This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a human 7- transmembrane receptor which has the greatest amino acid sequence homology to the human anaphylatoxin C5a receptor. The invention also relates to inhibiting 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 second messengers, e.g., cAMP (Lef owitz, Nature, 351:353-354 (1991)). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins. Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., PNAS, 84:46-50 (1987); Kobilka, B.K. , et al., Science, 238:650-656 (1987); Bunzow, J.R., et al., Nature, 336:783-787 (1988)), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein

kinase A and protein kinase C (Simon, M.I., et al. , Science, 252:802-8 (1991)) .

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 binding. A G-protein connects the hormone receptors to adenylate cyclase. G- protein was shown to exchange 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 membrane protein gene superfamily of G-protein coupled receptors have been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane α-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 neuro-receptors.

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. Examples of G-protein family of coupled receptors includes dopamine receptors, calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins and rhodopεins, odorant, cytomegalovirus receptors.

Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to

stabilize functional protein structure. The 7 transmembrane regions are designated as TMl, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 is implicated in signal transduction.

Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors. Most G- protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several G-protein coupled receptors, such as the 3-adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.

The ligand binding sites of G-protein coupled receptors are believed to comprise a hydrophilic socket formed by several G-protein coupled receptors transmembrane domains, which socket is surrounded by hydrophobic residues of the G- protein coupled receptors. The hydrophilic side of each G- protein coupled receptor transmembrane helix is postulated to face inward and form the polar ligand binding site. TM3 has been implicated in several G-protein coupled receptors as having a ligand binding site, such as including the TM3 aspartate residue. Additionally, TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated in ligand binding.

G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al . , Endoc, Rev., 10:317-331 (1989)). Different G-protein α- subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protein coupled receptors have been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors.

The anaphylatoxin C5a is a 74-amino acid polypeptide generated by cleavage of the alpha-chain of native C5 at a specific site by convertase of the blood complement system, as well as by enzymes of the coagulation system. In vivo, C5a is thought to play a significant role in the inflammatory response and in a number of clinical disorders (Goldstein, I.M., Inflammation: Basic Principles and Clinical Correlates, 309-323, Raven Press, New York (1988)). This peptide is a highly potent inflammatory agent, evoking dramatic responses in experimental animals (Bodammer, G. and Vogt, . , Int. Arch. Allergy Appl. Immunol., 33:417-428 (1967)), and stimulating pulmonary, cardiac, vascular and gastrointestinal tissues in vi tro (Stimler, N.P., et al., Am. J. Pathol., 100:327-348 (1980)). C5a is a potent activator of polymorphonuclear neutrophils and macrophages, stimulating chemotaxis, hydrolytic enzyme release, and superoxide anion formation (Ward, P.A. and Newman, L.J., J. Immunol., 102:93- 99 (1969) ) .

Several reports have additionally demonstrated actions of this peptide on eosinophils, including chemotaxis and increased hexose uptake, in addition to its actions on mast cells and basophilε (Hugli, T.E., Biological Response Mediators and Modulators, 99-116, Academic Press, New York (1983) ) . In addition, the anaphylatoxin has been shown to have a spasmogenic effect on various tissues; it stimulates smooth muscle contraction (Stimler, N.P., et al., J. Immunol., 126:2258-2261 (1981)); induces histamine release from mast cells, promotes serotonin release from platelets (Meuer, S., et al., J. Immunol., 126:1506-1509 (1981)), and increases vascular permeability (Jose, P.J., et al., J. Immunol., 127:2376-2380 (1981)).

The interaction of C5a with polymorphonuclear leukocytes and other target cells and tissues results in increased histamine release, vascular permeability, smooth muscle contraction, and an influx into tissues of

inflammatory cells, including neutrophils, eosinophils and basophilε (Hugli, T.E., Springer, Semin. Immunopathol. , 7:193-219 (1981)). C5a may also play an important role in mediating inflammatory effects of phagocytic mononuclear cells that accumulate at sites of chronic inflammation (Allison, A.C., et al., H.U. Agents and Actions, 8:27 (1978) ) . C5a can induce chemotaxis in monocytes and cause them to release lysoεomal enzymes in a manner analogous to the neutrophil responses elicited by these agents. C5a may have an immunoregulatory role by enhancing antibody, particularly as sites of inflammation (Morgan, E.L., et al., J. Exp. Med., 155:1412 (1982)).

