L* GTT G) Val GI

I:

ACA A Thr 1 145 TGG Al Trp A GAC C Asp H CCA C Pro L AAG A Lye T 2 IVal J1 225 GTT I Val T I GAC r Asp e Lye A COG u Arg A TOT Cys 305 LC AAA ip Lye 00 0 TAT Ly Tyr *C TC iu S4 11 GO T1 LU Tj 30 rA G Le V GG C rg H AC A is I TG G eu P CA I hr I '10 IAT C anf C 1CC jer rTI Phe

CAT

His

AAA

Lye 290

TOG

Trp

TOT

cym

CTC

Leu

:A

rr L5 kT fr

T

al

AC

is

TT

le

CA

4 a .95

,TA

.eu

;GC

kC) rhi

GA'

Asi GAl 27

GC

Al

CT

Le

AA

Ly

A)

Al GCT Al Ala II CCC A( Ala A TG A' Trp I CAA G' Gin G 1 GTT T Val S 180 TTG A Leu I TAC C Tyr I CAA C Gin

TCC

Ser 1AA p Lye 260 G AAA u Lys 5 A 0CC a Ala 1 CCT 'u Pro

LAA

9e Ile in Ser 340

AA

Le 10 rT ie

GA

ly 65

CC

er

,TT

le

:AC

lis

TC

lal rAl Tyz 24'

AT

ii

TC

Se

AC

Th Ph

TC

Se 32

CI

LI

-76- OCT TTG GA Ala Leu AS 12 AAA AGG AC Lye Arg Tk 135 ATA TCT G1 Ile Ser Vl 150 ACT ACC Al Thr Ser A ACC ATT T Thr Ile T TTG ATC C Leu Ile L 2 AAG AGA C Lys Arg G 215 CTT TTO C Leu Leu C 230 GTA CTA C Val Leu C F CAT AGC e His Ser 1 TOG AGA r Trp Arg T ACC CTG r Thr Leu 295 T TTT OTA e Phe Val 310 TNA GAA !r Giu Glu 55 1T ATA AAT ?u le Aen

TT

'p 0l

:A

r

T

iu

;A

rg

AC

yr 2T eu 00

AA

In

;AG

;lu Al 1% rhl

AC

Ar 28

GG

Gl

AA

Ly

AT

me P2 CGG TIJ Arg Ty CCA Al Pro Lj TTT A Phe I.

GAT G Asp A 1 TCA A Ser T 185 TAC T Tyr T GCA A Ala S ACT C Ser C

AAG

Lye k GTG r Val 265 G CAA 9 Gln 0 A TTA y Leu

SGAA

's Glu '0 TCC rt Ser :A CTG ro Leu 345

LT

rU Le le

AT

sp

CA

hr

AC

yr

LGT

ler ez

AA

Ly

AT

Ii

TT

Se

AG

Ar 3A Al

II

CGA

Arg

CAT

His

TCT

Ser 155

GAA

Glu

TTT

Phe

AAA

Lye

AGG

Arg

GAG

Glu 235

TTP

Let k AGI g Set

ATI

S I I C T71 e Le 'A OT u Va 31 LT TT In Ph I0 rT TP Le Ty

GCA

Ala

OCT

Ala 140

ATG

Met

TGC

Cys

GGA

Gly

ATA

Ile

ATT

Ile 220

AA

Lye

TCT

Ser

CTC

Leu

TCA

e Ser u Gly 300 T OTT 1 Val T TTG e Leu LC ACA ,r Thr 1227 1275 1323 1371 1419 1467 1515 1563 1611 1659 987 1035 1083 1131 -77- ATC TT AAT GAA GAC TTC hAG AAA GCA TTC CAA AAG CTT GTG CGA TGT Ile Phe Asn Glu Asp Phe Lys Lye Ala Phe Gin Lys Leu Val Arg Cy.

