CA2093506A1 - Serotonin receptor protein and related dna compounds - Google Patents

Abstract

Abstract This invention provides a serotonin receptor protein and related DNA compounds. The invention will further the characterization of serotonin-related behavior and disease.

Description

SERQTONIN R~EPTQR PROTE~ ND RELATED DNA COMPOUNDS

Description This invention relates to a novel serotonin receptor protein, and to novel nucleic acid compounds which encode the novel protein.
Since the discovery of serotonin (5-hydrox-ytryptamine, 5-HT) over four decades ago, the cumulative results of many diverse studies have indicated that 5-HT
plays a significant role in the functioning of the mammalian body, both in the central nervous system (CNS ) and in peripheral systems as well. Morphological studies of the CNS have shown that serotonergic neurons, which originate in the brain stem, form a very diffuse system that projects to most areas of the brain and spinal cord.
O'Brien, R.A.: Serotonin in Mental Abnormalities 1, 41i (1978); Steinbusch, H.W.M., Vol. 3, Part II,Handbook of Chemical Neuroanatomy 68 (1984). ~nden, N.E. et al., 67 Acta Physiol. Scand. 313 (1966). These studies have been complemented by biochemical evidence that indicates large concentrations of 5-HT exists in the brain and spinal cord.
Steinbusch, H.W.M., Vol. 3, Part II Handbook of Chemical Neuroanatomy, 68 (1984). With such a diffuse system, it is not surprising that 5-HT has been implicated as being involved in the expression of a number of behaviors, physiological responses, and diseases which originate in the CNS. These include such diverse areas as sleeping, ~fJ ~ ~J ~ ' . ~' ', x-8130 -2-eating, perceiving pain, controling body temperature, controling blood pressure, depression, schizophrenia, and other bodily states. Essman, W.B. Vol. I-V Serotonin in Health and Disease,; Fuller, R.W., Biology of Serotonergic Transmission, 221 (1982); soullin, D.J., Serotonin in Mental Abnormalities 1, 316 (1978); Barchas, J. et al.
Serotonin and Behavior 1 (1973).
Serotonin plays an important role in peripheral systems as well. For example, approximately 90~ of the bodyls 5-HT is found in the gastrointestinal system, and 5-HT has been found to mediate a variety of contractile, secretory, and electrophysiologic effects in this system.
Another example of a peripheral network that is very sensitive to serotonin is the cardiovascular system, which also contains its own source of 5-HT, i.e., the platelet. -Given the broad distribution of 5-HT within the body, it is understandable that tremendous interest in drugs that affect serotonergic systems exists. In particular, receptor specific agonists and antagonists are of interest for the treatment of a wide range of disorders, including anxiety, depression, hypertension, migraine, compulsive disorders, and cancer-chemotherapy-induced vomiting.
Gershon M.D. et al., The Peripheral Actions of.5-hydroxytryptamine, 246 (1989); Saxena, P.R. et al. 15 ~.
Cardiovasc. Pharmacol. (Suppl. 7 1990).
Serotonin produces its effects on cellular physiology by binding to specialized receptors on the cell X~ 813 0 --3-- r~ 3 surface. It is now recognized that multiple types of 5-HT
receptors exist. The existence of multiple, structurally distinct serotonin receptors has provided the possibility that subtypeselective pharmacologic agents can be produced.
The development of such compounds could result in new and increasingly selective therapeutic agents with fewer side effects, since individual receptor subtypes may function to affect specific actions of the different parts of the central peripheral serotonergic systems. An example of such specificity can be demonstrated by using the vascular system as an example: In certain blood vessels, stimulation of 5-HTl-like receptors on the endothelial cells produces vasodilation while stimulation of 5-HT2 receptors on the smooth muscle cells produces vasoconstriction. Currently, the major classes of serotonin receptors (5-HTl, 5-HT2, 5-HT3, and 5-HT4) contain some eight to ten separate receptors that have been formally classified based on their pharmacologic and/or structural differences. For an excellent review of the pharmacological effects and clinical implications of the various 5-HT receptor types, see Glennon, et al. 14 Neuroscience ar~d Behavioral Reviews 35 (1990). The discovery of novel serotonin receptors provides a necessary research tool for the development of selective pharmaceutical agents.
According to the present invention there is provided a novel 5-HT receptor. The present invention x-8130 4 ~ 3 provides an amino acid compound which comprises the isolated amino acid sequence SEQ ID No:2. In particular, the amino acid compound which ls SEQ ID NO:2 is preferred.
The invention also provides a nucleic acid compound that comprises an isolated nucleic acid sequence which encodes for the amino acid compounds provided.
Particularly, nucleic acid compounds which are DNA are preferred. Most preferred is the DNA compound SEQ ID NO:l.
However, also preferred are those nucleic acid compounds which are sense mRNA.
Also provided by the present invention are recombinant nucleic acid vectors comprising the nucleic acids which encode SEQ ID NO:2. The preferred nucleic acid vectors are those which are DNA. Most preferred are recombinant DNA vectors which comprise the isolated DNA
sequence which is SEQ ID NO:l. Moreover, recombinant DNA
vectors of the present invention preferably comprise a promoter positioned to drive expression of said isolated DNA sequence. Those vectors wherein said promoter functions in AV12 cells or E. coli are preferred. The recombinant DNA
expression vector most preferred is plasmid pHD5HT2f.
Restriction fragments of the preferred vectors are also provided. Particularly, the 1598 base pair Bgll/HgaI and the 1169 base pair ApaLI/BanI restriction fragment of a vector which comprises SEQ ID NO:l are provided.
The present invention also provides probes and primers useful for molecular biology techniques. A