A human C5a receptor cDNA clone has been isolated by expression cloning from a CDM8 expression library prepared from mRNA of Human myeloid HL-60 cells differentiated to the granulocyte phenotype with dibutyryladenosine cyclic monophosphate (Boulay, F. et al., Biochemistry, 30:2993-2999 (1991) ) . Also, the human C5a receptor was cloned from U937 and HL-60 cells and identified by high affinity binding when expressed in COS-7 cells, (Gerard, N.P. and Gerard, C. , Nature, 349:614-617 (1991)).

In accordance with one aspect of the present invention, there are provided novel G-protein coupled receptor polypeptides, as well as an isense analogs thereof and biologically active and diagnostically or therapeutically useful fragments and derivatives thereof. The 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 human G-protein coupled receptor, 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 is provided a process for producing such

polypeptides by recombinant techniques which comprises culturing recombinant prokaryotic and/or eukaryotic host cells, containing the human G-protein coupled receptor nucleic acid sequence, under conditions promoting expression of said protein and subsequent recovery of said protein.

In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides.

In accordance with another embodiment, there is provided a process for using the G-protein coupled receptors to screen for receptor antagonists and/or agonistε and/or receptor ligandε.

In accordance with still another embodiment of the present invention there is provided a process of using such agonists for stimulating the G-protein coupled receptor for the treatment of conditions related to the under-expression of the G-protein coupled receptors, for example, as a defense against bacterial infection, as a defense against viral infection, to stimulate the immunoregulatory effects of C5a, to treat immunodeficiency diseases and severe infections.

In accordance with another aspect of the present invention there is provided a process of using such antagonists for inhibiting the action of the G-protein coupled receptors for treating conditions associated with over-expression of the G-protein coupled receptors, for example, to treat asthma, bronchial allergy, chronic inflammation, εyεtemic lupuε erythematosiε, vasculitiε, rheumatoid arthritiε, osteoarthritis, gout, certain auto- allergic diseases, transplant rejection, ulcerative colitis, in certain shock states, myocardial infarction, hypertension, abnormal cell growth and post-viral encephalopathies.

In accordance with another aspect of the present invention there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to human G-protein coupled receptor sequences.

In accordance with still another aspect of the present invention there are provided synthetic or recombinant G- protein coupled receptor polypeptides, conservative substitution and derivatives thereof, antibodieε, anti- idiotype antibodieε, compoεitionε and methodε that can be uεeful as potential modulators of G-protein coupled receptor function, by binding to ligands or modulating ligand binding, due to their expected biological properties, which may be used in diagnostic, therapeutic and/or research applications.

In accordance with yet another object of the present invention, there is provided a diagnoεtic assay for detecting a diseaεe or εuεceptibility to a disease related to a mutation in the G-protein coupled receptor nucleic acid sequence.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

The following drawings are illustrative of embodimentε 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 putative mature G-protein coupled receptor of the present invention. The standard one- letter abbreviation for amino acids is used. Sequencing was performed using a 373 Automated DNA sequencer (Applied Bioεyεtemε, Inc.) . Sequencing accuracy iε predicted to be greater than 97% accurate.

Figure 2 illustrates an amino acid alignment of the G- protein coupled receptor of the present invention (top line) and a human C5a receptor (bottom line) .

In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodeε for the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID No. 2) or for the

mature polypeptide encoded by the cDNA of the clone depoεited as ATCC Deposit No. 75982 on December 16, 1994.

A polynucleotide encoding a polypeptide of the present invention is predominantly expressed in peripheral lymphocytes. The polynucleotide of this invention was discovered in a cDNA library derived from a human activated neutrophil. It is structurally related to the G protein- coupled receptor family. It contains an open reading frame encoding a protein of 482 amino acid residueε. The protein exhibits the highest degree of homology to a human C5a receptor with 26 % identity and 58 % similarity over the entire amino acid sequence.