350 355 360 CGA TGT TAGTTTTAAA AATGTTT Arg Cys 365 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 366 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 1707 1730 Het Asp Phe Lou Ann Ser Ser.Asp Gin Ann Leu Thr Ser Glu Glu a a a a a a *c a 1 Lou Lou 110 Lou 65 110 Asp HIs Val Thr 145 Trp Asp Pro en Ala Val 50 Ala Val lie Lou Glu 130 Ile Arg His Lou Arg Lou Thr Val Tyr Trp Scr 115 Tyr Val Hi~s Ala 195 Met met Ar; Thr Ilec Lou 100 Ala Ala Trp Gin Val1 180 Lou Pro Thr Lye Asp Val 85 Ser 110 Ar; Ile Gly 165 Ser Ile Ser Thr Lou Phe 70 Ar; Val Ala Lys Ile 150 Thr Thr Leu Lys Thr His 55 Lou Glu Asp Lou Arg 135 Ser Ser 11e Ile Ile Ile 40 His Val1 Ser Ile Asp 120 Thr Val Ar; Tyr Leu 20C Lou 25 Aen Pro Ala TrF Thr 105 Ar; Pro Phe Asp Ser 185 y r 10 Val Ser Ser Leu Ala Ann Val Lou 75 Ile met 90 Cyo Cym Tyr Ar; Lye HLs ile Ser 155 Asp Clu 170 Thr Phe Tyr Lys Lou Val Tyr Val Gly Thr Ala Ala 140 Met Cys dly Ile Thr Ile Leu Met Gin Cys Ile 125 Gly Pro Ile Ala Tyr 205 Lou Ala Ile Pro Val Ser 110 Thr Ile Pro Ile Phe 190 Ar; Ser Ala Cys Phe Val Ile Asp Met Lou Lys 175 Tyr Ala Leu Gly Ile Ser Ser so Cys Leu Ala Ile Phe 160 His Ile Ala Lye Thr Lou Tyr His Lys Gin Ala Ser Arg le Ala Lye Clu Glu 220 -78- Val Ann Gly Gin Val Leu Lou Glu Ser Gly Giu Lye Ser Thr Lye Ser 225 230 235 240 Val Ser Thr Ser Tyr Val Leu Giu Lys Ser Leu Ser Amp Pro Ser Thr 245 250 255 Amp Phe Asp Lys Ile His Ser Thr Val Arg Ser Leu Arg Ser Glu Phe 260 265 270 Lye His Giu Lys Ser Trp Arg Arg Gin Lys Ile Ser Gly Thr Arg Giu 275 280 285 Arg Lye Ala Ala Thr Thr Leu Gly Leu Ile Leu Gly Ala Phe Val lie 290 295 300 Cys Trp Lou Pro Ph. Phe Val Lys Glu Lou Val Val Ann Val Cys Asp 305 310 315 320 Lye Cym Lye le Ser Glu Glu Met Ser Asn Phe Leu Ala Trp Leu Gly 325 330 335 Tyr Lou Ann Ser Leu Ile Ann Pro Leu Ile Tyr Thr le Phe Ann Glu 340 345 350 Asp Ph. Lye Lye Ala Phe Gin Lye Leu Val Arq Cys Arg Cys 355 360 365 INFOAR1ATION FOR SEQ ID NO:3: Ci) SEQUENCE CHARACTERISTICS: JA) LENGTH: 422 amino acids TYPE: amino acid STRAJ4OEDNESS: unknown *i TOPOLOGY: Inear MOLECULE TYPE: protein (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

FRAGMENT TYPE: W-tormina.

(ViL) IKMEDIATE

SOURCE:

CLONE: (Xi) SEQUENCE DESCRIPTION:-SEQ :Z NO:3: Met Asp Val Lou Sor Pro Gly Crn Gly Ann Ann Thr Thr Ser Pro Pro 1 0 is Ala Pro Phe Glu Thr dly Cly Ann th.r Thr Gly Ile Ser Asp Val Thr :5 Val Ser Tyr Gin Vai 11e Trr Ser Le. Lek; Leu Gly Thr Leu Ile Phe Cys Ala Val Lou Gly Amn Ala Cys Val Val Ala Ala lie Ala Leu Glu 55 -79- Arg Thr Gin Ile Ala Val 145 Trp Thr His Lou Ar; 225 Ar; Glu Gly Ala Lou 305 Ph.

Lou Thr Pro Scr Lou Gin Asn @0

S.

0 0 S. S 0 S 00 4 500 0

S.