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compound which encodes for SEQ ID NO:2 or a part thereof and which is at least 20 base pairs in length is provided.
Preferably, the 20 base pair or more compound is DNA. Most preferred for this use are the DNA compounds which are SEQ
ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
Further, this invention provides cells in which the nucleic acid compounds of the invention may be ; harbored. An oocyte expressing nucleic acid compounds of the invention is provided. One preferred nucleic acid com-; 10 pound which may be harbored in the oocyte is DMA. Most preferred, however, is an oocyte which expresses sense mRNA. Other host cells include those that are transfected with a nucleic acid compound which encodes SEQ ID NO:2.
Preferred cells include a host cell transfected with a recombinant DNA vector. Preferably, the DNA vector comprises SEQ ID NO:1. The preferred transfected host cells which encode SEQ ID NO:2 are AV12 and E. coli. The most preferred transfected host cells are AV12/pHDSHT2f, E. coli/pS5HT2f or an E. coli cell transfected with a DNA
expression vector comprising SEQ ID NO:1.
Additionally, the invention provides a method for identifying, in a test sample, DNA homologous to a probe of the present invention, wherein the test nucleic acid is contacted with the probe under hybridizing conditions and identified as being homologous to the probe.
The preferred probes for use in this method are SEQ ID
NO:3, SEQ ID NO:4 or SEQ ID NO:5.

Assays utilizing the compounds provided by the present invention are also provided. Generally, an assay provided includes a method for determining whether a substance is a functional ligand for the compound of SEQ ID
NO:2, said method comprising contacting a functional compound of SEQ ID NO:2 with said substance, monitoring serotonergic activity by physically detectable means, and identifying those substances which effect a chosen response.
Preferably, the physically detectable means is competition with radiolabeled serotonin, hydrolysis of phosphatidylinositol or binding of ligand in an oocyte expression system. Most preferred is an assay wherein the radiolabeled serotonin is tritiated at the 3 position or wherein the oocyte expression system utili.zes sense mRNA.
The invention also provides a method for constructing a recombinant host cell capable of expressing a nucleic acid compound which encodes a compound which comprises SEQ ID No:2/ said method comprising transfecting a host cell with a recombinant DNA vector that comprises said nucleic acid compound. The preferred method utilizes AV12 or E. coli cells as the host cells. The preferred method may include a DNA vector which comprises SEQ ID
NO:l. The most preferred method utilizes the DNA vector pHD5HT2f.
Additionally, a method for expressing a nucleic acid sequence which encodes SEQ ID NO:2 in a recombinant X-8130 7 ~ J 3 ~; 3 host cell is provided. The method comprises culturing a transformed host cell provided by the present invention ; under conditions suitable for gene expression. The preferred method utilizes AV12 or E. coli cells as the host cell. The more preferred method utilizes a recombinant DNA
vector comprising SEQ ID NO:l. The most preferred method utilizes the recombinant D~A vector pHD5HT2f.
The following section provides a more detailed description of the present invention. For purposes of clarity and as an aid in understanding the invention, as disclosed and claimed herein, the following items are defined below.
5-HT2freceptor - the amino acid sequence SEQ ID
NO:2.
Amino terminal encoding region - The DNA
compound which is SEQ ID NO:4.
Carboxy terminal encoding region - The DNA
compound which is SEQ ID NO:5.
Functional compound of SEQ ID NO:2 - A compound comprising SEQ ID NO:2 which is capable of binding serotonin.
G loop encoding region - The DNA compound which is SEQ ID NO:3.
Homologous - fragments or complete nucleic acid compounds which will hybridize to other fragments or complete nucleic acid compounds.

Hybridization - physically detectable adherence of fragments or complete nucleic acids to other fragments or complete nucleic acids.
Isolated amino acid sequence - any amino acid sequence, however constructed or synthesized, which is locationally distinct from the naturally occurring sequence.
Isolated DNA sequence - Any DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location in genomic DNA.
Isolated nucleic acid sequence - Any RNA or DNA
sequence, however constructed or synthesized, which is locationally distinct from its natural location.
Primer - a nucleic acid fragment which functions as initial template for enzymatic or synthetic elongation.
; Promoter - a DNA sequence which directs transcription of DNA to RNA.
Probe - a nucleic acid compound or a fragment thereof which hybridizes with a nucleic acid compound which encodes either the entire sequence SEQ ID NO:2 or a part thereof.
Stringency - hybridization condition which may be varied in order to vary the degree of nucleic acid affinity for other nucleic acid.
Transfection - any transfer of nucleic acid into a host cell, with or without integration of said nucleic acid into genome of said host cell.

x 8130 -9-Figure I iS a restriction map of pS5HT2f, the recombinant DNA vector discussed herein. The restriction site information is not exhaustive; therefore, there may be more restriction sites of a given type on the vector than actually shown on the map.
The present invention provides an amino acid compound which comprises the isolated amino acid sequence SEQ ID NO:2. The preferred amino acid compound is SEQ ID
NO:2, which is the following sequence of amino acids:
Met Ala Ser Ser Tyr Lys Met Ser Glu Gln Ser Thr Ile Ser Glu His Ile Leu Gln Lys Thr Cys Asp His Leu Ile Leu Thr Asp Arg Ser Gly Leu Lys Ala Glu Ser Ala Ala Glu Glu Met Lys Gln Thr Ala Glu Asn Gln Gly Asn Thr Val His Trp Ala Ala Leu Leu Ile Phe Ala Val Ile Ile Pro Thr Ile Gly Gly Asn Ile Leu Val Ile Leu Ala Val Ser Leu Glu Lys Arg Leu Gln Tyr Ala Thr Asn Tyr Phe Leu Met Ser Leu Ala Val Ala Asp Leu Leu Val Gly Leu Phe Val Met Pro Ile Ala Leu Leu Thr Ile Met Phe Glu Ala Thr Trp Pro Leu Pro Leu Ala Leu Cys Pro Ala Trp Leu Phe Leu Asp Val Leu Phe Ser Thr Ala Ser Ile Met His Leu Cys Ala Ile Ser Leu Asp Arg Tyr Ile Ala Ile Lys Lys Pro Ile Gln Ala Asn Gln Cys Asn Ser Arg Thr Thr Ala Phe Val Lys Ile Thr Val Val Trp Leu Ile Ser Ile Gly Ile A.la Ile Pro Val Pro Ile Lys Gly Ile Glu Ala Asp Val Val Asn Ala His Asn Ile Thr CYS Glu Leu X-8130 -10- ~~5~

