WO2001010889A1 - Rat-g-protein coupled receptor brs3 - Google Patents

Abstract

Rattus Norvegicus BRS3 polypeptides and polynucleotides and method for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for screening for compounds which either agonize or antagonize Rattus Norvegicus BRS3. Such compounds are expected to be useful in treatment of human diseases, including, but not limited to: infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, delirium, dementia, and severe mental retardation; and dyskinesias, such as Huntington's disease or Gilles de la Tourett's syndrome.

Description

Rat G-Protein Coupled Receptor BRS3

This application claims the benefit of U.S. Provisional Application No. 60/147,107, filed August 4, 1999, the entire contents of which are incorporated by reference herein.

Field of the Invention This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in identifying compounds that may be agonists and/or antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.

Background of the Invention

The drug discovery process is currently undergoing a fundamental revolution as it embraces 'functional genormcs', that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on 'positional cloning'. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.

Functional genomics relies heavily on the vanous tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and charactense further genes and their related polypeptides/proteins, as targets for drug discovery.

It is well established that many medically significant biological processes are mediated by proteins participating m signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 1991, 351:353-354). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins Some examples of these proteins include the G-protem coupled (GPC) receptors, such as those for adrenergic agents and dopamme (Kobilka, B.K., et al., Proc. Natl Acad. Sci., USA, 1987, 84:46-50; Kobilka, B.K., et al., Science, 1987, 238:650-656; Bunzow, J.R., et al., Nature, 1988, 336:783-787), G-protems themselves, effector proteins, e.g., phospho pase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kmase A and protein kmase C (Simon, M.I., et al., Science, 1991, 252:802-8).

For example, in one form of signal transduction, the effect of hormone binding is activation of the enzyme, adenylate cyclase, mside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide, GTP. GTP also influences hormone binding. A G-protem connects the hormone receptor to adenylate cyclase. G-protem was shown to exchange GTP for bound GDP when activated by a hormone receptor. The GTP-carrymg form then binds to activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G-protem itself, returns the G-protem to its basal, mactive form. Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.

The membrane protein gene superfamily of G-protem coupled receptors has been charactenzed as having seven putative transmembrane domains. The domains are believed to represent transmembrane α-hehces connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically acttve receptors, such as hormone, viral, growth factor and neuroreceptors.

G-protein coupled receptors (otherwise known as 7TM receptors) have been charactenzed as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include, but are not limited to: calcitomn, adrenergic, endothelin, cAMP, adenosme, muscannic, acetylcholine, serotonin, histarmne, thrombm, kinin, follicle stimulating hormone, opsms, endothehal differentiation gene-1, rhodopsms, odorant, and cytomegalovirus receptors.

Most G-protem coupled receptors have single conserved cysteme residues in each of the first two extracellular loops which form disulfϊde bonds that are believed to stabilize functional protein structure. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7 TM3 has been implicated in signal transduction. Phosphorylation and hpidation (palmitylatton or famesylation) of cysteme residues can influence signal transduction of some G-protem coupled receptors. Most G-protein coupled receptors contain potential phosphorylation sites withm the third cytoplasmic loop and/or the carboxy terminus. For several G-protem coupled receptors, such as the β-adrenoreceptor, phosphorylation by protein kmase A and/or specific receptor kinases mediates receptor desensitizatton. For some receptors, the gand binding sites of G-protem coupled receptors are believed to compnse hydrophilic sockets formed by several G-protem coupled receptor transmembrane domains, said sockets being surrounded by hydrophobic residues of the G-protem coupled receptors. The hydrophilic side of each G-protein coupled receptor transmembrane helix is postulated to face mward and form a polar gand binding site. TM3 has been implicated m several G-protein coupled receptors as having a hgand binding site, such as the TM3 aspartate residue. TM5 sennes, a TM6 asparagme and TM6 or TM7 phenylalanmes or tyrosines are also implicated m hgand binding. G-protem coupled receptors can be mtracellularly coupled by heterotnmenc G-protems to vanous mtracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331). Different G-protem α-subumts preferentially stimulate particular effectors to modulate vanous biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protem coupled receptors has been identified as an important mechanism for the regulation of G-protem coupling of some G-protem coupled receptors. G-protem coupled receptors are found m numerous sites within a mammalian host.

Over the past 15 years, nearly 350 therapeutic agents targeting 7 transmembrane (7 TM) receptors have been successfully introduced onto the market. Summary of the Invention

The present invention relates to Rattus Norvegicus BRS3, in particular Rattus Norvegicus BRS3 polypeptides and Rattus Norvegicus BRS3 polynucleotides, recombmant matenals and methods for their production. In another aspect, the invention relates to methods for identifying agonists and antagonists/inhibitors of the Rattus Norvegicus BRS3 gene. This invention further relates to the generation of in vitro and in vivo compaπson data relating to the polynucleotides and polypeptides in order to predict oral absorption and pharmacokmetics in man of compounds that either agonize or antagonize the biological activity of such polynucleotides or polypeptides. Such a companson of data will enable the selection of drugs with optimal pharmacokmetics in man, i.e., good oral bioavailabihty, blood-bram barrier penetration, plasma half life, and minimum drug interaction.