OS

Amp Val Ala Ile 130 Ann Lou Pro Gly Lou 210 Ile His Sir Ala Lou 29-0 Pro Glu Ala Phe Phe Lou Lou Lou 115 Ala Lye le Glu Tyr 195 Met Ar; Giy Gly Lou 275 Glu Lou Arg Ar; le 355 Cys Hot Ann 100 Asp Lou Ar; Giy Asp 180 Thr Lou Lys Ala Ser 260 Cys Val Pro Lys Glu 340 Lou Glu Val Lys Val Asp Thr Phe 165 Ar; Ile Val Thr Ser 245 Arg Ala le Ser An 325 Ar; Cys Ser Va1 70 Ser Trp Lou Ar; Pro 150 Lou Ser Tyr Leu Val 230 Pro An Ann Glu Glu 310 Glu Lys Trp Ser Tyr 390 Val Thr Cys Tyr 135 Arg lIle Asp Ser Tyr 215 Lys Ala Trp Gly Val 295 Ala Arg Thr Leu Cys 375 Leu Leu Cys 120 Trp Ar; Ser Pro Thr 200 Gly Lys Pro Arg Ala 280 His Gly Asn Val Pro 36C His Ala Ann Tyr Lou Ile Giy Ser Lou Ala Val Val Gly 105 Thr Ala Ala lle Asp 185 Phe Arg Val Gin Leu 265 Val Ar; Pro Ala Lys 345 Phe Met 75 Lou Pro 90 Gin Val Ser Ser Ile Thr Ala Ala 155 Pro Pro 170 Ala Cys Gly Ala Ile Ph, Glu Lys 235 Pro Lys 250 Gly Val Arg Gln Val Gly Thr Pro 315 Glu Ala 330 Thr Lou Phe Ile Pro Tmr Met Thr Ile Asp 140 Leu Met Thr Pho Ar; 220 Thr Lys Glu Gly Asn 300 Cys Lye dly Val Leu 380 Ala Cys Leu 125 Pro Ile Leu lIle Tyr 205 Ala Gly Ser Ser Asp 285 5cr Ala Ar; Ile Ala 365 Leu Ala Asp 110 His Ile Ser Gly Ser 190 Ile Ala Ala Val Lys 270 Asp Lys Pro Lys Ile 350 Leu Gly Lou Leu Leu Asp Leu Trp 175 Lys Pro Ar; Asp An 255 Al a Gly Glu Ala Met 335 Met Val Ala Tyr Phe Cys Tyr Thr 160 Arg Asp Leu Ph, Thr 240 Gly Gly Ala His Ser 320 Ala Gly Leu Ile Tyr 400 370 Ile Ann Trp Lou Gly 385 5cr Asn Ser Lou Lou Asn Pro Val Ile Ala Tyr Phe Aen Lys Asp Phe Gin Asn Ala Phe Lys Lye Ile Ile Lye 405 410 415 Cys Leu Phe Cys Arg Gin 420 INFORMATION FOR SEQ ID NO:4: SEQUENCE

CHARACTERISTICS:

LENGTH: 460 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

FRAGMENT TYPE: N-terminal (vii) IMMEDIATE

SOURCE:

CLONE: 5-HT1C (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Val Ann Leu Gly Asn Ala Val Arg Ser Leu Leu Met His Leu le 1 5 10 Gly Leu Leu Val Trp Gin Phe Asp Ile Ser Ile Ser Pro Val Ala Ala 20 25 Ile Val Thr Asp Thr Phe Asn Ser Ser Asp Gly Giy Arg Leu Phe Gin 35 40 Phe Pro Asp Gly Val Gln Aen Trp Pro Ala Leu Ser Ile Val Val Ile 55 Ile Ile Met Thr Ile Gly Gly Asn lie Leu Val Ile Met Ala Val Ser 70 75 Met Glu Lys Lys Leu His Asn Ala Thr Asn Tyr Phe Leu Met Ser Leu 90 Ala le Ala Asp Met Leu Val Gly Leu Leu Val Met Pro Leu Ser Leu 100 105 110 Leu Ala Ile Leu Tyr Asp Tyr Val Trp Pro Leu Pro Arg Tyr Leu Cys 115 120 125 Pro Val Trp lie Ser Leu Asp Val Leu Phe Ser Thr Ala Ser Ile Met 130 135 140 His Leu Cys Ala Ile Ser Leu Asp Arg Tyr Val Ala Ile Arg Aen Pro 145 150 155 160 Ile Glu His Set Arg Phe Awn Set Arq Thr Lye Ala Ile Met Lye 11e 165 170 175 -81- Ala lie Val Trp Ala Ile Ser Ile Gly Val ISO 185 Ser Val Pro Ile Pro Val 190 Ile Oly LOu Arg I 195 Val Lou Ann Asp 210 Phe Ile Pro Leu 225 Val Lou Ar; Arg Glu Lou Ala Amn I 260 Ann Gly Gly Glu 275 Pro Arg Ar; Lys 290 lie Ann Asn Glu 305 Val Ph. Leu Ile Val Lou Cys Gly 340 Aen Val Phe Val 355 Val Tyr Thr Lou 370 Lou Arg Cym Asp 385 Pro Arg Val Ala Ile Tyr Ar; His 420 Giu Pro Oly Ile 435 Pro 5cr Ann Val 450 INFORMATION FOR rsp ?ro rhr Gln 245 Met Glu Lys Lys Met 325 Lys Trp Phe Tyr Ala 405S Glu Run I lie I 230 Thr 5cr Glu Lys Lys 310 Trp Ala Ile Amn Lye 390 Thr 5er Phe 215 Met Leu Leu Aen Glu 295 Ala cy Cys Gly Lys 375 Prc Al Lys 200 Val Val Met AEn Ala 280 Lys Ser Pro An Tyr 360 Ile Amp a Lou jal Lou le Leu Ph* 265 Pro Arg Lys Phe Gin 345 Val Tyr Lys Set Phe N Ile C Thr Leu 250 Leu Asn Pro Val Phe 330 Lys Cys Arg Lys Gly 410 lal ;ly ryr 235 Arg Aun Pro Ar; Leu 315 Ile Leu Ser Arg Pro 395 Asn Ser 220 Phe Gly Cys Asn Gly 300 Gly Thr Met Gly Ala 380 Pro Asn 205 Phe Leu His Cys Pro 285 Thr Ile Asn Glu Ile 365 Phe Va] Thr Val Thr Thr Cy.