Thr Lys Asp Arg Phe Gly Ser Phe Met Leu Phe Gly Ser Leu Ala Ala Phe Phe Ala Pro Leu Thr Ile Met Ile Val Thr Tyr Phe Leu Thr Ile His Ala Leu Arg Lys Lys Ala Tyr Leu Val Arg Asn Arg Pro Pro Gln Arg Leu Thr Arg Trp Thr Val Ser Thr Val Leu Gln Arg Glu Asp Ser Ser Phe Ser Ser Pro Glu Lys Met Ala Met Leu Asp Gly Ser His Lys Asp Lys Ile Leu Pro Asn Ser Ile Asp Glu Thr Leu Met Arg Arg Met Ser Ser Ala Gly Lys Lys Pro Ala Gln Thr Ile Ser Asn Glu Gln Arg Ala Ser Lys Val Leu Gly Ile Val Phe Leu Phe Phe Leu Leu Met Trp Cys Pro Phe Phe Ile Thr Asn Val Thr Leu Ala Leu Cys Asp Ser Cys Asn Gln Thr Thr Leu Lys Thr Leu Leu Gln Ile Phe Val Trp Val Gly Tyr Val Ser Ser Gly Val Asn Pro Leu Ile Tyr Thr Leu Phe Asn Lys Thr Phe Arg Glu Ala Phe Gly Arg Tyr Ile Thr Cys Asn Tyr Gln Ala Thr Lys Ser Val Lys Val Leu Arg Lys Cys Ser Ser Thr Leu Tyr Phe Gly Asn Ser Met Val Glu Asn Ser Lys Phe Phe Thr Lys His Gly Ile Arg Asn Gly Ile Asn Pro Ala Met Tyr Gln Ser Pro Val Arg Leu Arg Ser Ser Thr Ile Gln Ser Ser Ser Ile Ile Leu Leu Asn Thr Phe Leu 450 455 4~0 Thr Glu Asn Asp Gly Asp Lys Val Glu Asp Gln Val Ser Tyr Ile .

Skilled artisans will recognize that this protein can be synthesized by a number of different methods. All of the amino acid compounds of the invention can be made by chemical methods well known in the art, including solid phase peptide synthesis or recombinant methods. Both methods are described in U.S. Patent 4,617,149, herein incorporated by reference. Recombinant methods are preferred if a high yield is desired. A
general method for the construction of any desired DNA
sequence is provided in Brown et al., 68 Methods in Enzymology 109 (1979). Skilled artisans also recognize that some alterations of SEQ ID NO:2 will fail to change the function of the amino acid compound. For instance, some hydrophobic amino acids may be exchanged for other hydrophobic amino acids. Those altered amino acid compounds which confer substantially the same function in substantially the same manner as the exemplified amino acid compound are also included in the present invention.
Moreover, alterations of SEQ ID NO:2 may be induced by alterations of a nucleic acid compound which encodes SEQ ID
NO:2. Those nucleic acid compounds which confer substantially the same function in substantially the same manner as the exemplified nucleic acid compounds are also included in the present invention.
Other routes of production are well known to skilled artisans. Expression in eucaryotic cells can be achieved via SEQ ID NO:l. For example, the amino acid compounds can be produced in eucaryotic cells using Sv40-derived expression vectors comprising DNA which encodes for SEQ ID NO: 2. As well known in the art, some viruses are also appropriate vectors. Eor example, the adenovirus, the adeno associated virus, the vaccinia virus, the herpes virus, the baculovirus, and the rous sarcoma virus are useful. Such a method is described in U.S. Patent 4, 775, 624, herein lncorporated by reference. Several alternate methods of expression are described in J.
Sambrook, E.F. Fritsch & T. Maniatis, Molecula.r Cloning: A
Laboratory Manual 16.3-17.44 (1989).
Other embodiments of the present invention are nucleic acid compounds which comprise isolated nucleic acid sequences which encode SEQ ID NO:2. As skilled artisans will recognize, the amino acid compounds of the invention can be encoded by a multitude of different nucleic acid sequences because most of the amino acids are encoded by more than one nucleic acid triplet. ~ecause these alternate nucleic acid sequences would encode the same amino acid sequences, the present invention further comprises these alternate nucleic acid sequences.
Preferably, the nucleic acid compound is DNA or sense mRNA.
A most preferred embodiment of a DNA compound encoding the 5-HT2f compound has this sequence:

~ ~ S ~

; GTG GCT GAT TTG CTG GTT GGA TTG TTT GTG ATG CCG ATT GCT CTC TTA 336 CTC l'GT GCC ATT TCC CTG GAT CGC TAT ATA GCC ATC AAA AAG CCA ATT 480 : 25 GAT AAA ATT CTA CCT AAC TCA ATT GAT GAG ACA CTG ATG AGA AGA ATG 912 ACT GAA AAC GAT GGT GAC AAA GTA.GAA GAC CAA GTC AGC TAC ATA. 1437 This is the sequence identified as SEQ ID NO:l.
Z;~. Coli/ps5HT2f~ which contains a cloning vector comprising SEQ ID NO:l, was deposited and made part of the stock culture collection of the Northern Regional Research Laboratories (NRRL), Agricultural Research Service, U.S.

