CA2221616A1 - Human g-protein receptor hibef51 - Google Patents

Abstract

Human G-protein coupled receptor polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptides for identifying antagonists and agonists to such polypeptides and methods of using the agonists and antagonists therapeutically to treat conditions related to the underexpression and overexpression of the receptor polypeptides, respectively. Also disclosed are diagnostic methods for detecting a mutation in the receptor nucleic acid sequences and detecting a level of the soluble form of the receptors in a sample derived from a host.

Description

}~AN G-PROTEIN K ~;-cr . OK ~IBEF51 This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use o~ such polynucleotides and polypeptides, as well as the produc~ion of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a human 7-tr~n~memhrane receptor. The tr~ncm~mhrane receptor is a G-protein coupled receptor. The invention also relates to ; nh; h;ting the action of such polypeptides.
It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or ~econd messengers, e.g., cAMP (Lefkowitz, Nature, 351:353-354 (1991)). Herein these proteins are referred to as protein~ participating in pathways with G-protein~ or PPG
proteins. Some examples of these proteins include the GPC
receptors, such as those for adrenergic agents and ~or~m;ne (~h;lka, B.g., et al., PN~S, 84:46-50 (1987); Xobilka, B.K., et al., Science, 238:650-656 (1987); Bunzow, J.R., et al., Nature, 336:783-787 (1988)), G-proteins themselves, ef~ector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein W096/39441 PCT~S95/07Z25 kinase A and protein kinase C (Simon, M.I., et al., Science, 252:802-8 (l99l)).
For example, in one form of signal transduction, the effect of hormone binding is activation of an enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP, and GTP also influences hormone b;n~ing. A G-protein connects the hormone receptors to adenylate cyclase. G-protein was shown to ~ch~nge GTP for bound GDP when activated by hormone receptors. The GTP-carrying form then binds to an activated adenylate cyclase. Hydrolysis of GTP
to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive 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 me.,~lane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative tr~ns~~~hrane ~nm~;n~, The ~m~lnC are believed to represent tr~n~ hrane ~-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
G-protein coupled receptors have been characterized 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 dop~m;ne receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include calcitonin, adrenergic, endothel;n, cAMP, adenosine, muscarinic, acetylcholine, serotonin, hist ~m; ne, thrombin, kinin, follicle stimulating hormone, opsins and rhodopsins, odorant, cytomegalovirus receptors, etc.

Most GPRs have single conserved cysteine residues in each of the ~irst two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure. The 7 tr~n~mhrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 is also implicated in signal transduction.
Phosphorylation and lipidation (palmitylation or ~arnesylation) o~ cysteine residues can influence signal transduction of some GPRs. Most GPRs contain potential phosphorylation sites within the third cytoplasmic loop and/or the c~ho~y terminus. For se~eral GPRs, such as the ~-adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor ~inases mediates receptor desensitization.
The ligand binding sites of GPRs are believed to comprise a hydrophilic socket formed by several GPR
tr~n~mPmhrane ~m~;n~, which socket is surrounded by hydrophobic residues of the GPRs. The hydrophilic side of each GPR tr~n~m~mhrane helix is postulated to face inward and form the polar ligand bt n~; ng site. TM3 has been implicated in several GPRs as having a ligand binding site, such as including the TM3 aspartate residue. Additionally, TM5 serines, a TM6 asparagine and TM6 or TM7 phenyl~l ~n; ne~ or tyrosines are also implicated in ligand hi n~ ng, GPRs can be intracellularly coupled by heterotrimeric G-prcteins to various intracellular enzymes, ion ch~nn~ls and transporters (see, John on et al ., Endoc., Rev., 10:317-331 (lg89)). Different G-protein ~-su~units preferentially 8~ te particular effectors to modulate various biological fu~ctions in a cell. Phosphorylation of cytoplasmic residues of GPRs has been identified as an important merh~n~ ~m for the regulation of G-protein coupling of some GP~s.
G-protein coupled receptors are ~ound in numerous sites within a ~ lian host, for example, dor~m~ne i~ a critical neurotransmitter in the central nervous system and is a G-protein coupled receptor ligand.
In accordance with one aspect of the present invention, there are provided novel mature receptor polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The receptor polypeptides of the present invention are of human origin.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the receptor polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA as well as antisense analogs thereof and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with a further aspect of the present invention, there are provided processes for producing such receptor polypeptides by recomh;n~nt techniques comprising culturing recom~inant prokaryotic and/or eukaryotic host cells, cont~;n;ng nucleic acid sequences encoding the receptor polypeptides of the present invention, under conditions promoting expression of said polypeptides and subsequent recovery of said polypeptides.
In accordance with yet a further aspect of the present invention, there are provided antibodies against such receptor polypeptides.
In accordance with another aspect of the present invention there are provided methods of screening for compounds which bind to and activate or inhibit activation of the receptor polypeptides of the present invention.
In accordance with still another embodiment of the present invention there are provided processes of ~mi n; stering compounds to a host which bind to and activate the receptor polypeptide of the present invention which are useful in the prevention and/or treatment of upper respiratory conditions, for example, allergic rhinitis, hay WO 96139441 PCT/US95/0722~;

fever, acute coryza and sinusitus, to promote uterine inhibition, to stimulate platelet aggregation, regulate lipid metabolism, and inhibit glucose-stimulated insulin release ~ from the pancreas.
In accordance with another aspect of the present invention there is pro~ided a method o~ ~m;n; stering the receptor polypeptides of the present invention via gene therapy to treat conditions related to underexpression of the polypeptides or underexpression o~ a ligand to the receptor polypeptide.
In accordance with still another e~odiment of the present invention there are provided processes o~
~mi n; stering compounds to a ho~t which bind to and inhibit activation of the receptor polypeptides of the present invention which are useful in the prevention and/or treatment of hyperten~ion and other myocardial di~ease and other diseases relating from vasoconstriction.
In accordance with yet another aspect of the present invention, there are provided nucleic acid probes compri~ing nucleic acid molecules of sufficient length to specifically hy~ridize to the polynucleotide sequences of the present inv~ention.
In accordance with still another aspect of the present in~ention, there are provided diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences encoding such polypeptides and for detecting an altered level of the soluble fonm of the receptor polypeptide~.
In accordance with yet a further aspect of the present invention, there are provided processes for utilizing such receptor polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.
These and other aspects of the present invention should be apparent to those skilled in the art from the te~chings herein.

WO 96139441 PCT/US95/0722~;

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figure 1 shows the cDNA sequence and the corresponding deduced amino acid sequence of the G-protein coupled receptor of the present invention. The st~n~Ard one-letter abbreviation for amino acids is used. Sequencing was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.).
Figure 2 illustrates an amino acid alignment of the G-protein coupled receptor of the present invention and the human adrenergic a,A receptor.
In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. on June 1, 1995.
A polynucleotide encoding a polypeptide of the present invention may be found in skeletal muscle, heart and brain.
The polynucleotide of this invention was discovered in a cDNA
library derived from a human infant brain. It is structurally related to the G protein-coupled receptor family. It contains an open reading frame encoding a protein of 349 amino acid residues. The protein exhibits the highest degree of homology to a human a A adrenergic receptor with 25.387~ identity and 51.084% similarity over a 331 amino acid stretch.
The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEQ ID