While the G-protein coupled receptor has the highest degree of amino acid sequence homology to a human C5a receptor, there is alεo a significant degree of amino acid sequence homology to the human receptors for other ligands, for example N-formyl peptide, angiotensin, somatostatin, opioid, interleukin-8 (IL-8) , bradykinin, thrombin and ATP receptors. Accordingly, while Applicant does not wish to limit the scientific theory underlying the present invention, the G-protein coupled receptor of the present invention may bind any one or a combination of the ligands identified above.

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 coding sequence which coding sequence, as a result of the redundancy 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 for 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 for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence εuch aε 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 εequence 5' and/or 3' of the coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well aε a polynucleotide which includeε additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID No. 2) or the polypeptide encoded by the cDNA of 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 (SEQ ID No. 2) or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotideε which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID No. 2) or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide 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 (SEQ ID No. 1) or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotideε, which does not substantially alter the function of the encoded polypeptide.

The present invention also includeε polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expreεεion and εecretion of a polypeptide from a hoεt cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues. A mature protein having a prosequence is a proprotein and iε an inactive form of the protein. Once the proεequence iε cleaved an active mature protein remainε.

Thus, for example, the polynucleotide of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence) .

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa- histidine tag supplied by a pQE-9 vector to provide for 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 mammalian host, e.g. COS-7 cells, is used. The HA tag correspondε to

an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)) .

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there iε at leaεt 50% and preferably 70% identity between the sequences. The present invention particularly relateε to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term "stringent conditions" meanε hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figure 1 (SEQ ID No. 1) or the deposited cDNA, i.e. function as a G-protein coupled receptor or retain the ability to bind the ligand for the receptor even though the polypeptide does not function as a G-protein coupled receptor, for example, a εoluble form of the receptor.

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 purposeε of Patent Procedure. Theεe depoεitε are provided merely as convenience to those of skill in the art and are not an admisεion that a depoεit iε required under 35 U.S.C. §112. The sequence of the polynucleotideε contained in the deposited materials, as well aε 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 iε hereby granted.

The present invention further relates to a G-protein coupled 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," "derivative" and "analog" when referring to the polypeptide of Figure 1 (SEQ ID No. 2) or that encoded 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 G-protein coupled receptor, or retains the ability to bind the ligand or the receptor even though the polypeptide does not function as a G-protein coupled receptor, for example, a soluble form of the receptor. An analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID No. 2) or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are subεtituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such subεtituted 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 subεtituent group, or (iii) one in which the mature polypeptide is fuεed with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. 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 polynucleotideε of the preεent invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the material iε 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 animal is not isolated, but the same polynucleotide or polypeptide, separated from εome or all of the coexiεting materialε in the natural εyεtem, 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 environment.

The present invention alεo relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Hoεt cellε are genetically engineered (tranεduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expreεεion vector. The vector may be, for example, in the form of a plaεmid, a viral particle, a phage, etc. The engineered hoεt cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the G- protein coupled receptor geneε. The culture conditions, such aε temperature, pH and the like, are thoεe previouεly uεed with the host cell selected for expression, and will be apparent to the ordinarily skilled 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, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids,- phage DNA; baculoviruε; yeast plaεmidε; vectorε derived from combinationε of plaεmidε 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 endonuclease 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 εequence in the expreεsion vector is operatively linked to an appropriate expression control sequence(ε) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P _L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector alεo containε a riboεome binding εite for tranεlation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expresεion vectorε preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such aε tetracycline or ampicillin reεistance 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 transform an appropriate hoεt to permit the host to express the protein.

As representative examples of appropriate hostε, there may be mentioned: bacterial cellε, εuch aε E. coli. Streptomvces, Salmonella tvphimurium; fungal cells, εuch as yeast; insect cells εuch aε Drosophila S2 and Spodootera Sf9; animal cellε εuch as CHO, COS or Bowes melanoma; adenoviruεeε; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequenceε aε broadly described above. The constructε compriεe a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inεerted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further compriseε regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen) , pbε, pDlO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia) . Eukaryotic: pW NEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) . However, any other plaεmid or vector may be used aε long aε they are replicable and viable in the hoεt.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P _R/ P _L and trp. Eukaryotic promoterε include CMV immediate early, HSV

thymidine kinaεe, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to hoεt cellε containing the above-deεcribed conεtructs. The host cell can be a higher eukaryotic cell, such as a mammalian 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, DEAE- 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 manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizerε.