Department of Agriculture, Peoria, Illinois, 61604 on March 24, 1992, under the accession number NRRL B18952. SEQ ID
NO:l can be isolated from the plasmid, for example, as a 1169 base (1.2 kb) ApaLI/sanI restriction fragment. Other fragments are useful in obtaining SEQ ID NO:l. A
restriction site and function map of plasmid pS5HT2f is presented in Figure I of the accompanying drawings.
Additionally, the DNA sequences can be synthesized using automated DNA synthesizers, such as the ABS (Applied siosystems, 850 Lincoln Centre Drive, Foster City, CA 9~404) 380B DNA synthesizer. The DNA sequences can also be generated by the polymerase chain reaction as described in U.S. Patent No. 4,889,818, herein incorporated by reference.
Because skilled artisans will recognize that many vectors are available for expression and cloning, those expression and cloning vectors which comprise nucleic acids which encode SEQ ID NO:2 are included in the present invention. The preferred nucleic acid vectors are those which are DNA. The most preferred recombinant DNA vector comprises the isolated DNA sequence S~Q ID NO:l. Plasmid pS5HT2f is a preferred DNA vector of the present invention.
Other preferred DNA vectors include those which comprise a promoter positioned to drive expression of SEQ
ID NO:l. Preferred expression vectors include those which function in AV12 or ~. coli cells. Most preferred for expression in AV12 cells is the expression vector pHD5HT2f.

G~

x-8130 -15-Restriction fragments of these vectors are also provided. The preferred fragments are the 1598 base pair sglI/HgaI restriction fragment and the 1169 base pair ApaLI/BanI restriction fragment of a vector which comprises SEQ ID NO:1~
Plasmid pS5HT2f may be isolated from ~he deposited E. coli/pS5HT2f, using an ordinary cesium chloride DNA lsolation procedure. Plasmid pS5HT2f is readily modified to construct expression vectors that produce 5-HT2f receptors in a variety of organisms, including, for example, E. coli, Sf9 (as host for bac-ulovirus), Spodoptera and saccharomyceyes . The current literature contains techniques for constructing AV12 expression vectors and for transforrning AV12 host cells.
For example, U.S. Patent No. 4,992,373, herein incorporated by reference, explains these techniques.
The construction protocols utilized for AV12 can be followed to construct analogous vectors for other organisms, merely by substituting, if necessary, the appropriate regulatory elements using techniques well known to skilled artisans. Prornoters which may be used, for example, are the thymidine kinase promoter, the metallothionin promoter or various viral and immunoglobulin promoters.
The DNA compounds of the present invention also include primers or probes. A nucleic acid compound of at least 20 base pairs which encodes SEQ ID NO:2 is included ; x-8130 -16-in the present invention. DNA is the preferred nucleic acid used as a probe or primer. Most preferred DNA compounds useful as probes or primers are: SEQ ID No:3, SEQ ID NO:4 and SEQ ID NO:5. A skilled artisan would recognize the techniques associated with probes and primers as well known in the art. Any sequence of at least 20 base pairs in length of the nucleic acids of the present invention may be used to screen any other nucleic acid. For example, all or part of SEQ ID NO:~ or SEQ ID NO:5 may be used to hybridize to the terminal ends of the coding sequence. Then, through PC~ amplification, the full length sequence may be generated. The full length sequence can be subsequently subcloned into any vector of choice.
Alternatively, SEQ ID NO:3, SEQ ID No:4~ or SEQ
ID NO:5 may be radioactively labeled at the 5' end in order to screen cDNA libraries by conventional means.
Furthermore, any piece of 5-HT2f DNA which has been bound to a filter may be flooded with total mRNA transcripts, in order to then reverse-transcribe the mRNA transcripts which bind.
Primers and probes may be obtained by means well known in the art. For example, once pS5HT2f is isolated, restriction enzymes and subsequent gel separation may be used to isolate the fragment of choice.
Host cells which harbor the nucleic acids provided by the present invention are also provided. A
preferred host cell is an oocyte which has been injected ~ , x-8130 -17-with RNA or DNA compounds of the present invention. Mostpreferred oocytes of the present invention are those which harbor sense mRNA. Other preferred host cells include AV12 and E. coli cells which have been transfected and/or transformed with a vector which comprises SEQ ID NO:l.
Most preferred AV12 and E. coli host cells are AV12/p~D5HT2f, E. coli/pS5HT2~, and an E. coli/E. coli ex-pression vector which comprises SEQ ID NO:1.
The oocyte expression system can be constructed according to the procedure described in Lubbert, et al. 84 Proc. Nat. Acad. Sci. 4332 (1987). Other host cell transfection and transformation is well known in the art.
Also provided by the present invention is a method for expressing a gene which encodes SEQ ID No:2 in a recombinant host cell, said method comprising culturing said transformed host cell under conditions suitable for gene expression. The preferred method utilizes AV12 or E. coli cells. The most preferred method utilizes AV12 cells as host cells for pHD5HT2f or E. coli cells as host cells for a recombinant DNA vector comprising SEQ ID NO:1.
Expression in host cells may be accomplished according to the procedures outlined in Sambrook, et al., Molecular Cloning: A Laboratory Manual 16-17 (1989).
The present invention also provides a method for constructing a recombinant host cell capable of expressing SEQ ID NO:2, said method comprising transforming a host cell with a recombinant DNA vector that comprises an isolated DNA sequence which encodes SEQ ID NO:2. The preferred host cell is AV12, which may be obtained from the ATCC under the accession number ATCC C~L 9595. The preferred vector for expression is one which comprises SEQ
ID NO:l. Especially preferred for this purpose is pHD5HT2f. Another preferred host cell for this method is E. coli. An especially preferred expression vector in E. coli is one which comprises SEQ ID NO:1. Transformed host cells may be cultured under conditions well known to skilled artisans such that SEQ ID NO:2 is expressed, thus producing 5-HT2f activity in the recombinant host cell.
Additionally, the invention provides a method for identifying, in a test sample, DNA homologous to a probe of the present invention, wherein the test nucleic acid is contacted with the probe under hybridizing conditions and identified as being homologous to the probe.
The preferred probes for use in this method are SEQ ID
NO:3, SEQ ID NO:4 or SEQ ID NO:5. Hybridization techniques are well known in the art. Sambrook, et al., Molecular Cloning: A Laboratory Manual 11 (1989) describe such procedures.
Assays utilizing the compounds provided by the present invention are also provided. Generally, an assay provided includes a method for determining whether a substance is a functional ligand for the compound of SEQ ID
NO: 2, said method comprising contacting a functional compound of SEQ ID NO: 2 with said substance, monitoring serotonergic activity by physically detectable means, and identifying those substances which effect a chosen response. Preferably, the physically detectable means is competition with radiolabeled serotonin, hydrolysis of phosphatidylinositol or binding of ligand in an oocyte expression system. Most preferred is an assay wherein the radiolabeled serotonin is tritiated at the 3 position or wherein the oocyte expression system utilizes sense mRNA.
The oocyte expression system can be constructed according to the procedure described in L~bbert, et al. 84 Proc. Nat. Acad. Sci. 4332 (1987). The radiolabeled 5-~T
competition assay may be accomplished according to Nelson, et al., 41 Life Sciences 1567 (1567). The assay which measures serotonergic activity via phosphatidylinositol hydrolysis may be accomplished according to Berridge M., 212 Biochem. J. 849 (1983).
Skilled artisans will recognize that desirable Ki values are dependent on the selectivity of the compound tested. For example, a compound with a Ki which is less than 10 nM is generally considered an excellent candidate for drug therapy. However, a compound which has a lower affinity, but is selective for the particular receptor, may be an even better candidate. The present invention, however, provides radiolabeled competition assays, whether results therefrom indicate high affinity or low affinity to 5-HT2f receptor, because skilled artisans will recognize that any information regarding binding or selectivity of a x-8130 -20- '~