270 Asp Met Val Ile Lys 350 Aen Ser Ar; Thr Ala Ile Glu 255 Lys Gin Gin Phe Leu 335 Lou Pro Lys Gln Cys Phe Tyr 240 Glu Lys Lys Ala Ph.

320 Ser Lou Leu Tyr Ile 400 a a Arg Glu Lau Asn Val Ann .415 Thr Asn Glu Ar; Val Ala Arg Lys Ala Glu Met Gin Val Ser Glu 455 SEQ ID Val 440 Ar; Amn Ser Leu Ser Glu Lou 445 Val 460 Ann 430 Pro Asp Val Pro hen SEQUENCE CHARACTERISTICS: LENGTH: 376 amino acids TYPE: amino acid STPANDEDNESS: unknown TOPOLOGY: linear -82- (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

FRAGMENT TYPE: N-terminal (vii) IMMEDIATE

SOURCE:

CLONE: 5-HT1DA (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ser Pro Lou Amn Gln Ser Ala Glu Gly Leu Pro Gin Glu Ala Ser 1 5 10 Aen Arg Ser Leu Asn Ala Thr Glu Thr Ser Glu Ala Trp Asp Pro Ar; 20 25 Thr Lou Gin Ala Leu Lys Ile Ser Lou Pro Val Lou Leu Ser Val Ile 35 40 Thr Lou Ala Thr Val Lou Ser Asn Ala Phe Val Leu Thr Thr Ile Lou 55 Leu Thr Arg Lye Lou His Thr Pro Ala Asn Tyr Lou Ile Gly Sor Lou 70 75 Ala Thr Thr Asp Lou Lou Val Ser Ile Lou Val Hot Pro le Ser Met ag 90 Ala Tyr Thr Ile Tr Hs Thr Trp Asn Phe Gly Gin le Lou Cys Ap 100 105 110 lie Trp Lou Sr Sor Amp Ile Thr Cys Cys Thr Ala 5cr Ile Lou Him 115 12012 Lou Cym Val Ilo Ala Lou Asp Arg Tyr Trp Ala Ile Thr Amp Ala Lou 130 135 140 Glu Tyr 5cr Lys Arg Ar; Thr Ala Gly His Ala Ala Thr Met Ile Ala 145 150 155 160 Ile Val Trp Ala Ile Ser :ke Cys Ile Ser Ile Pro Pro Lou Phe Trp 00165 170 175 Arg Gin Glu Lym Ala Gin Glu Cl- met Ser Asp Cys Lou Val Asn Thr ISO:18 185 190 Se 5r GIn Ile Sr Tyr Thr Ile Tyr Ser Thr Cym Gly Ala Pho Tyr Ile 195 200 205 Pro Sor Val Lou Lou Ile Ile Lou Tyr Gly Arg Ile Tyr Arg Ala Ala 210 215 220 Arg Amn Arg Ile Lou Amn Pro Pro Ser Leu Sor Gly Lys Ar; Pho Thr 225 230 235 240 Thr Ala His Lou Ile ThE Cly Se Ala Gly Ser Val Cys Ser Lou Amn 245 250 255 -83- Ser Ser Lou Him Glu Gly His Ser His Ser Ala Gly Ser Pro Leu Phe 260 265 270 Ph. hen His Val Lys Ile Lys Lou Ala Asp Ser Ala Lou Giu Kr; Lys 275 280 285 Ar Ilie Ser Ala Ala Arg Giu Ar; Lys Ala Thr Lys Ile Lou Gly Ile 290 295 300 Ile Lou Gly Ala Phe Ile Ile cym Trp Leu Pro Phe Phe Val Val Ser 305 310 315 320 Lou Val Lou Pro Ile Cys Ar; Asp Ser Cys Trp Ile His Pro Gly Leu 325 330 335 Ph& Asp Ph. Phe Thr Trp Lou Gly Tyr Leu Asn Ser 1.eu Ilie Aen Pro 340 345 350 Ile Ile Tyr Thr Val Phe Asn Giu Giu Phe Arg Gln Ala Phe Gln Lye 355 360 365 Ile Val Pro Phe Ar; Lys Ala Ser 370 375 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 390 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:

NO

ANTI-SENSE: NO FRAGMENT TYPE: N-terminal :(vii) IMMEDIATE SOURCE: CLONE: S5HT103 (xL4 SEQUENCE DESCR:PTON: SEQ :Z NO:6: Met Giu Giu Pro Cly Ala Gin. Cym Aia Prc Pro Ala Pro Ala Gly Ser 10 is Giu Thr Trp Val Pro Gin Ala Asn Lo.6 Ser Ser Ala Pro Ser Gin Aen 25 Cys 5cr Ala Lys Asp Tyr lie Tyr Gin Asp Ser Ile Ser Lou Pro Trp 4045 Lys Val Lew Leu Val Met Low Lew Ala Lew Ilie Thr Lou Ala T-hr Thr so 55 Lou 5cr hAn Ala Ph. Val Ile Ala Thr Val Tyr Ar; Thr Ar; Lys Leu 70 '5 s0 -B4- Ala Ser Leu Asp Val Thr Him Thr Pro Ala Air' Tyr Leu Ile as Asp Leu 95 Leu Asp Amp Lou 145 Arg Giu Thr Ile 225 Lye Ile Arq G1l Mel 301 G1 Val Arg Ile 130 Asp Thr Ile Giu Val1 210 Ala Gin Thr Val SVal 290 t Ala

S

Y Ala ;or Ile Leu Val Ile Pro Ile 1 fl, Ser Thr met Tyr Thr Val Thr Lrp rhr PArg Pro Ser Giu 195 Tyr Lou Thr Asp: Pro 275 Lys Aid Phi 100 Thr Cys Tyr Lys Ile 180 Val Ser Tyr Pro 5cr 260 Asp Val Arg RIlie '-Cu :ys rrp Arg 165 Scr Ser Thr Gly Asrn 245 Pro Val Arg G1'L Va.

32' Ser Thr Ala 150 Ala Lou Glu Val Arg 230 Arg Giy Pro pVal Arg 31C 1Cyl Gin Val 120 Ala Ser 135 Ile Thr Ala Val Pro Pro Cym Val 200 Gly Ala 215 Ile Tyr Thr Gly 5cr Thr 5cr Glu 280C Ser Asp 295 L~ys Ala Trp Let Val Cys Amp Phe Ilie Leu His Asp Ala Val 155 Met Ile Ala 170 Phe Ph. Trp 185 Val Ahen Thr Phe Tyr Phe Val Giu Ala 235 Lys Arg Lou 250 5cr 5cr Val 265 Scr Gly 5cr Ala Lou Lou .e u 140 Glu Leu Arg Asp Pro 220 Trp 125 Cym Ty r Val Gin His 205 Thr Leu Val1 Ser Trp Al a 190 Ile Leu Ser Ile Ala Val1 Ly s Leu Leu Scr Ala Lys 160 Ph.

Ala Tyr Leu Arg Ser Ar Ile Leu S S S a Thr Thr Pro Giu 300 Arg 5cr Val 285 Lys Ala Ile 270 Ty r Lys Gin 255 Ann Val Lys Lou Ann Lou Lou 320 Val1 Amp Thr Lys Thr Lou Gly Ile Ile 315 Pro Ph. Phe Ile Ile 5cr Lou 330 335 9 Trp Phe His Leu Ala Ile Phe 345 350 met Pro 110 Cys Lys 340 Amp Ala Cyl Pho Phs, Thr Trp Lou dly Tyr Lou Air' 5cr Lou Ile Asnn 365 Pro Ile Ile 355 360 Tyr Thr Met 5cr Annl Glu Amp 370 375 Arg Ph. Lys Cys Thr Ser 385 390 INFORMATION FOR SEQ ID NO:1:, Pme Lys Gir' Ala Phe 380 Hi.s Lys Lou Ile SEQUENCE CHARACTERISTICS: LENGTH: 366 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO FRAGMENT TYPE:, N-terminal (vii) IMMEDIATE SOURCE: CLONE: 5-HT1F (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7: Met Asp Phe Leu Ann Ser Ser Asp Gin Ann Lou Thr Ser Glu Glu, Leu 1 5 10 Lou Ann Arg Met Pro Ser Lys Ile Leu Val Ser Lou Thr Lou Ser Gly 25 Lou Ala Lou Met Thr Thr Thr Ile Ann Ser Leu Val Ile Ala Ala Ile 40 Ile Val Thr Arg Lys Lou Him His Pro Ala Ann Tyr Lou Ile Cym Ser Lou Ala Val Thr Asp Phe Lou Val Ala Val Lou Val Met Pro Phe Ser 70 75 so I le Val Tyr Ile Val Ar; Giu Ser Trp Ile met Gly Gin Val Val Cys Asp ile Trp Lou Ser Val Asp Ilie Thr Cys Cys Thr Cys Ser Ile Lou 100 105 110 *Him Lou Ser Ala Ile Ala Lou Amp Ar; Tyr Arg Ala Ilie Thr Amp Ala 115120 125 Val Giu Tyr Ala Arg Lys Ar; Thr Pro Lye Him Ala Gly Ile met Ile go130 135 140 Thr 11e Val Tip Ile, Ile Ser Val Ph Ilie Ser Met Pro Pro Lou Ph.