The present invention further relates to methods for creating transgenic animals, which overexpress or underexpress or have regulatable expression of a BRS3 gene and "knock-out" animals, preferably mice, in which an animal no longer expresses a BRS3 gene. Furthermore, this invention relates to transgenic and knock-out animals obtained by using these methods. Such animal models are expected to provide valuable insight into the potential pharmacological and toxicological effects in humans of compounds that are discovered by the aforementioned screening methods as well as other methods. An understanding of how a Rattus Norvegicus BRS3 gene functions m these animal models is expected to provide an insight into treating and preventing human diseases including, but not limited to: infections such as bacteπal, fungal, protozoan and viral infections, particularly infections caused by HΓV-1 or HIV-2; pam; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; unnary retention; osteoporosis; angina pectons; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, dehnum, dementia, and severe mental retardation; and dyskmesias, such as Huntmgton's disease or Gilles dela Tourett's syndrome, hereinafter referred to as "the Diseases", amongst others.

Description of the Invention

In a first aspect, the present invention relates to Rattus Norvegicus BRS3 polypeptides. Such polypeptides include isolated polypeptides composing an ammo acid sequence having at least a 95% identity, most preferably at least a 97-99% identity, to that of SEQ ID NO.2 over the entire length of SEQ ID NO:2. Such polypeptides include:

(a) an isolated polypeptide compnsing the ammo acid of SEQ ID NO:2.

(b) an isolated polypeptide encoded by a polynucleotide comprising the sequence contained ιn SEQ ID NO:l;

(c) an isolated polypeptide comprising a polypeptide sequence having at least a 95%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;

(d) an isolated polypeptide comprising the polypeptide sequence of SEQ ID NO:2;(d) an isolated polypeptide having at least a 95%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;

(e) the polypeptide sequence of SEQ ID NO:2; and

(f) vanants and fragments thereof; and portions of such polypeptides in (a) to (e) that generally contain at least 30 ammo acids, more preferably at least 50 ammo acids, thereof.

Polypeptides of the present invention are believed to be members of the 7-transmembrane receptor (G-protem coupled) family of polypeptides. They are, therefore, of interest, because understanding of the biological activities of BRS3 in rat would help to understand the biological function of its human counterpart, human BRS3, and other related genes. In addition, G-protem coupled receptors, more than any other gene family, are the objects of pharmaceutical intervention. Furthermore, the polypeptides of the present invention can be used to establish assays to predict oral absorbtion and pharmacokmetics m man and thus enhance compound and formulation design, among others. These properties, either alone or in the aggregate, are hereinafter referred to as "Rattus Norvegicus BRS3 activity" or "Rattus Norvegicus BRS3 polypeptide activity" or "biological activity of BRS3." Preferably, a polypeptide of the present invention exhibits at least one biological activity of Rattus Norvegicus BRS3. Polypeptides of the present invention also include vanants of the aforementioned polypeptides, including alleles and splice vanants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative. Particularly preferred vanants are those m which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acids are inserted, substituted, or deleted, in any combination. Particularly preferred pnmers will have between 20 and 25 nucleotides.

Preferred fragments of polypeptides of the present invention include an isolated polypeptide compπsmg an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO:2, or an isolated polypeptide compnsing an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO:2.

Also preferred are biologically active fragments which are those fragments that mediate activities of BRS3, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or lmmunogemc in an animal, especially in a human. Particularly preferred are fragments composing receptors or domains of enzymes that confer a function essential for viability of Rattus Norvegicus or the ability to initiate, or maintain cause the Diseases in an individual, particularly a human.

Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these vanants may be employed as intermediates for producing the full-length polypeptides of the invention. The polypeptides of the present invention may be in the form of a "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional ammo acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid m punfication, for instance, multiple histidine residues, or an additional sequence for stability during recombinant production. The present invention also includes vanants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like charactenstics. Typical substitutions are among Ala, Val, Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are vanants in which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added in any combination. Polypepttdes of the present invention can be prepared m any suitable manner. Such polypeptides include isolated naturally occumng polypeptides, recombmantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods Means for prepaπng such polypepttdes are well understood in the art. In a further aspect, the present invention relates to Rattus Norvegicus BRS3 polynucleotides.

Such polynucleotides include isolated polynucleotides compnsing a nucleotide sequence encoding a polypeptide having at least a 95% identity, to the ammo acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO.2. In this regard, polypeptides which have at least a 97% identtty are highly preferred, while those with at least a 98-99% identity are more highly preferred, and those with at least a 99% identity are most highly preferred. Such polynucleotides include a polynucleotide compnsing the nucleotide sequence contained in SEQ ID NO: 1 encoding the polypeptide of SEQ ID NO.2.

Further polynucleotides of the present invention include isolated polynucleotides compnsing a nucleotide sequence having at least a 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, over the entire coding region. In this regard, polynucleotides which have at least a

91% identity are highly preferred, while those with at least a 98-99% identity are more highly preferred, and those with at least a 99% identity are most highly preferred.

Further polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence having at least a 95% identity, to SEQ ID NO: 1 over the entire length of SEQ ID NO 1. In this regard, polynucleotides which have at least a 97% identity are highly preferred, while those with at least a 98-99% identify are more highly preferred, and those with at least a 99% identity are most highly preferred. Such polynucleotides include a polynucleotide composing the polynucleotide of SEQ ID NO: 1 , as well as the polynucleotide of SEQ ID NO: 1.

The invention also provides polynucleotides which are complementary to all the above described polynucleotides.