particular compound is beneficial in the pharmaceutical development of drugs.
The following are examples oE the present lnvention:

Exam~le 1 Production of_5-HT~ece~E_in AV12/pHD5HT~ cells A lyophil of E. coli pS5HT2f can be obtained from the Northern Regional Research Laboratories, Peoria, IL
61604 under the accession number NRRL B18952 and used directly as the culture in the process described below.
Plasmid pS5HT2f was isolated from E. coli/pS5HT2f by procedures well known by those skilled in the art. Once pS5HT2f was isolated from the deposited strain, the plasmid was altered using conventional methods to produce a SalI
restriction site in addition to the preexisting SalI
restriction site. The plasmid was then digested with Snasl restriction enzyme to produce a blunt end of DNA. The resulting fragment was linear. Ligase was used to ligate a SalI linker sequence to the blunt end created by SnaBl.
The plasmid was then recircularized with ligase to produce two SalI endonuclease sites at both flanking regions of SEQ
ID NO:l. After the plasmid was recircularized, the plasmid was digested with SalI restriction endonuclease and purified by gel purification and subsequent extraction.
The fragment comprising SEQ ID NO:l was then inserted into a modified pHD vector. The modified pHD vector is X-8130 -21- ~ ~ ~3 substantially the vector described in issued Patent No.
4,992,373, except that the plasmid is cleaved at the Bcl site, shortened by Klenow to form blunt ends, and ligated with a SalI linker to form a SalI restriction site. The resulting pHD vector contained a SalI restriction site, an adenovirus promoter, and an ampicillin resistance marker to provide selection in E. coli. The pHD vector was dephosphorylated in order to prevent recircularization.
Subsequently, the fragment comprising SEQ ID NO:1 was ligated into the pHD vector. Competent E. coli cells were then transformed with the pHD vector containing SEQ ID NO:1 and selected for those cells which contained the ampicillin resistance gene by growing on ampicillin-containing medium.
At this point, restriction digests were done to determine orientation of the gene. After transformation of the pHD
vector into E. coli, a subsequent plasmid preparation was made in order to isolate the pHD5HT2f vector. In order to transfect AV12 cells with the pHD5HT2f vector, the procedure according to issued Patent No. 4,992,373 was used. Selection on hygromycin was included in this step.
After selection on hygromycin, a pure culture was obtained by picking the colonies which grew in the presence of hygromycin. These colonies were then assayed to determine serotonin binding ability.

x 8130 22 Example 2 Rindi~q Assay for t,he ~-HT~~ Receptor The 5-HT2f receptor was defined by radioligand binding according to the following procedure:
S A. Tissu~ Preparation AV12/5-HT2f cells which had been grown in suspension were centrifuged at 1000 g to remove the growth medium, and the resulting pellet was washed by resuspending (using vortexing) in ice-cold Tris(Sigma)-HCl buffer (50 mM, pH 7.4). The cells were then centrifuged at 39,800 g for 10 minutes. This washing procedure was repeated for a total of three times with a 10 minute incubation at 37C between the first and second washes.
The final pellet was resuspended in ice-cold Tris-HCl buffer (67 mM, pH 7.4) to achieve a final concentration of approximately 2.5-9 million cells per 200 ~1, depending on the degree of receptor expression in the cells. The final resuspension was effected by using a Tissumizer (Tekmar) at setting 65, for 15 seconds, to thoroughly disrupt the cèll membranes.
B. Saturation A$say to Determine Affinity for ~3Hl5-HT
Tubes for the saturation binding assay had a final volume of 800 ~1 and contained the following: Tris-HCl (500 mM), pargyline (10 ~M), ascorbic acid (5.7 ~nM), CaCl2 (3 mM), various concentrations of [3H]5-HT
(concentrations ranged from approximately 0.5 to 35 nM), and 200 ~1 of cell membrane resuspension for a final pH of X-8130 -23- ~i~S.,~