NO:1) or that of the deposited clone or may be a different cod- ~ sequence which coding sequence, as a result of the r~ n~ncy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1 (SEQ ID NO:1) or the deposited cDNA.
The polynucleotide which encodes ~or the mature polypeptide of Figure 1 (SEQ ID NO:2) or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence ~or the mature polypeptide; the coding sequence for the mature polypeptide and additional co~i n~
sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5~ and/or 3 o~ the coding sequence ~or the mature polypeptide.
Thus, the tenm "polynucleotide PnroA~ng a polypeptide"
Pnco~r?sses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional r~A;n~ and/or non-coding sequence.
The present invention ~urther relates to variants o~ the hereinabove described polynucleotides which encode for fragments, analog~ and derivatives of the polypeptide having the deduced amino acid equence of Figure 1 (S~Q ID NO: 2 ) or the polypeptide Pnco~e~ by the cDNA o~ the deposited clone.
The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides whi.c.A variants encode ~or a ~ragment, derivative or analog o~
the polypeptide of Figure 1 (S~Q ID NO: 2 ) or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide W096/39441 PCT~S95/07225 variants include deletion variants, substitution variants and addition or insertion variants.
As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 (SFQ ID
N0:1) or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternate form o~
a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
The polynucleotides may also encode for a soluble form of the receptor polypeptide which is the extracellular portion of the polypeptide which has been cleaved from the TM
and intracellular ~om~in of the full-length polypeptide of the present invention.
The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for puri$ication of the polypeptide o~ the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide ~or purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mAmm~lian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
The term "gene n means the segment of DNA involved in proAncing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
Fragments of the full length receptor gene may be used as a hybridization probe ~or a cDNA library to isolate the full length gene and to isolate other genes which have a high W096/39441 PCT~S95/07225 sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomlc clone or clones th~t contain the complete receptor gene including regulatory ancl promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence con~l~m~nt~ry to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the pro~e hybridizes to.
The present invention further relates to polynucleotides which hybridize to the here~n~hove-described sequences if there is a~ lea~t 70%, preferably at least 90%, an~l more preferably at least 95~ identity between the se~nc~s. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the herP;n~hove-described polynucleotides. A~ herein used, the term "stringent conditions~ means hybridization will occur only if there is at least 95% and preferably at least 97~ identity between ~he sequences. The polynucleotides which hybridize to the here;n~hove described polynucleotides in a preferred ~mho~;m~nt encode polypeptides which either retain subst~nt;~lly the same biological function or activity as the mature polypeptide ~nco~ed by the cDNAs o~ Figure 1 (SBQ ID NO:1) or the depo~ited cDNA(s).
Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as herp~n~hove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for CA 0222l6l6 lgg7-ll-l9 WO96/39441 PCT~S95/072Z5 the polynucleotide of SEQ ID NO:l, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR
primer.
Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90~ and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.
The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. 112.
The sequence of the polynucleotides cont~ine~ in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.
The present invention further relates to a receptor polypeptide which has the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
The terms "fragment, n ~derivative" and ~analog~ when referring to the polypeptide of Figure 1 (SEQ ID NO:2) or that Pnro~ed by the deposited cDNA, means a polypeptide which either retains substantially the same biological function or activity as such polypeptide, i.e. functions as a receptor, or retains the ability to bind the ligand or the receptor even though the polypeptide does not function as an receptor, CA 02221616 1997-ll-lg 096/39441 PCT~S95/07225 ~or example, a soluble ~orm o~ the receptor. An analog includes an extracellular portion which can be cleaved from the tr~ncm~mhrane ~om~n and intracellular portion to produce a solu~l~ active peptide.
The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, pre~erably a recombinant polypeptide.
The fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID N0:2) or that encoded by ~he deposited cDN~ may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and ~uch substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent grollp, or (iii) one in which the mature polypeptide is fused wit~ another compound, such as a c~ ~o~d to increase the halE-life of the polypeptide (for example, polyethylene gly~ol), or (iv) one in which the additional amino acids are fused to the ma~ure polypeptide which is employed for purification of the mature polypeptide, or (v) one in which a fragment of the polypeptide is soluble, i.e. not .,.~..~Ldne bound, yet still bind6 ligands to the ...~..~L~ne bound rec~eptor. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated fonm, and preferably are purified to ho...oye.~eity.
The polypeptides of the present invention include the polypeptide of SEQ ID N0:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70%
similarity (preferably at least a 70% identity) to the polypeptide of S~Q ID N0:2 and more preferably at least a 90~
similarity (more preferably at least a 90% identity) to the WO96/39441 PCT~S95/07225 polypeptide of SEQ ID NO:2 and still more preferably at least a 9S~ similarity (still more preferably at least a 95%
identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally cont~n~ng at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "similarity" between two polypeptides ifi determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize ~ull-length polynucleotides of the present invention.
The term "gene" mean~ the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region "leader and trailer" as well as intervening sequences (introns) between individual coding segments (exons).
The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living ~n~-l iS not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environm~nt~

The polypeptides of the present invention include the polypeptide of SBQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70~
- similarity (preferably a 70% identity) to the polypeptide of S~Q ID NO:2 and more preferably a 90% s~m;l~rity (more preferably a 90~ identity) ~o the polypeptide of SEQ ID NO:2 and still more prefera~ly a 95% similarity (still more preferably a 90~ identity) ~o the polypeptide o~ SBQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide general~y cont~in;ng at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "similarity" between two polypeptides is determlned by comparing the amino acid sequence and its conser~red amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present in~ention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fra~onts or portions of the polynucleotides of the present invention may be used to sy~thesize full-length polynucleotides of the present in~ention.
The present inven~ion also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production o~ polypeptides of the invention by recombinant techniques.
Host cells are genetically engineered (transduced or tran~for~ed or transfected) with the vectors of this in~rention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the fo~n of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as a~l~riate for activating WO 96/39441 PCT/US9JI~ / ~2~

promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily ~killed artisan.
The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonrh~omosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
However, any other vector may be used as long as it is replicable and viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA
sequence is inserted into an appropriate restriction ~n~QnllClease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the ~. coli. lac or tr~, the phage lambda PL
promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of trans~ormed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resi~tance in E. coli.
The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to trans~orm an appropriate ho~t to permit the host to express the protein.
As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as B. coli, strePtomyces~ Salmonella t~~himurium; fungal cells, such as yeast; insect cells such as Droso~hila and S~odoptera Sf9;
~n;m~l cells such as CHO, COS or Bowes melanoma; adenovirus;
plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art ~r~m the teachings herein.
More particularly, the present invention also includes recombinant constructs co~prising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embo~im~nt, the construc~ further comprises regulatory sequences, including, for example, a promoter, operably lir~ed to the sequence. Large n-lmh~rs of suitable vectors and promoters are known to those of skill in the art, and are co~mercially aV~;lAhle~ The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbs" pD10, phagescript, psiX174, pbluescript SK, pbsks, pN~8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSVZCAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pS~TL (Pharmacia). However, any other plasmid or vector may WO96139441 PCT~S95/07225 be used as long as they are replicable and viable in the host.
Promoter regions can be selected from any desired gene using CAT (chloramphenicol trans~erase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp.
Eukaryotic promoters include CMV imm~t~te early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection o~ the appropriate vector and promoter is well within the level of ordinary skill in the art.
In a further embo~;m~nt, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a m~tnm~ lian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, D~AB-Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional m~nner to produce the gene product encoded by the recomh~n~n~ sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in m~mmAlian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sa-m-brook~
et al., Molecular Cloning: A Laboratory ~;tnn~l, Second CA 02221616 1997-ll-l9 Edition, Cold Spring Harbor, N.Y., (1989), the disclosure o~
which is hereby incorporated by reference.
Transcription of the DNA encoding the polypeptides of - the present invention by higher eukaryotes is increased by inserting an ~nh~ncer sequence into the vector. Rnh~ncer~
~ are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription.
~xamples including the Sv40 ~nh~ncer on the late side o~ the replication origin bp 100 to 270, a cytomegalovirus early pr~moter ~nh~ncer, the polyoma ~nh~ncer on the late side of the replication origin, and adenovirus ~nh~ncers.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation o~ the host cell, e.g., the ampicillin recistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct tra~scription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enz:y~es such as 3-phosphoglycerate kinase (PGR), ~-~actor, aci.d pho~phatase, or heat shock proteins, among others. The heterologous structural sequence is asfiembled in appropriate phase with translation initiation and termination sequences, and pre~erably, a leader ~equence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can ~ncoAe a fusion protein including an N-terminal identification peptide imparting desired charac~eristics, e.c~., stabilization or simplified purification of expressed reromh;n~nt product.
Use~ul expression ~ector~ for bacterial use are constructed by inserting a structural DNA sequence ~nro~;ng a desired protein together with suitable translation in:Ltiation and termination signals in operable reading phase wi~h a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin o~

replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella t~phimurium and various species within the genera Pse~l~o~onAs~ Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from comm~cially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC
37017). Such commercial vectors include, for example, pKK223-3 ~Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone"
sections are co~h~ne~ with an appropriate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate -ell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the re~ulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted by any con~enient method, including freeze-thaw cycling, sonication, mechanical di~ruption, or use of cell lysing agents, such methods are well ~now to those skilled in the art.
Various ~mm~lian cell culture systems can also be employed to express recombinant protein. Examples of m~ lian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a CA 02221616 1997-ll-lg WO96/39441 PCT~S95/07225 compatible vector, for example, the C127, 3T3, CHO, HeLa and BH~ cell lines. ~mm~lian expression ~ectors will comprise an origin of replication, a suitable promoter and ~nh~ncer, - and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination ~ecuences, and 5/ flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
The receptor polypeptides can be recovered and purified from recomhin~nt cell cultures by methods including ~mmonium su].fate or ethanol precipi~ation, acid ex~raction, anion or cat;ion exchange chromatography, phosphocellulo~e chxomatography, hydrophobic interaction chromatography, a~inity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding step~ can be used, as necessary, in completing configuration of the mature prc~tein. Finally, high performance liquid chromatography (HPLC) can be employed for final puri~ication steps.
The polypeptides of the present invention may be a nat:urally purified product, or a product of chemical synthetic procedures, or produced by recomh;n~nt techniques ~rom a prokaryotic or eukaryotic host (~or example, by bacterial, yeast, higher plant, insect and m~ l ian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present in~ention may be glycosylated or may be non-glycosylated.
Polypeptides of the i~ven~ion may also include an initial me~hionine amino acid residue.
The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease.
The receptor of the present invention may be employed in a process for screening ~or compounds which bind to and activate (agonists) or bind to and ~nh;h~ t activation --lg -CA 0222l6l6 lgg7-ll-l9 WO96/39441 PCT~S95/07225 (antagonists) of the receptor polypeptide of the present invention.
In yeneral, such screening procedures involve providing appropriate cells which express the receptor on the surface thereof. In particular, a polynucleotide encoding the receptor of the present invention is employed to transfect cells to thereby express the receptor. Such transfection may be accomplished by procedures as hereinabove described.
One such screening procedure involves the use of the m~1~nophores which are transfected to express the receptor of the present invention. Such a screening technique is described in PCT WO 92/01810 published February 6, 1992.
Thus, for example, such assay may be employed for screening for a receptor antagonist by contacting the m~l ~nophore cells which encode the receptor with both the receptor ligand and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i.e., inhibits activation of the receptor.
The screen may be employed for determining an agonist by contacting such cells with compounds to be screened and determ;n~ng whether such compound generates a signal, i.e., activates the receptor.
Other screening technigues include the use of cells which express the receptor (for example, transfected CHO
cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science, volume 246, pages 181-296 (~ctober 1989). For example, potential agonists or antagonists may be contacted with a cell which expresses the receptor and a second messenger response, e.g. signal transduction or pH changes, may be measured to determine whether the potential agonist or antagonist is effective.
Another such screening technigue involves introducing RNA PnCo~;ng the receptor into xenopus oocytes to transiently WO96/39441 PCT~S95/07225 e~ress the receptor. The receptor oocytes may then be contacted in the case of antagonist screening with the receptor ligand and a compound to be screened, followed by detection of inhibition of a calcium signal.
Another screening technique involves expressing the ~ receptor in which the receptor is linked to a phospholipase C or D. As representative examples of such cells, there may be mentioned endothelial cells, smooth muscle cells, embryonic kidney cells, etc. The screening for an antagonist or agonist may be accomplished as here~n~hove described by detecting activation of the receptor or inhibition of activation o~ the receptor from the phospholipase second signal.
Another method involves screening for compounds which inhibit activation of the receptor polypeptide of the present invention by determining ;nhth;tion of htn~tng of labeled ligand to cells which have the receptor on the surface thereof. Such a method involves transfecting a eukaryotic cell with DNA ~ncoAtng the receptor such that the cell expresses the receptor on its surface and contacting the cell with a potential antagonist in the presence of a labeled form of a known ligand. The ligand can be labeled, e.g., by radioactivity. The a~ount of labeled ligand bound to the receptor~ is measured, e.g., by measuring radioactivity of the receptors. If the potential antagonist binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited.
In general, antagonists for G-protein coupled receptors which are determined by screening procedures may be employed for a variety of therapeutic purposes. For example, such antagonists have been employed for treatment of hypertension, angina pectoris, myocardial infarction, ulcers, asthma, allergies, psychoses, depression, migraine, vomiting, stroke, CA 0222l6l6 lgg7-ll-l9 W096/39441 PCT~S95/07225 eating disorders, migraine headaches, cancer and benign prostatic hypertrophy.
Agonists for G-protein coupled receptors are also useful for therapeutic purposes, such as the treatment of asthma, Parkinson's disease, acute heart failure, hypotension, urinary retention, and osteoporosis.
Examples o~ G-protein coupled receptor antagonists include an antibody, or in some cases an oligonucleotide, which binds to the G-protein coupled receptor but does not elicit a second messenger response such that the activity of the G-protein coupled receptor is prevented. Antibodies include anti-idiotypic antibodies which recognize unique determinants generally associated with the antigen-binding site of an antibody. Potential antagonists also include proteins which are closely related to the ligand of the G-protein coupled receptor, i.e. a fr~gmPnt of the ligand, which have lost biological function and when hinAing to the G-protein coupled receptor, elicit no response.
A potential antagonist also includes an antisense construct prepared through the use of antisense technology.
Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on hinA;ng of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complPm~nt~y to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988);
and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of G-protein coupled receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vi~o and blocks translation of the WO 9~/39441 PCT/US95/07225 mRNA molecule into the G-protein coupled receptor (antisense - O~ano, ~. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Tnh; hl tors of Gene ~xpression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells ~uch that the antisense RNA or DNA
may be expressed in vivo to inhibit production of G-protein coupled receptor.
Another potential antagonist is a small molecule which binds ~o the G-protein coupled receptor, making it inaccessible to ligands such that normal biological activity is prevented. Bxamples o~ small molecules include but are not limited to small peptides or peptide-like molecules.
Potential antagonists also include a soluble form of a G-protein coupled receptor, e.g. a fragment of the receptor, which binds to the ligand and prevents the ligand from interacting with ...e..~Lane bound G-protein coupled receptor~.
The G-protein coupled receptor and antagonists or agonists may be employed in comh;n~tion with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but i8 not limited to ~l;nP, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of ~; n; ~tration.
The invention also provides a pharmaceutical pack or kit comprising one or more cont~;ners filled with one or more of the ingredient~ of the pharmaceutical composition~ of the in~ention. Associated with such contAiner(s) carl be a notice in the form prescribed by a govern~~nt~l agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency o~
m~lufacture, use or sale for human ~m; n;stration. In addition, the pharmaceutical compositions may be employed in conjunction with other therapeutic compound~.