:::145 150 155 160 Trp Ar; His Gin Gly Thr Sex Ar; Asp Amp Glu Cym Ile Ile Lye Him *165 170 175 Asp Him Ile Val Ser Thr Ilie Tyr Ser Thr Phe Gly Ala Phe Tyr Ile 1830 185 190 Pro Lou Ala Lou Ile Lou Ile Lou Tyr Tyr Lye Ile Tyr Ar; Ala Ala 195 200 205 Lys Thr Lou Tyr Hism Lym Arg Gin Ala Ser Ar; Ilie Ala Lym Giu Glu 210 215 220 -86- Val Ann Gly Gin Val Leu Leu Giu Ser Gly Glu Lys Ser Thr Lys Ser 225 230 235 240 Val Ser Thr Ser Tyr Val Leu Giu Lys Ser Leu Ser Asp Pro Ser Thr 245 250 255 Asp Phe Asp-Lys Ile His ser Thr Val Arg Ser Leu Ar; Ser Glu Phe 260 265 270 Lys His Giu Lys Ser Trp Ar; Ar; Gin Lys Ile Ser Gly Thr Arg Glu 275 280 285 Arg Lys Ala Ala Thr Thr Leu Giy Leu Ile Leu Giy Ala Phe Val Ile 290 295 300 Cys Trp Lou Pro Ph. Ph. Val Lys Glu Leu Val Val Asn Val Cys Asp 305 310 315 320 Lys Cys Lys Ile Ser Glu Giu Met Ser Asf Phe Leu Ala Trp Leu Gly 325 330 335 Tyr Leu Ann Ser Leu Ile Ann Pro Leu Ile Tyr Thr Ile Phe Asn Glu 340 345 350 Asp Phe Lys Lys Ala Phe Gin Lye Leu Val Arg Cys Arg Cys 355 360 365 INFORMATION FOR SEQ ID NO:8: SEQUENCE

CHARACTERISTICS:

LENGTH: 471 aminno acids TYPE: aino acid STRANDEDNESS: unknown TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

FRAGMENT TYPE: N-ter inal (vii) IMMEDIATE

SOURCE:

CLONE: 5-HT2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Asp Ile Lou Cys Giu Glu Asn Thr Ser Lou Ser Ser Thr Thr Asn 5 10 Ser Lou Met Gin Lou Ann Asp Asp Thr Arg Leu Tyr Ser Ann Asp Phe 20 25 Ann Ser Gly Giu Ala Ann Thr Ser Asp Ala Phe Asn Trp Thr Val Asp 35 40 Ser Ciu &an Ar; Thr Ann Leu Ser Cys Giu Gly Cys Leu Ser Pro Ser 50 55 -8'7- Glu Lys Ann Trp Ser Ala Leu cym Lou Ser Lou Leu His Leu Gin Thr A met A Lou I Pro Sor 145 Ala Ile Phe Pro Gly 225 Val3 S. Thr Gly Lou So r 305 Al a l a aot 7al -ye 5e r In Ile 210 Ser S. r Tb: So Ty~ cly Val Val I Ser 115 ser Lou Ile Ann Lys 195 Pro Cy U Phe Lys -Arq 275 Ser r Thr a Lys ~al er

LOO

.eu 4et :ys Met Pro 180 Ile Val Lou Phe So r 260 Ala Gl'.