7.4. The assay tubes were incubated for 15 minutes at 37C
(or in some cases 2 hours at 0C), and the contents were then rapidly filtered through GF/B filters (Whatman) (pretreated with 0.5% polyethylenimine), and followed by four 1 ml washes with ice-cold buffer (50 mM Tris-HC1, pH
7.4). The radioactivity trapped by the filters was quantitated by liquid scintillation spectrometry, and specific [3H]5-HT binding was defined as the difference between [3H]5-HT bound in the presence and absence of 10 ~M
unlabeled 5-HT. The affinity of [3H]5-HT for the 5-HT2f receptor was calculated from the saturation curves by use of nonlinear regression analysis (SYSTAT, SYSTAT, Inc., Evanston, IL). Using this method, Kd values calculated for [3H]5-HT at the 5-HT2f receptor ranged from approximately 5-8 nM with a mean of 6.9 nM at 37C and 0.15-0.3 nM with a mean of 0.19 nM at 0C. S~illed artisans will recoynize the resultant Kd as indicative of a serotonergic receptor.

Exam~le 3 Com~tition sindinq Assays A. 13Hl5-HT Assay To determine the affinities of various other compounds at the 5-HT2~ receptor, the assays were carried out as described in Example 2, except that a fixed concentration of [3H]5-HT (2 nM) was used and the tubes contained appropriate dilutions of the drugs of interest, x-8130 -24-for example methoxytryptamine or ritanserin, in order to generate competition curves.
The EC50 values from the competition curves were determined by nonlinear regression (SYSTAT, SYSTAT, Inc., Evanston, IL) and converted to Ki values by use of the Cheng-Prusoff equation discussed in Cheng, Y. et al., 22 Biochem. Pharmacol. 3099 (1973).
B. Phos~ha~idylinositol Hydrolysis Assav Wells of a 12 well plate were seeded with enough AVl2/pHD5HT2f cells in 1.0 ml of media containing ~.0 ~ Ci of 3H-myoinositol such that confluency was obtained in ~8 hours. (for AV12 cells this was 5 x 105 cells). After 48 hours in a tissue culture incubator, the media was aspirated, and the cells were washed two times with serum-free media containing 10 mM myoinositol and 10 mM LiCl.
After the final wash, 1.0 ml of the serum free wash media containing 10 mM myoinositol an 10 mM LiCl was added.
Dilutions of appropriate compounds were added to the wells and the plate was floated in a 37C water bath for 1 hour.
; 20 The reaction was terminated by aspirating the media and adding 0.5 ml of ice-cold acetone:methanol (1:1, v:v). The plate was then left on a rocking platform for 20 minutes at 4C.
The extract was harvested into an eppendorf tube and the well was rinsed with 0.5 ml water and combined with the extract. Samples were centrifuged in an eppendorf centrifuge for approximately 10 minutes in order to pellet x-8130 -25- ~3 ,.~

the cellular residue. The supernatants were loaded onto SepPak Waters Accell P1US QMA cartridges which were previously treated by adding 10 ml of lM ammonium formate, 0.1 M formic acid to the cartridge and then drawing the solution through with vacuum. Next, the cartridge was washed two times with 10 ml water. Five ml water was added, and then the sample was added. The vacuum manifold was then used to draw samples through the cartridges.
After the sample went through the cartridges, 10 ml of 5.0 mM sodium tetraborate was added to each cartridge and drawn through.
The manifold was loaded with 20 ml scintillation vials and 4.0 ml 0.1 M ammonium formate, 0.01 M formic acid and 5 mM Na tetraborate was added to the cartridges and the eluates were drawn into the vials using vacuum. Next, 18 ml Ready Solv HP was added to each vial and the radioactive inositol phosphate eluted from the cartridges was quantitated by scintillation spectroscopy. Curves which illustrate the ECso of 5-HT at various concentrations of the antagonist were generated, and a Shield Plot was used to calculate Ki, as accomplished in serridge M., 212 Biochem. J. 849 ~1983).

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Baez, Melvyn Kursar, Jonathan David (ii) TITLE OF INVENTION: SEROTONIN RECEPTOR PROTEIN AND RELATED DNA
COMPOUNDS
(iii) NUMBER OF SEQUENCES: 5 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Patent Division/~PL
(B) STREET: Lilly Corporate Center (C) CITY: Indianapolis (D) STATE: Indiana (E) COUNTRY: U.S.A.
(F) ZIP: 46285 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: diskette, 3.5 inch, 1.44 Nb (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WordPerfect version 5.1 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 07/864,005 (B) FILING DATE: 04-APR-92 (C) CL~SSIFICATION: 435 (viii) ATTO~NEY/AGENT INFORMATION:
(A) NAME: Leeds, James P.
(B) REGISTRATION NUMBER: 35,241 (C) REFERENCE/DOCKET NUMBER: X-8130 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 317-276-1667 (8) TELEFAX: 317-27~-3861 (2~ INFORMATION FOR SEQ ID NO:l:
(i) SEQULNCE CHA~ACTERISTICS:
(A) LENGTH: 1437 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