The nucleotide sequence of SEQ ID NO- 1 shows homology with human BRS3. The nucleotide sequence of SEQ ID NO:l is a cDNA sequence and composes a polypeptide encoding sequence (nucleotides 1 to 1149) encoding a polypeptide of 382 ammo acids, the polypeptide of SEQ ID NO.2. The nucleotide sequence encoding the polypeptide of SEQ ID NO.2 may be identical to the polypeptide encoding sequence of SEQ ID NO: 1 or it may be a sequence other than SEQ ID

NO:l, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO.2. The polypeptide of the SEQ ID NO'2 is structurally related to other proteins of the 7-transmembrane receptor family, having homology and/or structural similaπty with Human BRS3.

Preferred polypeptides and polynucleotides of the present mvention are expected to have, mter aha, similar biological functions/properties to their homologous polypepttdes and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one BRS3 activity.

Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library deoved from mRNA in cells of Rattus Norvegicus rat testis, using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252: 1651-1656; Adams, M.D. et al , Nature (1992) 355:632-634; Adams, M.D., et al , Nature (1995) 377 Supp.: 3-174). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbraoes or can be synthesized using well known and commercially available techniques.

When polynucleotides of the present invention are used for the recombmant production of polypeptides of the present invention, the polynucleotide may include the codmg sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypepttde m reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence that facilitates puoficatton of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histtdme peptide, as provided in the pQE vector (Qiagen, Inc.) and descobed m Gentz, et al , Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also compose non-codmg 5 ' and 3 ' sequences, such as transcobed, non-translated sequences, splicing and polyadenylation signals, obosome binding sites and sequences that stabilize mRNA.

Further embodiments of the present invention include polynucleotides encoding polypeptide vanants that compose the ammo acid sequence of SEQ ID NO:2 and in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 1 to 1 or 1 ammo acid residues are substituted, deleted or added, in any combination Particularly preferred probes will have between 30 and 50 nucleotides, but may have between 100 and 200 contiguous nucleotides of the polynucleotide of SEQ ID NO.1. A preferred embodiment of the invention is a polynucleotide consisting of or composing nucleotide ATG to the nucleotide immediately upstream of or including nucleotide TAG set forth m SEQ ID NO: 1, both of which encode a BRS3 polypepttde. The invention also includes a polynucleotide consisting of or compnsing a polynucleotide of the formula:

X-(Rι )_m-(R₂)-(R₃)_n-Y wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occunence of Rj and R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero, n is an integer between 1 and 3000 or zero, and R₂ is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly the nucleic acid sequence set forth in SEQ ID NO: 1 or a modified nucleic acid sequence thereof. In the polynucleotide formula above, R₂ is ooented so that its 5' end nucleic acid residue is at the left, bound to R1 and its 3' end nucleic acid residue is at the oght, bound to R3. Any stretch of nucleic acid residues denoted by either R1 and/or R₂, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Where, in a preferred embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary. In another preferred embodiment m and or n is an integer between 1 and 1000. Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500. Polynucleotides that are identical, or are substantially identical to a nucleotide sequence of

SEQ ED NO.1 , may be used as hybndization probes for cDNA and genomic DNA or as pnmers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than Rattus Norvegicus) that have a high sequence identity to SEQ ED NO: 1. Typically these nucleotide sequences are 95% identical to that of the referent. Preferred probes or pnmers will generally compnse at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides, and may even have at least 100 nucleotides. Particularly preferred pnmers will have between 20 and 25 nucleotides

A polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from a species other than Rattus Norvegicus, may be obtained by a process composing the steps of screening an appropoate library under stringent hybodizatton conditions with a labeled probe having the sequence of SEQ ED NO:l or a fragment thereof, preferably of at least 15 nucleotides m length; and isolating full-length cDNA and genomic clones composing said polynucleotide sequence Such hybodizatton techniques are well known to the skilled artisan. Preferred stongent hybodizatton conditions include overnight incubation at 42°C in a solution composing 50% formamide, 5xSSC (150mM NaCl, 15mM tosodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in O.lx SSC at about 65°C. Thus, the present invention also includes isolated polynucleotides, preferably of at least 100 nucleotides in length, obtained by screening an appropoate library under stongent hybodizatton conditions with a labeled probe havmg the sequence of SEQ ED NO:l or a fragment thereof, preferably of at least 15 nucleotides.

The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA.

This is a consequence of reverse transcoptase, an enzyme with inherently low processivity' (a measure of the ability of the enzyme to remain attached to the template duong the polymerisation reaction), failing to complete a DNA copy of the mRNA template duong 1st strand cDNA synthesis. There are several methods available and well known to those skilled in the art to obtain full- length cDNAs, or extend short cDNAs, for example, those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al , Proc Natl Acad Sci , USA 85, 8998-9002, 1988) Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech Laboratooes Inc.), for example, have significantly simplified the search for longer cDNAs. In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end. Nucleic acid amplification (PCR) is then earned out to amplify the 'missing' 5' end of the cDNA using a combination of gene specific and adaptor specific ohgonucleotide primers The PCR reaction is then repeated using 'nested' pomers, that is, pomers designed to anneal within the amplified product (typically an adaptor specific pnmer that anneals further 3' in the adaptor sequence and a gene specific pomer that anneals further 5' in the known gene sequence) The products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer Recomb ant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells compnsing expression systems. Accordingly, m a further aspect, the present invention relates to expression systems compnsing a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polypepttdes of the invention by recombmant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs denved from the DNA constructs of the present invention.