WO96139441 PCT~S95/072Z5 The pharmaceutical compositions may be ~mi nl stered in a convenient m~nn~r such as by the topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are A~m; n~ stered in an amount which is ef~ective for treating and/or prophylaxis of the specific indication. In general, the pharmaceutical compositions will be ~m; n; stered in an amount of at least about lO ~g/kg body weight and in most cases they will be ~mjn; stered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage is from about lO ~g/kg to about l mg/kg body weight daily, taking into account the routes of ~m; n; stration, symptoms, etc.
The G-protein coupled receptor polypeptides and antagonists or agonists which are polypeptides, may be employed in accordance with the present invention by expression of such polypeptides iR vivo, which is often referred to as "gene therapy."
Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle cont~;n;ng RNA encoding a polypeptide of the present invention.
S;m; 1 ~rly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle cont~; n; ng RNA
encoding the polypeptide of the present invention may be ~m; n; stered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods ~or ~m; n; stering a polypeptide of the present invention by such method should be apparent to those skilled CA 02221616 1997-ll-l9 WO 96/39441 PCT/US9~/07225 in ~he art ~rom the teachings o~ the present invention. For exal~ple, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo a~ter combination with a suitable delivery vehicle.
~ Retroviruses ~rom which the retroviral plasmid vectors herein~hove mentioned may be derived include, but are not limited to, Moloney Murine ~eukemia Virus, spleen necro~i~
virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human ;mmnno~eficiene~ viru8, adenovirus, Myeloproli~erative Sarcoma Virus, and m~ ry tumor virus.
In one embodiment, the retroviral plasmid vector is derived ~rom Moloney Murine Leul~emia Virus.
The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Bictechnic~ues, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the hiEtone, pol III, and ~-actin promoters). Other viral promoter~ which may be employed include, but are not limited to, ade~ovirus promoters, thymidine kinase (TK) promoters, ancl B19 parvovirus promoters. The selection o~ a suitable prc~moter will be apparent to those skilled in the art from the teachings ront~;nP~ herein.
The nucleic acid secluence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but: are no~ limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologou~ promoters, such as the cytomegalo~irus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; ~n~r~hle promoters, such as the ~MT promoter, the metallothionein promoter; heat ~hock CA 0222l6l6 lgg7-ll-l9 096/39441 PCT~S9S/07225 promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such a~
the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LT~s her~in~hove described); the ~-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the gene encoding the polypeptide.
The retroviral plasmid vector i8 employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ~-2, ~-AM, PA12, T19-14X, VT-19-17-H2, ~CRE, ~CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Thera~y, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP04 precipitation. In one alternative, the retroviral plasmid vector may be ~ncArsulated into a liposome, or coupled to a lipid, and then ~mi n~ stered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will expres~ the nucleic acid sequence(s) encoding the polypeptide. ~ukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carr~ n9,m~ cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronrh~ Al epithelial cells.
G-protein coupled receptors are ubiquitous in the m~mm~lian host and are responsible for many biological functions, including many pathologies. Accordingly, it is W096/39441 PCT~S95/07225 desirous to find compounds which stimulate a G-protein coupled receptor and compounds which antagonize a G-protein coupled receptor.
This invention further provides a method of identifying compounds which specifically interact with, and bind to, the human G-protein coupled receptors on the surface of a cell which comprises contacting a m~mm~l ian cell comprising an isolated DNA molecule ~nCo~;ng the G-protein coupled receptor with a plurality of compounds, determining those which bind to the m~mm~ lian cell, and thereby identifying compounds which specifically interact with and bind to a human G-protein coupled receptor of the present invention.
This invention also provides a method of detecting expression o~ the G-protein coupled receptor on the sur~ace of a cell by detecting ~he presence of mRNA coding for a G-protein coupled receptor which comprises obt~;n-ng total mRNA
from the cell and contacting the mRNA so obtA~ne~ with a nucleic acid probe compri8ing a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a F,equence included within the sequence of a nucleic acid molecule ~nro~;ng a human G-protein coupled receptor under hybridizing conditions, de~ecting the presence of mRNA
hybridized to the probe, and thereby detecting the expression of the G-protein coupled receptor by the cell.
This invention is also related to the use of the G-protein coupled receptor gene as part of a diagnostic assay for detecting diseases or su~ceptibility to diseases related to the presence of mutated G-protein coupled receptor genes.
Such diseases are related to cell trans~ormation, such as tumors and cancers.
Indiv~ ~n~l s carrying mutations in the human G-protein co~1pled receptor gene may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obt~n~ from a patient' 8 cells, such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA

CA 0222l6l6 Isg7-ll-ls WO96/39441 PCT~S95/07225 may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding the G-protein coupled receptor protein can be used to identify and analyze G-protein coupled receptor mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled G-protein coupled receptor RNA or alternatively, radiolabeled G-protein coupled receptor antisense DNA sequences.
Perfectly matched sequences can be distinguished from mismatched duplexes by RWase A digestion or by differences in melting temperatures.
Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresi~. DNA
fra~n~s of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al ., Science, 230:1242 (1985)).
Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and Sl protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA.

WO 96t39441 PCT/US95/0i225 In addition to more conventional gel-electrophoresis and DNA se~uencing, mutations can al80 be detected by in si tu analysis.
~ The present invention also relates to a diagnostic assay for detecting altered levels of ~oluble ~orms of the receptor po]ypep~ides of the present invention in various tissues.
Assays used to detect levels of the soluhle receptor polypeptides in a sample derived from a host are well known to those of skill in the art and include radioimmnnoassays, co~petitive-h; n~ ng assays, Western blot analysis and preferably as ELISA assay.
An ELISA assay initially comprises preparing an antibody specific to antigens of the G-protein coupled receptor polypeptides, pre~erably a mono~'onal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioa.ctivity, fluorescence or in this example a horseradish peroxidase enzyme. A sample is now removed from a host and incuhated on a solid support, e.g. a pol~ el.e dish, that binds the proteins in the sample. Any free protein b1 n~; n~ sites on the dish are then covered by ;ncllh~ting with a non-speci~ic protein such as bovine serum m~ n . Next, the monoclonal ~nt; ho~y iS incubated in the di,sh during which time the monoclonal antibodies attach to any G-protein coupled receptor proteins attached to the polystyrene dish. All nnhound monoclonal antibody is washed out with buffer. The reporter ~nt;ho~y linked to horseradish peroxidase is now placed in the dish resulting in h;nd;ng of the reporter antibody to a~y monoclonal antibody bound to G-protein receptor proteins. Unatt~rhe~ reporter ~nt; ho~y is then washed out. Peroxidase su-hstrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of G-protein coupled receptor proteins present in a gi~en volume of patient sample when c~..,~ared against a st~n~rd curve.