X-8l30 27 ~ Ji ~ J

(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
tB) LOCATION: 1..1437 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Met Ala Ser Ser Tyr Lys Met Ser Glu Gln Ser Thr Ile Ser Glu His Ile Leu Gln Lys Thr Cys Asp His Leu Ile Leu Thr Asp Arg Ser Gly Leu Lys Ala Glu Ser Ala Ala Glu Glu Met Lys Gln Thr Ala Glu Asn Gln Gly Asn Thr Val His Trp Ala Ala Leu Leu Ile Phe Ala Val Ile Ile Pro Thr Ile Gly Gly Asn Ile Leu Val Ile Leu Ala Val Ser Leu Glu Lys Ars Leu Gln Tyr Ala Thr Asn Tyr Phe Leu Met Ser Leu Ala Val Ala Asp Leu Leu Val Gly Leu Phe Val Met Pro Ile Ala Leu Leu Thr Ile Met Phe Glu Ala Thr Trp Pro Leu Pro Leu Ala Leu Cys Pro GCC TGG TTA TTC CTT GAT GTT Cl'C TTT TCA ACT GCC TCC ATC ATG CAT 432 Ala Trp Leu Phe Leu Asp Val Leu Phe Ser Thr Ala Ser Ile Met His 13~ 135 140 Leu Cys Ala Ile Ser Leu Asp Arg Tyr Ile Ala Ile Lys Lys ~ro Ile ln Ala Asn Gln Cys Asn Ser Arg Thr Thr Ala Phe Val Lys Ile Thr Val Val Trp Leu Ile Ser Ile Gly Ile Ala Ile Pro Val Pro Ile Lys Gly Ile Glu Ala Asp Val Val Asn Ala His Asn Ile Thr Cys Glu Leu Thr Lys Asp Arg Phe Gly Ser Phe Met Leu Phe Gly Ser Leu Ala Ala Phe Phe Ala Pro Leu Thr Ile Met Ile Val Thr Tyr Phe Leu Thr Ile His Ala Leu Arg Lys Lys Ala I'yr Leu Val Arg Asn Arg Pro Pro Gln Arg Leu Thr Arg Trp Thr Val Ser Thr Val Leu Gln Arg Glu Asp Ser Ser Phe Ser Ser Pro Glu Lys Met Ala Met Leu Asp Gly Ser His Lys Asp Lys Ile Leu Pro Asn Ser Ile Asp Glu Thr Leu Met Arg Arg Met Ser Ser Ala Gly Lys Lys Pro Ala Gln Thr Ile Ser Asn Glu Gln Arg `305 310 315 320 Ala Ser Lys Val Leu Gly Ile Val Phe Leu Phe Phe Leu Leu Met Trp Cys Pro Phe Phe Ile Thr Asn Val Thr Leu Ala Leu Cys Asp Ser Cys Asn Gln Thr Thr Leu Lys Thr Leu Leu Gln Ile Phe Val Trp Val Gly 29 ~ J !;~
Tyr Val Ser Ser Gly Val Asn Pro Leu Ile Tyr Thr Leu Phe Asn Lys Thr Phe Arg Glu Ala Phe Gly Arg Tyr Ile Thr Cys Asn Tyr Gln Ala 385 390 3~5 400 ACA AAG TCA GTA AAA GTG CTT AGA AAG TGT TCT AGT ACA CTC TAT TTT l248 Thr Lys Ser Val Lys Val Leu Arg Lys Cys Ser Ser Thr Lau Tyr Phe ~05 410 415 GGG AAT TCA ATG GTA GAA AAC TCT AAA TTT TTC ACA AAA CAT GGA ATT l296 Gly Asn Ser Met Val Glu Asn Ser Lys Phe Phe Thr Lys His Gly Ile 420 42.5 430 CGA AAT GG& ATC AAC CCT GCC ATG TAC CAG AGC CCA GTA AGG CTC CGA l344 Arg Asn Gly Ile Asn Pro Ala Met Tyr Gln Ser Pro Val Arg Leu Arg AGT TCA ACC ATT CAG TCT TCA TCC ATC ATT CTC CTC AAT ACA TTT CTC l392 Ser Ser Thr Ile Gln Ser Ser Ser Ile Ile Leu Leu Asn Thr Phe Leu ACT GAA AAC GAT GGT GAC AAA GTA GAA GAC CAA GTC AGC TA ATA l437 Thr Glu Asn Asp Gly Asp Lys Val Glu Asp Gln Val Ser Tyr Ile t2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 479 amino acids (B) TYPE: amirlo acid (D) TOPOLOGY: linear - .
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Ser Ser Tyr Lys Met Ser Glu Gln Ser Thr Ile Ser Glu His l 5 l0 15 le Leu Gln Lys Thr Cys Asp His Leu Ile Leu Thr Asp Arg Ser Gly Leu Lys Ala Glu Ser Ala Ala Glu Glu Met Lys Gln Thr Ala Glu Asn . 45 Gln Gly Asn Thr Val His Trp Ala Ala Leu Leu Ile Phe Ala Val Ile Ile Pro Thr Ile Gly Gly Asn Ile Leu Val Ile Leu Ala Val Ser Leu i~--8130 ~ ~ eJ c3 . ~; ~ J