For recombmant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells can be effected by methods descnbed in many standard laboratory manuals, such as Davis, et al, BASIC METHODS EN MOLECULAR BIOLOGY (1986) and Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spnng Harbor Laboratory Press, Cold Spnng Harbor, N.Y. (1989). Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, catiomc hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropnate hosts include bactenal cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophύa S2 and Spodoptera Sf9 cells; animal cells such as

CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

A great vanety of expression systems can be used, for instance, chromosomal, episomal and virus-denved systems, e.g , vectors denved from bactenal plasmids, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids. The expression systems may compose control regions that regulate as well as engender expression. Generally, any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropoate nucleotide sequence may be inserted into an expression system by any of a vaoety of well-known and routine techniques, such as, for example, those set forth m Sambrook, et al., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

If a polypepttde of the present invention is to be expressed for use m screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use m the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced mtracellularly, the cells must first be lysed before the polypeptide is recovered Polypeptides of the present invention can be recovered and punfϊed from recombmant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapattte chromatography and lecttn chromatography. Most preferably, high performance liquid chromatography is employed for puoficatton. Well known techniques for refolding proteins may be employed to regenerate acttve conformation when the polypepttde is denatured duong isolation and/or puoficatton.

Rattus Norvegicus BRS3 gene products can be expressed m transgenic animals. Animals of any species, including, but not limited to: mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human pomates, e g , baboons, monkeys, chimpanzees, may be used to generate BRS3 transgenic animals.

This invention further relates to a method of producing transgenic animals, preferably Rattus Norvegicus, over-expressing BRS3, which method may compose the introduction of several copies of a segment composing at least the polynucleotide sequence encoding SEQ ED NO: 2 with a suitable promoter into the cells of a Rattus Norvegicus embryo, or the cells of another species, at an early stage.

This invention further relates to a method of producing transgenic animals, preferably Rattus Norvegicus, under-expressing or regulatably expressing BRS3, which method may compose the introduction of a weak promoter or a regulatable promoter (e g. , an mducible or repressible promoter) respectively, expressibly linked to the polynucleotide sequence of SEQ ED NO: 1 into the cells of a

Rattus Norvegicus embryo at an early stage.

This invention also relates to transgenic animals, characteozed in that they are obtained by a method, as defined above.

Any technique known in the art may be used to introduce a Rattus Norvegicus BRS3 transgene into animals to produce a founder line of animals. Such techniques include, but are not limited to: pronuclear micromjection (U.S. Patent No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten, et al , Proc Natl Acad Sci , USA 82: 6148-6152 (1985); gene targeting m embryonic stem cells (Thompson, et al , Cell 56. 313-321 (1989); elecrropolatton of embryos (Lo, Mol Cell Biol. 3: 1803-1814 (1983); and sperm-mediated gene transfer (Lavitrano, et al , Cell 57. 717-723 (1989); etc For a review of such techniques, see Gordon, Ml Rev Cytol. 115

171-229 (1989). A further aspect of the present invention involves gene targeting by homologous recombination in embryonic stem cells to produce a transgenic animal with a mutation in a BRS3 gene ("knock-out" mutation). In such so-called "knock-out" animals, there is inactivation of the BRS3 gene or altered gene expression, such that the animals are useful to study the function of the BRS3 gene, thus providing animals models of human disease, which are otherwise not readily available through spontaneous, chemical or irradiation mutagenesis. Another aspect of the present invention involves the generation of so-called "knock-m" animals m which a portion of a wild-type gene is fused to the cDNA of a heterologous gene.

This invention further relates to a method of producing "knock-out" animals, preferably mice, no longer expressing BRS3. By using standard cloning techniques, a Rattus Norvegicus BRS3 cDNA

(SEQ ED NO: 1) can be used as a probe to screen suitable hbraoes to obtain the muone BRS3 genomic DNA clone. Using the muone genomic clone, the method used to create a knockout mouse is characteozed in that: a suitable mutation is produced in the polynucleotide sequence of the muone BRS3 genomic clone, which inhibits the expression of a gene encoding muone BRS3, or inhibits the activity of the gene product; said modified muone BRS3 polynucleotide is introduced into a homologous segment of muone genomic DNA, combined with an appropoate marker, so as to obtain a labelled sequence composing said modified muone genomic DNA; said modified muone genomic DNA composing the modified polynucleotide is transfected into embryonic stem cells and correctly targeted events selected in vitro; then said stem cells are remjected into a mouse embryo; then said embryo is implanted into a female recipient and brought to term as a chimera which transmits said mutation through the germline; and homozygous recombmant mice are obtained at the F2 generation which are recognizable by the presence of the marker

Vanous methods for producing mutations in non-human animals are contemplated and well known m the art In a preferred method, a mutation is generated m a muone BRS3 allele by the introduction of a DNA construct composing DNA of a gene encoding muone BRS3, which muone gene contains the mutation. The mutation is targeted to the allele by way of the DNA construct. The

DNA of the gene encoding muone BRS3 composed in the construct may be foreign to the species of which the recipient is a member, may be native to the species and foreign only to the individual recipient, may be a construct composed of synthetic or natural genetic components, or a mixture of these. The mutation may constitute an insertion, deletion, substitution, or combination thereof. The DNA construct can be introduced into cells by, for example, calcium-phosphate DNA co-precipitatton. It is preferred that a mutation be introduced into cells using electroporation, microinjection, virus infection, gand-DNA conjugation, virus-ligand-DNA conjugation, or posomes.