CA 02221616 1997-ll-l9 WO96/39441 pcT~s9s/o722s The present invention also provides a method for determining whether a ligand not known to be capable o~
binding to a receptor can bind to such receptor which comprises contacting a m~mm~l ian cell which expresses a recepto~ with the ligand under conditions permitting binding of ligands to the receptor, detecting the presence of a ligand which binds to the receptor and thereby determining whether the ligand binds to the receptor. The systems herein~hove described for deter~i ni ng agonists and/or antagonists may also be employed for deter~i n; ng ligands which bind to the receptor.
This invention also provides a method of detecting expression of a receptor polypeptide of the present invention on the surface of a cell by detecting the presence of mRNA
coding for the receptor which comprises obt~ining total mRNA
from the cell and contacting the mRNA ~o obt~i n~ with a nucleic acid probe comprising a nucleic acid molecule of at least 10 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding the receptor under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the receptor by the cell.
The present invention also provides a method for identifying receptors related to the receptor polypeptides of the present invention. The~e related receptors may be identified by homology to an receptor polypeptide of the present invention, by low stringency cross hybridization, or by identifying receptor~ that interact with related natural or synthetic ligands and or elicit similar behaviors after genetic or pharmacological blockade of the receptor polypeptides of the present invention.
The sequences of the present invention are al~o valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current W096/39441 PCT~S95/07225 need ~or identi~ying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking ~ chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with difiease .
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
Co~puter analysis of the cDNA is used to rapidly select primers that do not sp~n more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used ~or PCR screening of somatic cell hybrids cont~intng individual human chromosomes. Only those hybrids cont~in~ng the human gene corresponding to the primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
U~ing the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous ~nne~. Other mapping strategies that can ~imilarly be used ~o map to its chromosome include in sit:u hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA
clo~e to a met~ph~e chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases.
For a review of this technique, see Verma et al., Human Chromosomes: a ~nll~ 1 of Basic Techniques, PeL~dlllO~l Press, New York (1988).
Once a sequence ha~ been mapped to a preci~e chromosomal location, the physical position of the sequence on the 096/39441 PCT~S95/07225 chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expres~ing them can be used as an ;mmllnogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies.
The present invention also includes chim~ric~ single chain, and h~ n;zed antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such ant; ho~; es and fragments.
~ nt; ho~; es generated against the polypeptides COrre8pQn~; ng to a sequence of the present invention can be obtained by direct injection of the polypeptides into an ~n;m~l or by ~ n~ ~tering the polypeptides to an ~n;m~l, preferably a nonhllm~n The antibody 80 obtained will then bind the polypeptides itself. In this m~nn~r, even a sequence encoding only a fragment of the polypeptides can be WO 96~39441 PCT/US95/07225 used to generate antibodies ~nAtn~ the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. ~xamples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique ~Kozbor et al., 1983, T~llnology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal ~nt~hodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain ant:ibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to im~l~nogenic polypeptide products o~ this invention. Also, transgenic mice may be used to express hl~mAnized antibodies to ;m~llnogenic polypeptide products of this invention.
The present invention will be further described with reiEerence to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.
In order to facilitate underst~n~; n~ of the following examples certain frequently occurring methods and/or terms will be described.
"Plasmids" are designated by a lower case p preceded and/or $ollowed by capital letters and/or nllmh~rs~ The starting plasmids herein are either co~~rcially available, publicly aV~ hl e on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addi~ion, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

-WO96/39441 PCT~S95/07225 "Digestion" of DNA re~ers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, co~actors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically l ~g of plasmid or DNA
fragment is used with about 2 units of enzyme in about 20 ~l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 ~g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about l hour at 37 C are ordinarily used, but may vary in accordance with the supplier's instructions. A~ter digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D.
et al., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" re~ers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without AAA~ng a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buf~ers and conditions with lO units to T4 DNA ligase WO 96/39441 PCT/US9~,J~ 25 ("ligase") per 0.5 ~g of approximately equimolar amounts o~
the DNA fra~nents to be ligated.
Unless otherwise stated, trans~ormation was perfonmed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

Exam~le 1 Bacterial Ex~ression and Purification of G-protein Coupled Rece~tor The DNA sequence encoding the receptor, ATCC # _ is initially amplified using PCR oligonucleotide primers corresponding to the S~ and sequences of the processed receptor pro~ein (minus the ~ignal peptide sequence) and the vec:tor sequences 3' to ~he receptor gene. Additional nucleotides correspon~i n~ to receptor were added to the ~' and 3~ sequences respectively. The 5~ oligonucleotide primer ha~ the sequence 5'-CGGAA~ ~ATGAACTCCACCTTGGAT contains a Eco RI restriction enzyme site followed by 18 nucleotides of receptor coding sequence starting from the presumed te~inal amino acid of the processed protein codon. The 3' sequence 5'-CGGAAGGTTCGTCAGATATGACATCCATT contains co~plementary sequences to HindIII site and is followed by 18 nucleotides of receptor. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQ~-9. ~Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311). pQE-9 encodes antibiotic resistance (~npr), a bacterial origin of replication (ori), an IPT&-regulatable ~-~...oLer operator (P/0), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQ~-9 was then digested with Eco RI and HindIII. The amplified sequences were ligated into pQE-9 and were inserted in frame with the ~equence encoding for the histidine tag and the RB'3. The ligation mixture was then used to transform E. coli strain available from Qiagen under the trA~mArk M15/rep 4 by the procedure de cribed in Sambrook, J. et al., Molecular CA 0222l6l6 lgg7-ll-l9 096/39441 PCT~S95/07225 Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pRBP4, which expresses the la~I repressor and also confers kanamycin resistance (Kanr). Trans~ormants are identi~ied by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis.
Clones cont~ nt ng the desired constructs were grown overnight (0/N) in li~uid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The 0/N
culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical density 600 (O.D.~) of between 0.4 and 0.6. IPTG (nIsopropyl-B-D-thiogalacto pyranoside") was then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/0 leA~;ng to increased gene expression. Cells were grown an extra 3 to 4 hours. Cells were then harvested by centrifugation. The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HCl.
After clarification, solubilized receptor was purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight h; n~; ng by proteins ~ontA;ntng the 6-His tag. Hochuli, ~. et al., J.
Chromatography 411:177-184 (1984). The receptor protein was eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in this solution for 12 hours the protein was dialyzed to 10 mmolar sodium phosphate.

Exam~le 2 Bx~ression of Recomhinant Receptor in COS cells The expression of plasmid, G-protein coupled receptor HA
is derived from a vector pcDNAI/Amp (Invitrogen) contAtntng WO96t39441 PCT~S95/07225 1) Sv40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site.
~ A DNA fragment encoding the entire receptor precursor and a HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hem~gglutinin protein as previously described (I. Wilson, H.
Nim~n, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 198g, Cell 37, 767). The infusion of HA tag to our target protein allows easy detection of the recomhin~nt protein with an antibody that recognizes the ~A epitope.
The plasmid construction strategy is described as follows:
The DNA se~uence encoding for the receptor, ATCC #
was constructed by PCR on the original EST cloned using two p r i m e r s : t h e 5 ' p r i m e r ( 5 ' -GTCCAAGCTTGCCACCATGAACTCCA~ w AT-3~) - (SEQ ID NO:5) contains a HindIII site followed by 18 nucleotides of receptor coding sequence starting from the initiation codoni t h e 3 ' 8 e q u e n c e 5~-CTAGCTCGAGTCAAGCGTA~l~-l~A~L~lAl~ w lAGCAGATATGACATCCA
TTA~AG-3' (SEQ ID NO:6) cont~n~ complPmPnt~ry sequences to Xho I site, translatio~ stop codon, HA tag and the last 18 nucleotides of the receptor coding sequence (not including the stop codon). Therefore, the PCR product cont~nc a HindIII site, receptor co~ng sequence followed by HA tag ~u~ed in ~rame, a translation termination stop codon next to the HA tag, and an Xho I site. The PCR amplified DNA
fragment and the ~ector, pcDNAI/Amp, were digested with HindIII and Xho I restriction enzyme and ligated. The ligation mixture wa~ tran~formed into E. coli strain SURE
(a~ailable from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the trans~ormed WO96/39441 PCT~S95/07225 culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformants and e~mined by restriction analysis for the presence of the correct fragment. For expression of the recombinant receptor, COS cells were transfected with the expression vector by D~AE-D~XTRAN method. (J. Sambrook, E.
Fritsch, T. Maniatis, Molecular Cloning: A Laboratory M~n~
Cold Spring Laboratory Press, (1989)). The expression of the receptor HA protein was detected by radiolabelling and ;~m-lnQprecipitation method. (~. Harlow, D. Lane, Antibodies:
Laboratory ~nll~l, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with 35S-cysteine two days post transfection. Culture media were then collected and cells were ly~ed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM
Tris, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media were precipitated with a HA
specific monoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAG~ gels.