Ci, Va Ile I Lou G Aia I Lou IJ Ala Hi.s 165 Ile Ile Phe Lou Ile 245 Lou Lys aLys Arg ILou 325 le u le 'hr ~al

LSO

-Cu His Leu Lys Al a Ile 135 Trp Cys His Thr Ile Lys Lou 105 Asp Met 120 Leu Tyr Ile Tyr Ala Ile 5cr Arg 185 Ala Gly An Ile Leu Val Ile Gin Leu Gly Lou Ser 170 Phe Aunn Lou Tyr Asp 155 Leu Ann Ala Gly Arg 140 Val Asp Ser Val1 Lys 220 Lou Thr Phe 125 Trp Lou Arg Ar; Gly 205 Val Ile nn 110 Lou Pro Phe Tyr Thr 190 Ile Phe G 1) Tyr Val Lou Ser Val.

175 Lye Ser Lys Ser P he Met Pro Thr 160 Ala Ala Met Giu Phe 240 Ala Val Gly Lou 215 Al a Asp 230 Pro Lou Gin Lys Lou Ala Lou Pne 295 Ar; Thr 310 Cly I.

Trp 200 Gin Asp Thr GIlu 5cr 280 Gin met Va.

Thr Ile Ser Asp Asp Asn Phe Ile Met 250 Ala Thr 265 Ph. Ser Arg Ser Gin Ser Phe Phe Ser Val3 235 Val Ile Thr Tyr Phe Leu 255 Lou Cym Phe Leu Ilie Hism Ile Ser 315 Lou Phe Val Ser 270 Pro Gin 285 Ar; Glu Asn Giu Val Val Asp So r Pro Gln Met 335 Lau Ser Gly Lys 320 T rp 330 Ph Ile Thr Ann Ile Met Ala Val. Ile Cys Cys cys dly Lys 385 Pro Ann Ty r 370 Thr Ph* Glu 355 Lou Tyr 340 Asp Val Ser Ser Arg Ser Ile Ala Ala 390 Cly Val 375 P he Ala 360 Aer.

Ser Lou Pro Arg Lou Leu Tyr hen Val Ilie 395 Val1 Tyr 380 Gin Phe 365 Thr Cy s Lys Glu Ser 350 Val Trp Ile Lou Phe Ann Gin Tyr Lys 400 -88- Glu Ann Lys Lys Pro Leu Gln Leu Ile Leu Val hun Thr Ile Pro Ala 405 410 415 Lou Ala Tyr Lys Ser Ser Gln Leu Gin Met Gly Gin Lys Lys Asfn Ser 420 425 430 Lys Gln Amp Ala Lys Thr Thr Asp hAn Asp Cys Ser Met Val Ala Leu 435 440 445 Gly Lys GIn His Ser Glu Glii Ala Ser Lys Asp Ann Ser Asp Gly Val 450 455 460 Ann Glu Lys Val Ser Cys Val 465 470 INFORMATION FOR SEQ ID NO:9: SEQUENCE

CHARACTERISTICS:

LENGTH: 45 base pairs TYPE: nucleic acid STRANDEONESS: both TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

YES

(vi) ORIGINAL

SOURCE:

ORGANISM: ANTISENSE

OLIGO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TCTCACCACT CTCCAAAAGG ACTTCCCCAT TCACCTCCTC

CTTTG

0* 9 9 9* 9 9 9 9*9 9 9 9 9~ 9 9*9* 9.

99 9*9999 9 1 P:\OPER\MR\56212-96.021 27/1199 -89- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A method of identifying a chemical compound which binds specifically to a human 5-HTi, receptor, said method involving competitive binding and comprising: separately contacting nonneuronal cells expressing on their cell surface the human 5-HTI, receptor, with both said chemical compound and a second chemical compound known to bind to the human HTI receptor; and (ii) only the second chemical compound, wherein said contacting is performed under conditions suitable for binding of both of said compounds; and detecting specific binding of the chemical compound to the human 5-HT,1 receptor, wherein a decrease in binding of the second chemical compound to the human 5-HTI, receptor in the presence of said chemical compound indicates that said chemical compound specifically binds to the human 5-HTi, receptor.