Mature proteinε can be expreεsed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free tranεlation εystems can also be employed to produce such proteins uεing RNAε derived from the DNA constructs of the present invention. Appropriate cloning and expression vectorε for use with prokaryotic and eukaryotic hoεts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryoteε iε increaεed by inεerting an enhancer εequence into the vector. Enhancerε are cis-acting elements of DNA, uεually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the

replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancerε.

Generally, recombinant expreεsion vectors will include origins of replication and selectable markers permitting tranεformation of the host cell, e.g., the ampicillin resiεtance gene of E. coli and S. cereviεiae TRPl gene, and a promoter derived from a highly-expresεed gene to direct tranεcription of a downstream structural sequence. Such promoters can be derived from operonε encoding glycolytic enzymeε εuch aε 3-phosphoglycerate kinase (PGK) , α-factor, acid phosphatase, or heat shock proteinε, among otherε. The heterologouε εtructural sequence iε aεεembled in appropriate phaεe with tranεlation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic εpace or extracellular medium. Optionally, the heterologouε sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristicε, e.g., εtabilization or εimplified purification of expressed recombinant product.

Useful expreεsion vectors for bacterial use are constructed by inεerting a εtructural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic εelectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the hoεt. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various species within the genera Pseudomonaε, Streptomyceε, and Staphylococcuε, 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 commercially 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 GEMl (Promega Biotec, Madison, WI, USA) . These pBR322 "backbone" sections are combined 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 cell density, the selected promoter iε induced by appropriate meanε (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cellε are typically harvested by centrifugation, disrupted by phyεical or chemical meanε, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical diεruption, or uεe of cell lysing agents, such methods are well know to those skilled in the art.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression syεtemε include the COS-7 lineε of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lineε. Mammalian expreεεion vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necesεary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed εequences. DNA sequences derived from the

SV40 splice, and polyadenylation siteε may be uεed to provide the required nontranεcribed genetic elementε.

The G-protein coupled receptor polypeptideε can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phoεphocelluloεe chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) . Depending upon the host employed in a recombinant production procedure, the polypeptides of the preεent invention may be glycoεylated or may be non-glycoεylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

Fragments of the full length G-protein coupled receptor gene may be employed aε a hybridization probe for a cDNA library to iεolate the full length gene and to iεolate other geneε which have a high sequence similarity to the gene or similar biological activity. Probes of this type generally have at least 20 bases. Preferably, however, the probes have at least 30 bases and generally do not exceed 50 bases, although they may have a greater number of baseε. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete G-protein coupled receptor gene including regulatory and promotor regions, exons, and

intronε. As an example of a screen comprises isolating the coding region of the G-protein coupled receptor gene by using the known DNA sequence to syntheεize an oligonucleotide probe. Labeled oligonucleotideε having a εequence complementary to that of the gene of the preεent invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The G-protein coupled receptor of the present invention may be employed in a process for screening for antagonistε and/or agoniεts for the receptor.

In general, such screening procedureε involve providing appropriate cellε which express the receptor on the surface thereof. In particular, a polynucleotide encoding the receptor of the present invention is employed to transfect cellε to thereby expreεε the G-protein coupled receptor. Such transfection may be accompliεhed by procedures as hereinabove described.

One such screening procedure involves the use of the melanophores which are tranεfected to expresε the G-protein coupled receptor of the preεent invention. Such a screening technique iε 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 melanophore cells which encode the G-protein coupled 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 εcreen may be employed for determining an agonist by contacting such cellε with compounds to be εcreened and determining whether such compound generates a εignal, i.e., activateε the receptor.

Other screening techniques include the use of cells which express the G-protein coupled receptor (for example, transfected CHO cellε) in a εystem which measureε extracellular pH changes caused by receptor activation, for example, as described in Science, volume 246, pages 181-296 (October 1989) . For example, potential agonistε or antagonists may be contacted with a cell which expresses the G-protein coupled receptor and a second messenger response, e.g. signal transduction or pH changes, may be measured to determine whether the potential agoniεt or antagoniεt iε effective.