E~am~le 3 Cloninq and expression of G-protein Coupled receptor usinq the baculovirus ex~ression svstem The DNA sequence ~nCo~ing the full length receptor protein, ATCC # , was amplified using PCR
oligonucleotide primers corresponding to the 5~ and 3' se~l~nceC of the gene:
~ he 5' primer has the sequence 5'-CG&GAl~C~ ~ATGAACTCCAC~ AT (SEQ ID NO:7) and cont~in~ a Bam HI restriction enzyme site ~in bold) followed by 4 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (J. Mol. Biol. 1987, 196, 947-950, Xozak, M.), and just behind the first 18 nucleotides of the receptor gene (the initiation codon for translation "ATG" is underlined).

-The 3~ primer has the sequence 5'-CGG~TCCCGCTCAGATATGAGATCCATT-3' (SEQ ID NO:8) and contains the cleavage site for the restriction en~o~llclease Bam HI and 18 nucleotides complementary to the 3I non-translated se~lence o~ the receptor gene. The ampli~ied sequences were isolated from a 1~ agarose gel using a commercially available kit ("Geneclean," BI0 101 Inc., La Jolla, Ca.). The fragment was then dige~ed with the ~n~o~-lcleases Bam HI and then purified as described in Example 1. This fragment is deslgnated F2.
The vector pRG1 (modi~ication o~ pVL941 vector, discussed below) is used for the expression of the receptor pro~ein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A m~nll~l o~ method~
for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555).
This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the resl_riction Pn~sn~lcleases Bam HI. The polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinant viruses the beta-galactosidase gene ~rom ~.coli is inserted in ~he same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The pol~lledrin seguences are ~lanked at both sides by viral seq~ences for the cell-media~ed homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).
The plasmid was digested with the restriction enzyme Bam HI and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The DNA was then CA 0222l6l6 Isg7-ll-ls 096/39441 pcT~s9s/o7225 isolated from a 1~ agarose gel as described in ~xample 1.
This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. E.coli B 101 cells were then transformed and bacteria identified that contA; n~ the plasmid (pBac receptor) with the receptor gene using the enzymes Bam HI. The sequence of the cloned fragment was confirmed by DNA sequencing.
5 ~g of the plasmid pBac receptor were co-transfected with 1.0 ~g of a c~m~ercially available linearized baculovirus ("BaculoGold~ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al.
Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
l~g of BaculoGold~ virus DNA and 5 ~g of the plasmid pBac receptor were mixed in a sterile well of a microtiter plate contA;n;ng 50 ~l of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Afterwards 10 ~l Lipofectin plus 90 ~l Grace~s medium were added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture was added drop wise to the Sf9 insect cells (ATCC CRL 1711) ~eeded in a 35 mm tissue culture plate with 1 ml Grace' medium without serum. The plate was rocked back and ~orth to mix the newly added solution. The plate was then incubated for 5 hours at 27~C. After 5 hours the transfection solution was removed from the plate and 1 ml of Grace' 8 insect medium supplemented with 10% fetal calf serum was added. The plate was put back into an incubator and cultivation continued at 27~C for four days.
A~ter ~our days the supernatant was collected and a plaque assay performed s;m;lA~ as described by Summers and Smith (supra). As a modification an agarose gel with nBlue Gal" (Life Technologies Inc., Gaithersburg) was used which allows an easy isolation o~ blue s~A; n~ plaques. (A
detailed description of a "plaque assay" can al80 be found in the user's guide for insect cell culture and baculovirology -distributed by Life Technologies Inc., Gaithersburg, page 9-10) .
Four days after the serial dilution of the viruses was ~ added to the cells, blue stained plaques were picked with the tip of an Eppendorf pipette. The agar cont~ ni ng the recombinant viruses was then resuspended in an Eppendorf tube cont~;n~ng 200 ~1 o~ Grace~s medium. The agar was removed by a brie~ centri~ugation and the supernatant cQnta;n~ng the recombinant baculoviru~es was used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants o~ these culture dishes were har~ested and then stored at 4~C.
Sf9 cells were grown in Gracels medium supplemented with 10~ heat-inacti~ated FBS. The cells were infected with the recombinant baculovirus V-receptor at a multiplicity of infection (MOI) of 2. Six hours later the medium was removed and replaced with SF900 II medium minus methionine and cysteine (Li~e Technologies Inc., Gaither~burg). 42 hours later 5 ~Ci of 3~S-methionine and 5 ~Ci 35S cysteine (Amersham) were added. The cells were further ~nc~hAted for 16 hours be~ore they were har~ested by centri~ugation and the labelled proteins ~isualized by SDS-PAGE and autoradiography.

~xam~le 4 ~xDression pattern o~ G-~rotein Cou~led rece~tor in human tissue Northern blot analysis was carried out to ~Y~m;ne the levels of expression of receptor in human tissues. Total cellular RNA samples were isolated with RNAzol~ B system (Biotecx Laboratories, Inc. 6023 South Loop ~ast, Houston, TX
77033). About 10~g o~ total RNA isolated from each human tissue speci~ied was separated on 1% agarose gel and blotted onto a nylon ~ilter. (Sa..~ ook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Pre~s, (1989)). The labeling reaction was done according to the Stratagene Prime-It kit with 50ng DNA ~ragment. The labeled DNA was purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303). The filter was then hybridized with radioactive labeled $ull length receptor gene at 1,000,000 cpm/ml in 0.5 M NaPO4, pH 7.4 and 7% SDS
overnight at 65 C. After wash twice at room temperature and twice at 60 C with 0.5 x SSC, 0.1% SDS, the filter was then expo~ed at -70 C overnight with an intensifying screen. The message RNA for the receptor is abundant in skeletal muscle.

Example 5 ~xDression via Gene Therapy Fibroblasts are obt~nP~ from a subject by skin biopsy.
The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added.
Thi~ is then incubated at 37~C for approximately one week.
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger fla~ks.
pMV-7 (Rirschmeier, P.T. et al, DNA, 7:219-25 (1988) flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The 1 ;n~r vector is fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified u~ing PCR primer~ which correspond to the 5' and 3' end sequences respectively. The 5~ primer contains an EcoRI site and the 3' primer further includes a HindIII site.
Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII ~ragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is used to transform bacteria HB101, which are then plated onto agar-cont~;n;ng kanamycin for the purpose of confirming that the vector had the gene of interest properly in~erted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector cont~ining the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce in~ectious viral particles cont~lnin~ the gene (the packaging ce].ls are now referred to as producer cells).
Fresh media is added to the transduced pro~lc~r cells, ancl ~ubsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, cont~in;ng the infectious viral particles, is filtered through a millipore fi]ter to remove detached producer cells and this media i8 then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his.
The engineered fibroblasts are then injected into the ho;t, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product.

WO 96/39441 PCT/US95/0i225 Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

WO96/39441 PCT~S95/07Z25 SEQUENCE LISTING

(1) GENERAL INFORMATION:
~i) APPLICANT: Li, Y.