Another embodiment of the instant invention relates to "knock-out" animals, preferably mice, obtained by a method of producing recombmant mice as defined above, among others.

Another aspect of this invention provides for in vitro BRS3 "knock-outs", i.e., tissue cultures. Animals of any species, including, but not limited to: mice, rats, rabbits, guinea pigs, dogs, cats, pigs, micro-pigs, goats, and non-human pomates, e g., baboons, monkeys, chimpanzees, may be used to generate in vitro BRS3 "knock-outs". Methods for "knocking out" genes in vitro are descnbed in Galh-Tahadoros, et al. , Journal of Immunological Methods 181: 1-15 (1995).

Transgenic, "knock-m", and "knock-out" animals, as defined above, are a particularly advantageous model, from a physiological point of view, for studying 7 transmembrane receptors. Such animals will be valuable tools to study the functions of a BRS3 gene. Moreover, such animal models are expected to provide information about potential toxicological effects in humans of any compounds discovered by an aforementioned screening method, among others. An understanding of how a Rattus Norvegicus BRS3 gene functions these animal models is expected to provide an insight into treating and preventing human diseases including, but not limited to: infections such as bactenal, fungal, protozoan and viral infections, particularly infections caused by HJV-1 or HEV-2, pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; unnary retention; osteoporosis; angina pectons; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, dehnum, dementia, and severe mental retardation; and dyskinesias, such as Hunttngton's disease or Gilles dela

Tourett's syndrome.

Polypeptides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases mentioned herein. It is, therefore, an aspect of the invention to devise screening methods to identify compounds that stimulate (agonists) or that inhibit (antagonists) the function of the polypeptide, such as agonists, antagonists and inhibitors.

Accordingly, in a further aspect, the present invention provides for a method of screenmg compounds to identify those that stimulate or inhibit the function of the polypeptide. In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for the Diseases mentioned herein mentioned. Compounds may be identified from a vanety of sources, for example, cells, cell- free preparations, chemical branes, and natural product mixtures. Such agonists and antagonists so- ldentified may be natural or modified substrates, gands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Coligan, et al, CURRENT PROTOCOLS EN IMMUNOLOGY 1(2): Chapter 5 (1991)).

The screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes beanng the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, a screening method may involve measunng or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to the polypeptide with a labeled competitor (e g , agonist or antagonist). Further, screening methods may test whether the candidate compound results in a signal generated by an agonist or antagonist of the polypeptide, using detection systems appropriate to cells beanng the polypeptide. Antagonists are generally assayed in the presence of a known agonist and an effect on activation by the agonist by the presence of the candidate compound is observed. Further, screenmg methods may simply compose the steps of mixing a candidate compound with a solution composing a polypeptide of the present invention, to form a mixture, measunng Rattus Norvegicus BRS3 activity in the mixture, and companng a Rattus Norvegicus BRS3 activity of the mixture to a control mixture which contains no candidate compound.

Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats. Such HTS formats include not only the well-established use of 96- and, more recently, 384-well microtiter plates but also emerging methods such as the nanowell method described by Schullek, et al , Anal Biochem., 246, 20-29, (1997).

Fusion proteins, such as those made from Fc portion and Rattus Norvegicus BRS3 polypeptide, as herein described, can also be used for high-throughput screening assays to identify antagonists of antagonists of the polypeptide of the present invention (see D. Bennett, et al , J Mol Recognition, 8:52-58 (1995); and K Johanson, et al , J Bwl Chem., 270(16):9459-9471 (1995))

One screenmg technique includes the use of cells which express the receptor of this invention (for example, transfected CHO cells) in a system which measures extracellular pH or mtracellular calcium changes caused by receptor activation. In this technique, compounds may be contacted with cells expressing the receptor polypeptide of the present invention. A second messenger response, e.g., signal transduction, pH changes, or changes in calcium level, is then measured to determine whether the potential compound activates or inhibits the receptor Another method involves screening for receptor inhibitors by determining inhibition or stimulation of receptor-mediated cAMP and/or adenylate cyclase accumulation. Such a method involves transfectmg a eukaryotic cell with the receptor of this invention to express the receptor on the cell surface. The cell is then exposed to potential antagonists in the presence of the receptor of this invention. The amount of cAMP accumulation is then measured. If the potential antagonist binds the receptor, and thus inhibits receptor binding, the levels of receptor-mediated cAMP, or adenylate cyclase, activity will be reduced or increased.

Another method for detecting agonists or antagonists for the receptor of the present invention is the yeast based technology as described m U.S. Patent No. 5,482,835. Examples of potential polypeptide antagonists include antibodies or, in some cases, ohgopeptides or proteins that are closely related to ligands, substrates, receptors, enzymes, etc , as the case may be, of a BRS3 polypeptide, e g., a fragment of a hgand, substrate, receptor, enzyme, etc.; or small molecules which bind to a BRS3 polypeptide but do not elicit a response, so that an activity of a BRS3 polypeptide is prevented. Thus, in another aspect, the present invention relates to a screenmg kit for identifying agonists, antagonists, inhibitors, ligands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which compounds compnse a member selected from the group consisting of:

(a) a polypeptide of the present invention; (b) a recombmant cell expressing a polypeptide of the present invention; or

(c) a cell membrane expressing a polypeptide of the present invention; which polypeptide is preferably that of SEQ ED NO.2.