Another εuch εcreening technique involveε introducing RNA encoding the G-protein coupled receptor into xenopuε oocyteε to tranεiently expreεs the receptor. The receptor oocytes may then be contacted in the case of antagonist εcreening with the receptor ligand and a compound to be screened, followed by detection of inhibition of a calcium εignal.

Another εcreening technique involveε expreεεing the G- protein coupled 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 hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipaεe εecond signal.

Another method involveε screening for antagonists by determining inhibition of binding of labeled ligand to cells which have the receptor on the surface thereof. Such a method involves transfecting a eukaryotic cell with DNA encoding the G-protein coupled receptor such that the cell expresεeε the receptor on itε 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 amount of labeled ligand bound to the receptors 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.

G-protein coupled receptors are ubiquitous in the mammalian host and are responsible for many normal and pathological biological functions. Accordingly, it is desirous to find compounds and drugs which stimulate the G- protein coupled receptors on the one hand and which can antagonize a G-protein coupled receptor on the other hand when it is desirable to inhibit the G-protein coupled receptor.

For example, agonists for G-protein coupled receptors may be employed for therapeutic purposes, such as the treatment of asthma, Parkinson'ε disease, acute heart failure, hypotension, urinary retention, and osteoporosis.

In general, antagonists to the G-protein coupled receptors may be employed for a variety of therapeutic purposeε, for example, for the treatment of hypertenεion, angina pectoriε, myocardial infarction, ulcerε, asthma, allergieε, benign proεtatic hypertrophy and psychotic and neurological disorderε, including εchizophrenia, manic excitement, depression, delirium, dementia or severe mental retardation, dyskineεiaε, εuch as Hun ing on's disease or Gilles dila Tourett'ε εyndrome, among otherε. G-protein coupled receptor antagoniεts have also been useful in reversing endogenous anorexia and in the control of bulimia.

Exampleε of G-protein coupled receptor antagonists include antibodies, or in some cases oligonucleotides, which bind to the G-protein coupled receptorε but do not elicit a second mesεenger response such that the activity of the G- protein coupled receptors iε prevented. Antibodieε include anti-idiotypic antibodieε which recognize unique determinantε

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 receptors, i.e. a fragment of the ligand, which have lost biological function and when binding to the G-protein coupled receptors, elicit no response.

A potential antagonist also includes an antisenεe conεtruct prepared through the uεe of antiεense technology. Antisense technology can be used to control gene expresεion through triple-helix formation or antisenεe DNA or RNA, both of which methodε are baεed on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide εequence, which encodeε 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 complementary 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 receptors. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of mRNA molecules into G-protein coupled receptors (Antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotideε as Antisense Inhibitorε of Gene Expreεεion, CRC Preεε, Boca Raton, FL (1988) ) . The oligonucleotideε described above can alεo be delivered to cells εuch that the antisense RNA or DNA may be expresεed in vivo to inhibit production of G-protein coupled receptors.

Another potential antagonist is a small molecule which binds to the G-protein coupled receptor, making it inaccesεible to ligandε εuch that normal biological activity is prevented. Examples of small molecules include but are not limited to small peptideε or peptide-like molecules.

Potential antagonists also include a soluble form of a G-protein coupled receptor, e.g. a fragment of the receptors, which binds to the ligand and prevents the ligand from interacting with membrane bound G-protein coupled receptors.

Antagonists to the G-protein coupled receptor may also include a method of re-engineering the receptor such that the internal three-dimensional structure is maintained but the external structure is made hydrophilic.