2. A method of identifying a chemical compound that binds specifically to a human 5-HTF receptor, said method involving competitive binding and comprising: separately contacting a membrane fraction from a cell extract of nonneuronal cells expressing on their cell surface the human 5-HTF receptor, with both said chemical compound and a second chemical compound known to bind to the human HTiF receptor, and (ii) only the second chemical compound, wherein said contacting is performed under conditions suitable for binding of both compounds; and detecting specific binding of the chemical compound to the human 5-HTI, receptor,

Claims

3. A method of determining whether a chemical compound specifically binds to and activates a human 5-HT1F receptor, comprising: contacting nonneuronal cells producing a second messenger response and expressing on their cell surface the human 5-HTIF receptor, with the chemical compound under conditions suitable for activation of the human 5 -HT1F receptor; and measuring the second messenger response in the presence and in the absence of the chemical compound, wherein a change in second messenger response in the presence of the chemical compound indicates that the chemical compound activates the human 5-HT1F receptor.
4. A method of determining whether a chemical compound specifically binds to and activates a human 5-HTF receptor, "comprising: contacting a membrane fraction from a cell extract of *nonneuronal cells producing a second messenger response and expressing on their cell surface the human 5-HTiF receptor, with the chemical compound under conditions suitable for activation of the human receptor; and measuring the second messenger response in the presence and in the absence of the chemical compound, wherein a change in second messenger response in the presence of the chemical compound indicates that the chemical compound activates the human 5-HT1F receptor. A method of determining whether a chemical compound P:\OPER\MRO\56212-96.021 27/1/99 -91- specifically binds to and inhibits activation of a human HTi, receptor, comprising: separately contacting nonneuronal cells producing a second messenger response and expressing on their cell surface a human 5-HTI, receptor, with both said chemical compound and a second chemical compound that is known to activate the human 5-HTiF receptor, and (ii) only the second chemical compound, under conditions suitable for activation of the human receptor; and measuring the second messenger response in the S* presence of only the second chemical compound and in the presence of both the second chemical compound and said chemical compound, wherein a smaller change in second messenger response in the presence of both of said chemical compound and said second chemical compound than in the presence of said second chemical compound alone indicates that the chemical compound inhibits activation of the human 5-HT,1 receptor.
6. A method of determining whether a chemical compound specifically binds to and inhibits activation of a human HTIF receptor, comprising: separately contacting a membrane fraction from a cell extract of nonneuronal cells producing a second messenger response and expressing on their cell surface a human 5-HT, receptor, with both said chemical compound and a second chemical compound known to activate the human HT 1 receptor, and (ii) only the second chemical compound, under conditions suitable for activation of the human receptor; and measuring the second messenger response in the P:\OPER\MRO\56212-96.021 -27/1/99 -92- presence of only the second chemical compound and in the presence of both of said second chemical compound and said chemical compound, wherein a smaller change in second messenger response in the presence of both of said chemical compound and said second chemical compound than in the presence of only said second chemical compound indicates that the chemical compound inhibits activation of the human 5-HTIF receptor.
7. The method according to any one of claims 3 to 6, wherein the second messenger response comprises adenylate cyclase activity and the change in second messenger response is a decrease in adenylate cyclase activity. 9
8. The method according to claims 5 or 6, wherein the second messenger response comprises adenylate cyclase activity and the change in second messenger response is a smaller decrease in the level of adenylate cyclase activity in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound. *5*9S9
9. The method according to any one of claims 1 to 8, wherein the nonneuronal cell is a mammalian cell. The method according to claim 9, wherein the mammalian cell is an Ltk cell.
11. The method of claim 9, wherein the mammalian cell is an NIH 3T3 cell.
12. A method of preparing a pharmaceutical composition comprising identifying a compound by the method of any one of claims 1 to 11 and combining said compound with a pharmaceutically acceptable carrier. I P:\OPER\MRO\56212-96.021 27/1/99 -93-
13. A compound that specifically binds to a human receptor, wherein said compound is identified by the method according to any one of claims 1 or 2 and was not known previously to bind said receptor.
14. A compound that specifically binds to and activates a human receptor, wherein said compound is identified by the method according to any one of claims 3 to 8 and was not known previously to bind to and activate said receptor. The method according to any one of claims 1 to 11 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this TWENTY-SEVENTH day of JANUARY,
1999. Synaptic Pharmaceutical Corporation By its Patent Attorneys DAVIES COLLISON CAVE ABSTRACT This invention provides an isolated nucleic acid molecule encoding a human receptor, an isolated protein which is a human 5-HTIF receptor, vectors comprising an isolated nucleic acid molecule encoding a human 5-HTIF receptors, mammalian cells comprising such vectors, antibodies directed to the human 5-HTIF receptor, nucleic acid probes useful for detecting nucleic acid encoding human 5-HTi, receptors, antisense oligonucleotides complementary to any sequences of a nucleic acid molecule which encodes a human 5-HTi, receptor, pharmaceutical compounds related to human 5-HT, receptors, and nonhuman transgenic animals which express DNA a normal or a mutant human receptor. This invention further provides methods for determining ligand binding, detecting expression, drug screening, and treatment involving the human 5-HTiF receptor. o. a. o a oa t a o a a