It will be appreciated that in any such kit, (a), (b) or (c) may comprise a substantial component. It will also be readily appreciated by the skilled artisan that a polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by.

(a) determining in the first instance the three-dimensional structure of the polypeptide,

(b) deducing the three-dimensional structure for the likely reactive or binding sιte(s) of an agonist, antagonist or inhibitor; (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding or reactive site; and

(d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors

It will be further appreciated that this will normally be an iterative process. In an alternative preferred embodiment, the present invention relates to the use of Rattus

Norvegicus BRS3 polypeptides, polynucleotides, and recombmant matenals thereof m selection screens to identify compounds which are neither agonists nor antagonist/inhibitors of Rattus Norvegicus BRS3. The data from such a selection screen is expected to provide in vitro and in vivo compansons and to predict oral absorption, pharmacokmetics in humans. The ability to make such a companson of data will enhance formulation design through the identification of compounds with optimal development charactenstics, i e , high oral bioavailabihty, UTD (once a day) dosmg, reduced drug interactions, reduced variability, and reduced food effects, among others.

The following definitions are provided to facilitate understanding of certain terms used frequently herein. "Allele" refers to one or more alternative forms of a gene occumng at a given locus in the genome.

"Fragment" of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. "Fragment" of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ED NO: 1.

"Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. In one example, EP-A-0 464 discloses fusion proteins composing various portions of constant region of lmmunoglobuhn molecules together with another human protein or part thereof. In many cases, employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e g , EP-A 0232 262] On the other hand, for some uses, it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected, and purified.

"Homolog" is a generic term used m the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similarity between the two sequences as hereinbefore defined Falling withm this generic term are the terms, "ortholog", and "paralog". "Ortholog" refers to polynucleotides/genes or polypeptide that are homologs via speciation, that is closely related and assumed to have commend descent based on structural and functional considerations. "Paralog" refers to polynucleotides/genes or polypeptide that are homologs via gene duplication, for instance, duplicated variants withm a genome. "Identity" reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences, fri general, identity refers to an exact nucleotide to nucleotide or ammo acid to ammo acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared. For sequences where there is not an exact correspondence, a "% identity" may be determined. In general, the two sequences to be compared are aligned to give a maximum conelation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identtty may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.

"Similaoty" is a further, more sophisticated measure of the relationship between two polypepttde sequences. In general, "similaoty" means a comparison between the ammo acids of two polypepttde chains, on a residue by residue basis, taking into account not only exact coπespondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated 'score' from which the "% similarity" of the two sequences can then be determined.

Methods for companng the identity and similanty of two or more sequences are well known in the art. Thus for instance, programs available in the Wisconsin Sequence Analysis Package, version 9 1 (Devereux J., et al, Nucleic Acids Res, 12, 387-395, 1984, available from Genetics

Computer Group, Madison, Wisconsin, USA), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % similarity between two polypeptide sequences BESTFIT uses the "local homology" algorithm of Smith and Waterman (J Mol Bwl , 147: 195-197, 1981, Advances in Applied Mathematics, 2, 482- 489, 1981) and finds the best single region of similaoty between two sequences. BESTFIT is more suited to comparing two polynucleotide or two polypepttde sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer. In compaoson, GAP aligns two sequences, finding a "maximum similaoty", according to the algorithm of Neddleman and Wunsch (J Mol Bwl , 48, 443-453, 1970). GAP is more suited to compaong sequences that are approximately the same length and an alignment is expected over the entire length. Preferably, the parameters "Gap Weight" and "Length Weight" used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively. Preferably, % identities and similaoties are determined when the two sequences being compared are optimally aligned.

Other programs for determining identity and/or similaoty between sequences are also known m the art, for instance the BLAST family of programs (Altschul S.F., et al , J Mol Bwl , 215, 403-410, 1990, Altschul S.F., et al , Nucleic Acids Res , 25:389-3402, 1997, available from the National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA and accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, Methods in Enzymology, 183: 63-99 (1990); Pearson W R and Lipman DJ., Proc Nat Acad Sci USA, 85: 2444-2448 (1988) (available as part of the Wisconsin Sequence Analysis Package).

Preferably, the BLOSUM62 ammo acid substitution matrix (Henikoff S. and Henikoff J.G., Proc Nat Acad Sci USA, 89: 10915-10919 (1992)) is used in polypeptide sequence compansons including where nucleotide sequences are first translated into ammo acid sequences before companson.

Preferably, the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a polynucleotide or a polypeptide sequence of the present invention, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore described.

Alternatively, for instance, for the purposes of interpreting the scope of a claim including mention of a "% identity" to a reference polynucleotide, a polynucleotide sequence having, for example, at least 95% identity to a reference polynucleotide sequence is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference sequence Such point mutations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion These point mutations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. In other words, to obtain a polynucleotide sequence having at least 95% identity to a reference polynucleotide sequence, up to 5% of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descobed. The same applies mutatis mutandis for other % identities such as 96%, 97%, 98%, 99% and 100%.