The antagonistε may be uεed generally aε mediators of inflammatory responses, as immunoregulants and to treat all pathological conditions which result from anaphylaxiε stimulated by the C5a polypeptide and mediated by the G- protein coupled receptor. These pathological conditions include asthma, bronchial allergy, chronic inflammation, systemic lupus erythematosuε, vaεculitiε, εerum sicknesε, angioedema, rheumatoid arthritiε, osteoarthritis, gout, bullous εkin diεeaεeε, hypersensivity, pneumonitis, idiopathic pulmonary fibroεiε, immune complex-mediated glomerulonephritiε, psoriasis, allergic rhinitis, hypertension, adult respiratory distreεs syndrome, acute pulmonary disorders, endotoxin shock, hepatic cirrhosis, pancreatitiε, inflammatory bowel diεeases (including Crohn'ε disease and ulcerative colitis) , thermal injury, gram- negative sepsiε, necroεis in myocardial infarction, leukophoreεis, exposure to medical devices (including, but not limited to, hemodialyzer membranes and extracorpeal blood circulation equipment) , chronic hepatitis, transplant rejection, abnormal cell growth, for example tumorε and cancerε, poεt-viral encephalopathieε, and/or iεchemia induced myocardial or brain injury. Theεe antagoniεt may also be used aε prophylactics for such conditions aε shock accompanying Dengue Hemorrhagic fever.

The agonistε identified by the εcreening method as described above, may be employed to stimulate the G-protein coupled receptor to treat conditions related to an under-

expreεεion of the receptor, which include defense against bacterial infection, stimulation of the immunoregulatory effects of C5a, immunodeficiency diseaεeε, viral and other infectionε.

Thiε invention additionally provideε a method of treating abnormal conditionε related to an excess of G- protein coupled receptor activity which comprises administering to a subject the antagonist as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to block binding of ligands to the G- protein coupled receptors and thereby alleviate the abnormal conditionε.

The invention also provides a method of treating abnormal conditions related to an under-expresεion of G- protein coupled receptor activity which comprises administering to a subject a therapeutically effective amount of the agoniεt deεcribed above in combination with a pharmaceutically acceptable carrier, in an amount effective to enhance binding of ligandε to the G-protein coupled receptor and thereby alleviate the abnormal conditions.

The soluble form of the G-protein coupled receptors, antagonistε and agonists may be employed in combination with a suitable pharmaceutical carrier. Such compoεitionε comprise a therapeutically effective amount of the antagonist or agonist, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextroεe, water, glycerol, ethanol, and combinationε thereof. The formulation εhould εuit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Aεεociated with such container(s) can be a notice in the form preεcribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological

products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the pharmaceutical compositions may be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, the pharmaceutical compositions will be administered in an amount of at leaεt about 10 μg/kg body weight and in moεt cases they will be administered in an amount not in excesε of about 8 mg/Kg body weight per day. In most cases, the dosage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.

The G-protein coupled receptor polypeptides and antagonistε or agonists which are polypeptides, may be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to aε "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, cellε may be engineered by procedureε known in the art by uεe of a retroviral particle containing RNA encoding a polypeptide of the present invention.

Similarly, 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 containing RNA encoding the polypeptide of the present invention may be

administered to a patient for engineering cells in vivo and expresεion of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by εuch method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retroviruε, for example, an adenoviruε which may be uεed to engineer cells in vivo after combination with a suitable delivery vehicle.

The invention also provides a method for determining whether a ligand not known to be capable of binding to the G- protein coupled receptor can bind to such receptor which comprises contacting a mammalian cell which expresses a G- protein coupled receptor with the ligand under conditions permitting binding of ligands to the G-protein coupled receptor, detecting the preεence of a ligand which bindε to the receptor and thereby determining whether the ligand binds to the G-protein coupled receptor. The syεtems hereinabove described for determining agonists and/or antagonists may alεo be employed for determining ligands which bind to the receptor.

This invention further provides a method of screening drugs to identify drugs which specifically interact with, and bind to, the human G-protein coupled receptors on the surface of a cell which compriseε contacting a mammalian cell comprising an isolated DNA molecule encoding the G-protein coupled receptor with a plurality of drugs, determining those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with and bind to a human G-protein coupled receptor of the present invention.

This invention also provides a method of detecting expression of the G-protein coupled receptor on the surface of a cell by detecting the presence of mRNA coding for a G- protein coupled receptor which comprises obtaining total mRNA

from the cell and contacting the mRNA so obtained with a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human G-protein coupled receptor under hybridizing conditionε, detecting 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 alεo related to the use of the G- protein coupled receptor genes as part of a diagnostic assay for detecting diseaεeε or susceptibility to diseases related to the presence of mutations in the G-protein coupled receptor genes.