Glu Lys Arg Leu Gln Tyr Ala Thr Asn Tyr Phe Leu Met Ser Leu Ala al Ala Asp Leu Leu Val Gly Leu Phe Val Met Pro Ile Ala Leu Leu Thr Ile Met Phe Glu Ala Thr Trp Pro Leu Pro Leu Ala Leu Cys Pro Ala Trp Leu Phe Leu Asp Val Leu Phe Ser Thr Ala Ser Ile Met His 13~ 135 140 Leu Cys Ala Ile Ser Leu Asp Arg Tyr Ile Ala Ile Lys Lys Pro Ile ln Ala Asn Gln Cys Asn Ser Arg Thr Thr Ala Phe Val Lys Ile Thr 1~5 170 175 al Val Trp Leu Ile Ser Ile Gly Ile Ala Ile Pro Val Pro Ile Lys 180 18~ 190 Gly Ile Glu Ala Asp Val Val Asn Ala His Asn Ile Thr Cys Glu Leu Thr Lys Asp Arg Phe Gly Ser Phe Met Leu ~he Gly Ser Leu Ala Ala Phe Phe Ala Pro Leu Thr Ile Met Ile Val Thr Tyr Phe Leu Thr Ile is Ala Leu Arg Lys Lys Ala Tyr Leu Val Arg Asn Arg Pro Pro Gln rg Leu Thr Arg Trp Thr Val Ser Thr Val Leu Gln Arg Glu Asp Ser 260 2~5 270 Ser Phe Ser Ser Pro Glu Lys Met Ala Met Leu Asp Gly Ser His Lys 275 28~ 285 Asp Lys Ile Leu Pro Asn Ser Ile Asp Glu Thr Leu Met Arg Ar~ Met 2~0 295 300 Ser Ser Ala Çly Lys Lys Pro Ala Gln Thr Ile Ser Asn Glu Gln Arg la Ser Lys Val Leu Gly Ile Val Phe Leu Phe Phe Leu Leu Met Trp ys Pro Phe Phe Ile Thr Asn Val Thr Leu Ala Leu Cys Asp Ser Cys -31 ~ r3 Asn Gln Thr Thr Leu Lys Thr Leu Leu Gln Ile Phe Val Trp Val Gly Tyr Val Ser Ser Gly Val Asn Pro Leu Ile Tyr Thr Leu Phe Asn Lys 370 375 3~30 Thr Phe Arg Glu Ala Phe Gly Arg Tyr Ile Thr Cys Asn Tyr Gln Ala hr Lys Ser Val Lys Val Leu Arg Lys Cys Ser Ser Thr Leu Tyr Phe ly Asn Ser Met Val Glu Asn Ser Lys Phe Phe Thr Lys His Gly Ile Arg Asn Gly Ile Asn Pro Ala Met Tyr Gln Ser Pro Val Arg Leu Arg Ser Ser Thr Ile Gln SPr Ser Ser Ile Ile Leu Leu Asn Thr Phe Leu Thr Glu Asn Asp Gly Asp Lys Val Glu Asp Gln Val Ser Tyr Ile (2) INFORMATION FOR SEQ ID NO:3: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 141 base pairs (B) TYPE: nucleic ~cid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-S~NSE: NO

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1 - 141 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 Thr Ile His Ala Leu Arg Lys Lys Ala Tyr Leu Val Arg Asn Arg Pro Pro Gln Arg Leu Thr Arg Trp Thr Val Ser Thr Val Leu Gln Arg Glu Asp Ser Ser Phe Ser Ser Pro Glu Lys Met Ala Met Leu Asp Gly (2) INFORMATION FOR SEQ ID NO:4:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 165 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..165 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Ala Ser Ser Tyr Lys Met Ser Glu Gln Ser Thr Ile Ser Glu His l 5 10 15 Ile Leu Gln Lys Thr Cys Asp His Leu Ile Leu Thr Asp Arg Ser Gly Leu Lys Ala Glu Ser Ala Ala Glu Glu Met Lys Gln Thr Ala Glu Asn 35 40 ~5 CAG GGG AAT ACA GTG CAC TGG . 165 Gln Gly Asn Thr Val His Trp (2) INFORMATION FOR SEQ ID NO:5:

X~8130 ~r~t~ J

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 405 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1 - 405 (xi) SEQUENCE DESCRIPTION- SEQ ID NO:5:

Thr Leu Ala Leu Cys Asp Ser Cys Asn Gln Thr Thr Leu Lys Thr Leu Leu Gln Ile Phe Val Trp Val Gly Tyr Val Ser Ser Gly Val Asn Pro Leu Ile Tyr Thr Leu Phe Asn Lys Thr Phe Arg Glu Ala Phe Gly Arg Tyr Ile Thr Cys Asn Tyr Gln Ala Thr Lys Ser Val Lys Val Leu Arg Lys Cys Ser Ser Thr Leu Tyr Phe 6~ 65 70 GGG AAT TCA ATG GTA GAA AAC TCT AAA TTT TTC ACA A~A CAT GGA ATT 264 Gly Asn Ser Met Val Glu Asn Ser Lys Phe Phe Thr Lys His Gly Ile 7~ ~0 85 Arg Asn Gly Ile Asn Pro Ala Met Tyr Gln Ser Pro Val Arg Leu Arg Ser Ser Thr Ile Gln Ser Ser Ser Ile Ile Leu Leu Asn Thr Phe Leu Thr Glu Asn Asp Gly Asp Lys Val Glu Asp Gln Val Ser Tyr Ile

Claims (10)

1. An amino acid compound which comprises the isolated amino acid sequence SEQ ID NO:2.

2. The amino acid compound of Claim 1 which is SEQ ID NO: 2.

3. A nucleic acid compound which comprises an isolated nucleic acid sequence which encodes for the compound of Claim 2.

4. The DNA compound of Claim 4 which is SEQ ID
NO:1.

5. A recombinant nucleic acid vector which comprises the nucleic acid compound of Claim 3 or 4.

6. A host cell transfected with a recombinant nucleic acid vector of Claim 5.

7. The transfected host cell of Claim 30 which is E. coli/pS5HT2f.

8. A method for determining whether a substance is a functional ligand for the compound of SEQ ID
NO:2, said method comprising contacting a functional compound of SEQ ID NO:2 with said substance, monitoring serotonergic activity by physically detectable means, and identifying those substances which effect a chosen response.

9. A method for constructing a recombinant host cell capable of expressing the compound of Claim 3, said method comprising transforming a host cell with a recombinant nucleic acid vector that comprises the compound of Claim 3.

10. A method for expressing a nucleic acid sequence which encodes SEQ ID NO:2 in a recombinant host cell, said method comprising culturing said host cell under conditions suitable for gene expression.