For the purposes of interpreting the scope of a claim including mention of a "% identity" to a reference polypeptide, a polypeptide sequence having, for example, at least 95% identity to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include up to five point mutations per each 100 ammo acids of the reference sequence. Such point mutations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion. These point mutations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups withm the reference sequence. In other words, to obtain a sequence polypeptide sequence having at least 95% identity to a reference polypeptide sequence, up to 5% of the ammo acids of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descnbed. The same applies mutatis mutandis for other % identities such as 96%, 97%, 98%, 99%, and 100%.

A preferred meaning for "identity" for polynucleotides and polypepttdes, as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 95, 97 or 100% identity to the reference sequence of SEQ ED NO: 1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ED

NO:l or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and fransversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ED NO:l by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ED NO:l, or:

ι_n < x_n (*n • y). wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ED NO: l, y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-mteger product of x_n and y is rounded down to the nearest integer pnor to subtracting it from x_n. Alterations of a polynucleotide sequence encoding the polypepttde of SEQ ED NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.

(2) Polypeptide embodiments further include an isolated polypeptide compnsing a polypeptide having at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ED NO.2, wherein said polypepttde sequence may be identical to the reference sequence of SEQ ED NO.2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or m one or more contiguous groups withm the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ED NO:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ED NO:2, or:

n_a < x_a - (x_a • y),

wherein n_a is the number of ammo acid alterations, x_a is the total number of ammo acids in SEQ ID NO:2, y is 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-mteger product of x_a and y is rounded down to the nearest integer poor to subtracting it from x_a

"Isolated" means altered "by the hand of man" from its natural state, i e , if it occurs in nature, it has been changed or removed from its ooginal environment, or both. For example, a polynucleotide or a polypeptide naturally present m a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting mateoals of its natural state is "isolated", as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombmant method is "isolated" even if it is still present m said organism, which organism may be living or non-living. "Knock-in" refers to the fusion of a portion of a wild-type gene to the cDNA of a heterologous gene

"Knock-out" refers to partial or complete suppression of the expression of a protein encoded by an endogenous DNA sequence in a cell. The "knock-out" can be affected by targeted deletion of the whole or part of a gene encoding a protein, m an embryonic stem cell. As a result, the deletion may prevent or reduce the expression of the protein in any cell in the whole animal m which it is normally expressed.

"Splice Vanant" as used herein refers to cDNA molecules produced from RNA molecules initially transcnbed from the same genomic DNA sequence but which have undergone alternative RNA splicing. Alternative RNA splicing occurs when a pnmary RNA transcript undergoes splicing, generally for the removal of nitrons, which results in the production of more than one mRNA molecule each of that may encode different ammo acid sequences. The term splice vanant also refers to the proteins encoded by the above cDNA molecules.

"Transgenic animal" refers to an animal to which exogenous DNA has been introduced while the animal is still in its embryonic stage. In most cases, the transgenic approach aims at specific modifications of the genome, e.g., by introducing whole transcπptional units into the genome, or by up- or down-regulating pre-existing cellular genes. The targeted character of certain of these procedures sets transgenic technologies apart from expenmental methods in which random mutations are conferred to the germlme, such as administration of chemical mutagens or treatment with ionizing solution.

"Polynucleotide" generally refers to any polynbonucleotide or polydeoxobonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules composing DNA and RNA that may be smgle-sfranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNAs or RNAs comprising one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tπtylated bases and unusual bases such as mosme. A vanety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often refened to as oligonucleotides.

"Polypeptide" refers to any peptide or protein compnsing two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i.e , peptide isosteres. "Polypepttde" refers to both short chains, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may comprise ammo acids other than the 20 gene-encoded ammo acids. "Polypeptides" include ammo acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known m the art. Such modifications are well descnbed in basic texts and m more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chains and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may compose many types of modifications. Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-nbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or lipid denvative, covalent attachment of phosphotidylmositol, cross-linking, cyc zation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteme, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as argmylation, and ubiquitmation (see, for instance, PROTEINS - STRUCTURE AND

MOLECULAR PROPERTEES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, pgs. 1- 12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter, et al , "Analysis for protein modifications and nonprotein cofactors", Meth. Enzymol. (1990) 182:626-646 and Rattan, et al., "Protein Synthesis-

Post-translational Modifications and Aging", Ann NY Acad Sci (1992) 663:48-62).

"Vanant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the vanant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in ammo acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the vaoant are closely similar overall and, in many regions, identical. A vaoant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. A vanant of a polynucleotide or polypeptide may be a naturally occumng such as an allehc vaoant, or it may be a vaoant that is not known to occur naturally. Non-naturally occumng variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

All publications including, but not limited to, patents and patent applications, cited in this specification or to which this patent application claims poooty, are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

Examples

Example 1 Mammalian Cell Expression

The receptors of the present invention are expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. To maximize receptor expression, typically all 5 ' and

3 ' untranslated regions (UTRs) are removed from the receptor cDNA poor to insertion into a pCDN or pCDNA3 vector. The cells are transfected with individual receptor cDNAs by hpofectin and selected in the presence of 400 mg/ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis. HEK293 or CHO cells transfected with the vector alone serve as negative controls. To isolate cell lines stably expressing the individual receptors, about 24 clones are typically selected and analyzed by Northern blot analysis. Receptor mRNAs are generally detectable in about 50% of the G418-resιstant clones analyzed.