Individuals carrying mutations in the human G-protein coupled receptor genes may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cellε, such as from blood, urine, saliva, tisεue biopsy and autopsy material. The genomic DNA 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 proteins can be used to identify and analyze G-protein coupled receptor mutations. For example, deletions and insertionε can be detected by a change in εize of the amplified product in compariεon 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 RNaεe 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 fragmentε in gelε with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguiεhed on denaturing formamide gradient gelε in which the mobilitieε of different DNA fragmentε are retarded in the gel at different poεitions 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 SI protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)).

Thuε, the detection of a εpecific DNA εequence may be achieved by methodε 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.

In addition to more conventional gel-electrophoresiε and DNA sequencing, mutations can alεo be detected by in si tu analysis.

The sequences of the present invention are also valuable for chromosome identification. The sequence is εpecifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphismε) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the preεent invention is an important first step in correlating those sequenceε with geneε associated with diseaεe.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysiε of the 3' untranεlated region iε used to

rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing 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. Using the present invention with the same oligonucleotide primers, εublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategieε that can similarly be used to map to its chromosome include in si tu hybridization, preεcreening with labeled flow-εorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 bases,- however, cloneε larger than 2,000 bp have a higher likelihood of binding to a unique chromoεomal location with sufficient signal intensity for simple detection. FISH requires use of the clones from which the express sequence tag was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of this technique, see Verma et al., Human Chromosomeε: a Manual of Baεic Techniqueε, Pergamon Preεε, New York (1988) .

Once a εequence has been mapped to a precise chromosomal location, the physical position of the sequence on the 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 asεumes 1 megabase mapping reεolution and one gene per 20 kb) .

The polypeptideε, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, εingle chain, and humanized antibodieε, aε well as Fab fragments, or the product of an Fab expression library. Various procedureε known in the art may be used for the production of such antibodies and fragmentε.

Antibodies generated against the polypeptideε corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptideε into an animal or by adminiεtering the polypeptideε to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptideε can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodieε, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples 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, Immunology Today 4:72) , and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Lisε, Inc., pp. 77-96) .

Techniqueε deεcribed for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice may be uεed to expreεs humanized antibodies to immunogenic polypeptide products of this invention.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention iε not limited to εuch examples. All parts or amounts, unless otherwise specified, are by weight.

In order to facilitate understanding 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 followed by capital letters and/or numbers. The εtarting plaεmidε herein are either commercially available, publicly available on an unreεtricted basis, or can be constructed from available plasmids in accord with publiεhed procedures. In addition, equivalent plasmidε to those described are known in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various reεtriction enzymes used herein are commercially available and their reaction

conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment iε uεed with about 2 unitε of enzyme in about 20 μl of buffer εolution. For the purpoεe of isolating DNA fragments for plasmid construction, typically 5 to 50 μg 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 1 hour at 37'C are ordinarily used, but may vary in accordance with the supplier' ε instructionε . After digeεtion the reaction iε electrophoreεed directly on a polyacrylamide gel to iεolate the deεired 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" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically syntheεized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding 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" referε to the proceεs of forming phosphodieεter bonds between two double stranded nucleic acid fragments (Maniatis, T. , et al., Id., p. 146) . Unlesε otherwiεe provided, ligation may be accompliεhed uεing known buffers and conditions with 10 unitε to T4 DNA ligaεe ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

Unlesε otherwiεe εtated, tranεformation waε performed aε described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973) .