~ (ii) TITLE OF INVENTION: Human G-protein Coupled Receptor (iii) NUMBER OF SEQUENCES:

(iv) CO~R~-SPONDENCE ADDRESS:

(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OLSTEIN
(B) STREET: 6 BEC~ R FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) ~UUN-l-KY: USA
(F) ZIP: 0706~

(v) COM~ul~ READABLE FORM:
(A) M~DIUM TYPE: 3.5 INCH DIS ~ TTE
(B) COM~U-1~K: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS
(D' SOFTWARE: WORD PERFBCT 5.1 (vi) ~U~RNT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: Su~mitted Herewith (C) CLASSIFICATIO~:

(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBBR: None (B) FILING DATB: None WO96/39441 PCT~S95/07225 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: FERRARO, GREGORY D.
(B) REGISTRATION NUMBER: 36,134 (C) REFERENCE/DOCXET NUMBER: 325800-365 (viii) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700 (B) TELEFAX: 201-994-1744 (2) lN~-OKMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 2764 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STR~h~N~SS: SINGLE
(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

AATTACAGGT AACATTCTGA AATTGAACTA AACAGTAAAT ~ L~AAA ~ lCAAA 60 GTGGTAATTG TAGTGAAAGG ~-~-L-lC~-lAAA TATTATAAGC AAAl-~--l-l-l l~-lCCCCC~l 180 CTCAAATGAA AGGAAATGGG GGTAAATTAA TCTGACTGTG Al-~l-l-l-l~ TTTTAATGCTG 240 ATCTTGAAAG CTTGATGTTG CTGCTGCTCC Tr~T~r~r~TA CAGATCAGTT ~l~l~GG~lG 300 GCCTGAGCTA GCCAGGTTCT TTGATTAGGG CATTGGATGT GAAATGTAAA ATG~-l~-l~-lC 420 ~-1-~-~-1~-1-1~-1 ATCAGCTGTT CAaAGaAaAC TCATTACAAC TCCTGCTGAA GCTCCTAATC 480 ~l-~.--lCC~-~ ~-lACC ~-l~~-lCCCC~-l ACCCTCACTT GGC~-l~AAGA ~l-l~-lCCCC 540 AGAGTTTACC TTG~-lCCC~-l GGTGCTATGT GTATGGTGAA CCTGGCACTA TGGCCGCGTC 600 TGGGACTGGC r~r~ACTG CTGCTGGCTC TCCTTATTCC A~&AAGGATT TAAPr~G~A 660 TTGCACTGCA GGCAATGCAC CAGAGCAGCA GCATCAGGAG ~-l-l~GGGAGT AAGGCTCCTC 720 TGGCATTATT ACACACATGC AAAGCTGACC GCAATGACAG CAG--LG~-l~C TTTGAACTGT 780 Met Asn CA 0222l6l6 l997-ll-l9 W O 96/39441 PCT~US95/07225 Ser Thr Leu Asp Gly Aqn Gln Ser Ser His Pro Phe Cys Leu Leu Ala Phe Gly Tyr Leu Glu Thr Val Asn Phe Cys Leu Leu Glu Val ~eu Ile Ile Val Phe Leu Thr Val Leu Ile Ile Ser Gly Asn Ile Ile Val Ile Phe Val Phe His Cys Ala Pro ~eu Leu Asn His Hiq Thr Thr Ser Tyr Phe Ile Gln Thr Met Ala Tyr Ala ~sp Leu Phe Val Gly Val Ser Cys Val Val Pro Ser Leu Ser Leu Leu His His Pro Leu Pro Val Glu Glu Ser Leu The Cys Gln Ile Phe Gly Phe Val Val Ser Val Leu Lys Ser Val ser Met Ala Ser Leu Ala Cys Ile Ser Ile Asp Arg Tyr Ile Ala Ile Thr Lys Pro Leu Thr Tyr Asn Thr Leu Val Thr Pro Trp Arg Leu Arg Leu Cys Ile Phe Leu Ile Trp ~eu Tyr Ser Thr Leu Val Phe Leu Pro Ser Phe Phe His Trp Gly ~ys Pro Gly Tyr His Gly Asp Val Phe Gln Trp Cys Ala Glu Ser Trp Hi8 Thr Asp Ser Tyr Phe Thr Leu Phe Ile Val Met Met Leu Tyr Ala Pro Ala Ala Leu Ile Val Cys Phe Thr TAT TTC AAC ATC TTC CGC ATC TGC CAA CAG CAC ACA AAG GAT ATC ~GC 1509 Tyr Phe Asn Ile Phe Arg Ile Cy8 Gln Gln His Thr Lys Asp Ile Ser Glu Arg Gln Ala Arg Phe Ser Ser Gln Ser Gly Glu Thr Gly Glu Val CA 0222l6l6 l997-ll-l9 W O 96/39441 PCTrUS95/07225 CAG GCC TGT CCT GAT AAG CGC TAT GCC ATG GTC CTG TTT CGA ATC ACT 1605Gln Ala Cys Pro Asp Lys Arg Tyr Ala Met Val Leu Phe Arg Thr Thr AGT GTA TTT TAC ATC CTC TGG TTG CCA TAT ATC ATC TAC TTC TTG TTG 1653Ser Val Phe Tyr Ile Leu Trp Leu Pro Tyr Ile Ile Tyr Phe Leu Leu GAA AGC TCC ACT GGC CAC AGC AAC CGC TTC GCA TCC TTC TTG ACC ACC 1701Glu Ser Ser Thr Gly His Ser Asn Arg Phe Ala Ser Phe Leu Thr Thr TGG CTT GCT ATT AGT AAC AGT TTC TGC AAC TGT GTA ATT TAT AGT CTC 1749Trp Leu Ala Ile Ser Asn Ser Phe Cys Asn Cys Val Ile Tyr Ser Leu TCC AAC AGT GTA TTC CAA AGA GGA CTA AAG CGC CTC TCA GGG GCT ATG 1797Ser Asn Ser Val Phe Gln Arg Gly Leu Lys Arg Leu Ser Gly Ala Met TGT ACT TCT TGT GCA AGT CAG ACT ACA GCC AAC GAC CCT TAC ACA GTT 1845Cy5 Thr Ser Cy~ Ala Ser Gln Thr Thr Ala Asn Asp Pro Tyr Thr Val AGA AGC AAA GGC CCT CTT AAT GGA TGT CAT ATC TGAAGTGGCT 1888Arg Ser LYB Gly Pro Leu Asn Gly Cys His Ile CAGTTACGGG ~l-lCCC~L~ ~L~l~L~lG~l ~l'~'~'~'l~'l' ~l~L~L~L~l ATTTTATCTC 1948 TACAAATTAT TCGTATGGAT AC~-l-l~-lAAG TTTGTAGAAA ~ -LL-lCCC AAGTGCTTGT 2068 TTGGAAATGA CTACAGTTCT CAGATTTAAA ATGAATAAAG CCATATCTAA CAC~-l~-l-l-lC 2188 CAGcTGGcAT GACTGAACCT GAGTGTGAAA AGCGTCAGCA TTTTAAAAAG TCATCACTTT 2248 ~-l-l~L~ACTT TCTGGGCTCT TTCCAGCTAT TTGGGCGTCA TATGCAATTG AL-l-l~-iu--lA 2308 ACGGAATAGT AAAATATAAA TGAAAAGGTT TTAGAAATTA ~-lL-l-~-lATGT ATGCCAAAGC 2368 ATAACTACAC TGCAAGTTTC AACACT~TCA TTTAGAAAGC CAAATGTTCT ~l~L-l-l-lATT 2428 ~-l'~L-~AGAG AATTCTCAGT AGGGTGAATA ATGTGAACAC ATAAACATTA ATTTTAGAAT 2488 TA~-lL-ll-L~l GCCATGCTTC ACAGAGATCT AAAGATATGT ~L~LAGAA GTAATCGTGT 2608 AGTACTTTTG CCCATGCCTT i~L~i-lATGT CTATATTTAG AATATCTGAA TTGTTAGATT 2668 l~L~LlLLAC AGCAAAATGT GCTTAAGCTA AAAAGTAATT CAGGGAATTC GATATCAAGC 2728 TTATCGATAC CGTCGACCTC GAr~GGGGr~GC CCGGTA 2764 CA 02221616 1997-ll-l9 WO96/39441 PCT~S95/07225 (2) INFORMATION FOR SEQ ID NO:2:
(i) S~QUENCE CHARACT~RISTICS
(A) LENGTH: 349 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR

(i.i) MOLECULE TYPE: PROTEIN

(xi) SE~u~ DESCRIPTION: SEQ ID NO:2:

Met Asn Ser Thr Leu Asp Gly Asn Gln Ser Ser Hi8 Pro Phe Cys Leu Leu Ala Phe Gly Tyr Leu Glu Thr Val Asn Phe Cys Leu Leu Glu Val Leu Ile Ile Val Phe Leu Thr Val Leu Ile Ile Ser Gly Asn Ile Ile Val Ile Phe Val Phe His Cys Ala Pro Leu Leu Asn Hil3 His Thr Thr Ser Tyr Phe Ile Gln Thr Met Ala Iyr Ala Asp Leu Phe Val Gly Val Ser C~ys Val Val Pro Ser Leu Ser Leu Leu HiS HiS Pro Leu Pro Val Glu Glu Ser Leu The Cys Gln Ile Phe G~y Phe Val Val Ser Val Leu Lys Ser Val Ser Met Ala Ser Leu Ala Cys Ile Ser Ile Asp Arg Tyr Ile Ala Ile Thr Ly~ Pro Leu Thr Tyr Asn Thr Leu Val Thr Pro Trp Arg Leu Arg Leu Cy5 Ile Phe Leu Ile Trp Leu Tyr Ser Thr Leu Val Phe Leu Pro Ser Phe Phe Hi3 Trp Gly Lys Pro Gly Tyr HiS Gly Asp Val Phe Gln Trp Cys Ala Glu Ser Trp His Thr Asp Ser Tyr Phe Thr Leu Phe Ile Val Met Met Leu Tyr Ala Pro Ala Ala Leu Ile Val Cys Phe Thr Tyr Phe Asn Ile Phe Arg Ile Cy9 Gln Gln His Thr Lys Asp Ile Ser Glu Arg Gln Ala Arg Phe Ser Ser Gln Ser Gly Glu Thr Gly CA 0222l6l6 l997-ll-l9 Glu Val Gln Ala Cys Pro Asp Lys Arg Tyr Ala Met Val Leu Phe Arg Thr Thr Ser Val Phe Tyr Ile Leu Trp Leu Pro Tyr I' e Ile Tyr Phe Leu Leu Glu Ser Ser Thr Gly His Ser Asn Arg Phe Ala Ser Phe Leu Thr Thr Trp Leu Ala Ile Ser Asn Ser Phe Cys Asn Cys Val Ile Tyr Ser Leu Ser Asn Ser Val Phe Gln Arg Gly Leu Lys Arg Leu Ser Gly Ala Met Cy5 Thr Ser Cys Ala Ser Gln Thr Thr Ala Asn Asp Pro Tyr Thr Val Arg Ser Lys Gly Pro Leu Asn Gly Cys His Ile (2) INFORMATION FOR SEQ ID NO:3:

(i) SE~u~N~ CHARACTERISTICS
(A) LENGTH: 26 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQu~N~ DESCRIPTION: SEQ ID NO:3:

GCTAAGGATC ~-L~CTC AAGGTT 26 (2) lN~Okl ~TION FOR SEQ ID NO:4:

(i) SEQu~ CHARACTERISTICS
(A) LENGTH: 26 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR

CA 0222l6l6 l997-ll-l9 (ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GCI'A~TCTAG ATC~CATCTT GGGGAA 2 6 .

Claims (20)

WHAT IS CLAIMED IS:
.

1. An isolated polynucleotide comprising a member selected from the group consisting of:
(a) a polynucleotide encoding the polypeptide as set forth in SEQ ID NO:2;
(b) a polynucleotide encoding a mature polypeptide encoded by the DNA containing in ATCC Deposit No.________;
(c) a polynucleotide capable of hybridizing to and. which is at least 70% identical to the polynucleotide of (a) or (b); and (d) a polynucleotide fragment of the polynucleotide of (a), (b) or (c).

2. The polynucleotide of Claim 1 encoding the polypeptide as set forth in SEQ ID NO:2.

3. A vector containing the polynucleotide of Claim 1.

4. A host cell transformed or transfected with the vector of Claim 3.

5. A process for producing a polypeptide comprising:
expressing from the host cell of Claim 4 the polypeptide encoded by said polynucleotide.

6. A process for producing cells capable of expressing a polypeptide comprising transforming or transfecting the cells with the vector of Claim 3.

7. A receptor polypeptide selected from the group consisting of:

(i) a polypeptide having the deduced amino acid sequence of SEQ ID NO:2 and fragments, analogs and derivatives thereof; and (ii) a polypeptide encoded by the cDNA of ATCC
Deposit No.____ and fragments, analogs and derivatives of said polypeptide.

8. The polypeptide of Claim 7 wherein the polypeptide has the deduced amino acid sequence of SEQ ID
NO:2.

9. An antibody against the polypeptide of claim 7.

10. A compound which activates the polypeptide of claim 7.

11. A compound which inhibits activation the polypeptide of claim 7.

12. A method for the treatment of a patient having need to activate the polypeptide of Claim 7 comprising:
administering to the patient a therapeutically effective amount of the compound of claim 10.

13. A method for the treatment of a patient having need to inhibit the polypeptide of Claim 7 comprising:
administering to the patient a therapeutically effective amount of the compound of claim 11.

14. The method of claim 12 wherein said compound is a polypeptide and a therapeutically effective amount of the compound is administered by providing to the patient DNA
encoding said agonist and expressing said agonist in vivo.

15. The method of claim 13 wherein said compound is a polypeptide and a therapeutically effective amount of the compound is administered by providing to the patient DNA
encoding said antagonist and expressing said antagonist in vivo.

16. A method for identifying compounds which bind to and activate the receptor polypeptide of claim 7 comprising:
contacting a cell expressing on the surface thereof the receptor polypeptide, said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor polypeptide, with a compound under conditions sufficient to permit binding of the compound to the receptor polypeptide; and identifying if the compound is capable of receptor binding by detecting the signal produced by said second component.

17. A method for identifying compounds which bind to and inhibit activation of the polypeptide of claim 7 comprising:
contacting a cell expressing on the surface thereof the receptor polypeptide, said receptor being associated with a second component capable of providing a detectable signal in response to the binding of a compound to said receptor polypeptide, with a ligand known to bind to the receptor polypeptide and a compound to be screened under conditions to permit binding to the receptor polypeptide; and determining whether the compound inhibits activation of the polypeptide by detecting the absence of a signal generated from the interaction of the ligand with the polypeptide.

18. A method of screening compounds to identify those compounds which bind to the receptor polypeptide of claim 7 comprising:
contacting a cell expressing the receptor on the surface thereof with a compound and an analytically detectable ligand known to bind to the receptor, under conditions permitting binding to the receptor; and determining binding of the ligand to the receptor.

19. A process for diagnosing in a patient a disease or a susceptibility to a disease related to an under-expression of the polypeptide of claim 7 comprising:
determining a mutation in the nucleic acid sequence encoding said polypeptide in a sample derived from a patient.

20. A diagnostic process comprising:
analyzing for the presence of the polypeptide of claim 7 in a sample derived from a host.