Example 2 Ligand bank for binding and functional assays

A bank of over 600 putative receptor ligands has been assembled for screening. The bank composes- transmitters, hormones and chemokmes known to act via a human seven transmembrane

(7TM) receptor; naturally occumng compounds which may be putative agonists for a human 7TM receptor, non-mammalian, biologically active pepttdes for which a mammalian counterpart has not yet been identified; and compounds not found in nature, but which activate 7TM receptors with unknown natural ligands. This bank is used to initially screen the receptor for known ligands, using both functional (i.e . calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as binding assays. Example 3: Ligand Binding Assays

Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format. The punfied ligand for a receptor is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabelmg does not dimmish the activity of the ligand towards its receptor. Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell receptor sources. For these assays, specific receptor binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding. Example 4: Functional Assay in Xenopus Oocytes

Capped RNA transcnpts from lmeanzed plasmid templates encoding the receptor cDNAs of the invention are synthesized in vitro with RNA polymerases m accordance with standard procedures. In vitro transcnpts are suspended in water at a final concentration of 0.2 mg/ml. Ovanan lobes are removed from adult female toads, Stage V defolhculated oocytes are obtained, and RNA transcnpts (10 ng/oocyte) are injected m a 50 nl bolus using a micromjection apparatus.

Two electrode voltage clamps are used to measure the cuπents from individual Xenopus oocytes in response to agonist exposure. Recordings are made in Ca2+ free Barth's medium at room temperature. The Xenopus system can be used to screen known ligands and tissue/cell extracts for activating ligands. Example 5: Microphysiometoc Assays

Activation of a wide vanety of secondary messenger systems results m extrusion of small amounts of acid from a cell. The acid formed is largely as a result of the increased metabolic activity required to fuel the mtracellular signaling process The pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, CA) The CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilizing mtracellular signaling pathway such as the G-protem coupled receptor of the present invention. Example 6: Extract/Cell Supernatant Screenmg

A large number of mammalian receptors exist for which there remains, as yet, no cognate activating ligand (agonist). Thus, active ligands for these receptors may not be included with the ligands banks as identified to date. Accordingly, the 7TM receptor of the invention is also functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify natural ligands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified.

Example 7: Calcium and cAMP Functional Assays 7TM receptors which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimulation or inhibition. Basal calcium levels in the HEK 293 cells in receptor-transfected or vector control cells were observed to be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressing recombmant receptors are loaded with fura 2 and in a single day > 150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization. Similarly, HEK 293 cells expressing recombmant receptors are evaluated for the stimulation or inhibition of cAMP production using standard cAMP quantitation assays. Agonists presenting a calcium transient or cAMP fluctuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor.

Claims

What is claimed is:

1. An isolated polynucleotide selected from the group consisting of:

(1) an isolated polynucleotide compnsing a nucleotide sequence encoding a polypepttde having at least a 95% identity to the ammo acid sequence of SEQ ED NO:2, over the entire length of SEQ ED NO:2;

(π) an isolated polynucleotide composing a nucleotide sequence having at least a 95% identity over its entire length to a nucleotide sequence encoding the polypeptide of SEQ ID NO:2;

(in) an isolated polynucleotide composing a nucleotide sequence having at least a 95%o identity to that of SEQ ED NO: 1 over the entire length of SEQ ED NO: 1 ;

(IV) an isolated polynucleotide compnsing a nucleotide sequence encoding the polypepttde of SEQ D NO:2;

(v) an isolated polynucleotide that is the polynucleotide of SEQ ED NO: 1 ; or

(vi) an isolated polynucleotide with a nucleotide sequence of at least 100 nucleotides in length obtained by screening an appropnate library under stongent hybodizatton conditions with a labelled probe having the sequence of SEQ ID NO: 1 or a fragment thereof of at least 15 nucleotides; or a nucleotide sequence complementary to said isolated polynucleotide.

2. An isolated polypeptide selected from the group consisting of:

(I) an isolated polypepttde having at least a 95% identity to the ammo acid sequence of SEQ ED NO: 2 over the entire length of SEQ ED NO:2;

(n) an isolated polypeptide comprising the ammo acid sequence of SEQ ED NO:2; or

(in) an isolated polypeptide that is the ammo acid sequence of SEQ ID NO:2.

3. A method for screenmg to identify compounds that stimulate or that inhibit a function or level of the polypeptide of Claim 2, composing a method selected from the group consisting of: (a) measuong or, quantitatively or qualitatively, detecting the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound; (b) measuring the competition of the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor;

(c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropoate to the cells or cell membranes bearing the polypeptide;

(d) mixing a candidate compound with a solution composing a polypeptide of Claim 2, to form a mixture, measuring activity of the polypeptide in the mixture, and comparing the activity of the mixture to a to a control mixture which contains no candidate compound; or (e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells.

4. An agonist or an antagonist of the polypeptide of Claim 2.

5. An agonist or an antagonist of the Rattus Norvegicus BRS3 identified by the method of Claim 3.

6. An expression system comprising a polynucleotide capable of producing a polypeptide of Claim 2 when said expression system is present in a compatible host cell.

7. A process for producing a recombinant host cell composing the step of introducing the expression vector of Claim 6 into a cell, such that the host cell, under appropoate culture conditions, produces said polypeptide.

8. A recombinant host cell produced by the process of Claim 7.

9. A membrane of a recombinant host cell of Claim 8 expressing said polypeptide.

10. A process for producing a polypeptide comprising culturing a host cell of Claim 9 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.