Example 1 Bacterial Expression and Purification of G-protein coupled Receptor

The DNA εequence encoding the G-protein coupled receptor, ATCC # 75982, iε initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end sequenceε of the proceεsed G-protein coupled receptor nucleic acid sequence (minus the signal peptide sequence) and the vector sequenceε 3' to the gene. Additional nucleotides corresponding to the G-protein coupled receptor nucleotide sequence are added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence 5' GACTAAAGCπ ATGGCGTCrTTCTCTGCTGAG 3' (SEQ ID NO. 3) contains a HindiII restriction enzyme site followed by 18 nucleotides of G-protein coupled receptor coding sequence starting from the presumed terminal amino acid of the procesεed protein codon. The 3' εequence 5' GAACI CTAGAC TCACACΑGTTGTACTATTT 3' (SEQ ID No. 4) containε complementary εequenceε to an Xbal εite and iε followed by 21 nucleotideε of the gene. The restriction enzyme sites correspond to the restriction enzyme siteε on the bacterial expreεεion vector pQE-9 (Qiagen, Inc. Chatεworth, CA) . pQE-9 encodeε antibiotic resistance (AmpJ , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/0) , a ribosome binding site (RBS) , a 6- Hiε tag and reεtriction enzyme εiteε. pQE-9 iε then digested with Hindlll and Xbal. The amplified sequences are ligated into pQE-9 and are inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture is then used to transform E. coli strain available from Qiagen under the trademark Ml5/rep 4 by the procedure described in Sa brook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989) . M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lad repressor and alεo confers kanamycin resiεtance (Kan ^r ) . Tranεformantε are identified by their

ability to grow on LB plateε and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis. Clones containing the desired constructs are grown overnight (0/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) . The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D. ⁶⁰⁰ ) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") iε then added to a final concentration of l mM. IPTG induces by inactivating the lad repressor, clearing the P/0 leading to increased gene expression. Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HC1. After clarification, solubilized G-protein coupled receptor is purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984) ) . The G-protein coupled receptor is eluted from the column in 6 molar guanidine HC1 pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HC1, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this εolution for 12 hourε the protein iε dialyzed to 10 mmolar εodium phosphate.

Example 2 Expression of Recombinant G-protein coupled Receptor in COS cells

The expression of plasmid, pG-protein coupled receptor HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: l) 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 pG- protein coupled receptor protein and a HA tag fused in frame to its 3 ' end is 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 hemagglutinin protein as previouεly deεcribed (I. Wilεon, H. Niman, R. Heighten, A Cherenεon, M. Connolly, and R. Lerner, 1984, Cell 37, 767) . The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding for the G-protein coupled receptor, ATCC # 75982, iε constructed by PCR on the full- length gene cloned using two primers: the 5' primer 5' GTCCAAGCTTGCCΑCCΑTGGGTCrTTCTCTGCT 3' (SEQ ID No. 5) containε a Hindlll site followed by 18 nucleotides of G-protein coupled receptor coding sequence starting from the initiation codon; the 3' sequence 5' CTAGCTCGAGTCAAGCGTAGTCTGGGACGTCGT ATGGGTAGCACACAGTTGTACTATT 3' (SEQ ID No. 6) containε complementary εequenceε to an Xhol εite, tranεlation stop codon, HA tag and the last 15 nucleotides of the G-protein coupled receptor coding sequence (not including the stop codon) . Therefore, the PCR product contains a Hindlll site, G-protein coupled receptor coding sequence followed by HA tag fused in frame, a translation termination εtop codon next to the HA tag, and an Xhol εite. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with Hindlll and Xhol restriction enzymes and ligated. The ligation mixture iε transformed into E. coli strain SURE (Stratagene Cloning Systems, La Jolla, CA) the transformed culture is plated on ampicillin media plates and resiεtant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct

fragment. For expreεεion of the recombinant G-protein coupled receptor, COS cellε are tranεfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)) . The expresεion of the G-protein coupled receptor HA protein is detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodieε: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hours with ³⁵ S-cysteine two dayε poεt tranεfection. Culture media are then collected and cellε are lyεed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Triε, pH 7.5). (Wilεon, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media are precipitated with a HA specific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGE gels.

Example 3 Cloning and expression of G-protein coupled receptor using the baculovirus expression system

The DNA sequence encoding the full length G-protein coupled receptor protein, ATCC # 75982, is amplified using PCR oligonucleotide primers correεponding to the 5' and 3' εequences of the gene:

The 5' primer has the sequence 5' CGGGATCCCTCCATG GCX-xTCI TCTCTGCT 3' (SEQ ID No. 7) and contains a BamHI 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, Kozak, M.), which is just behind the first 18 nucleotides of the gene (the initiation codon for tranεlation "ATG" iε underlined) .

The 3' primer has the sequence 5' CGGGATCCCGCTCACACAGTTGTACTATT 3' (SEQ ID No. 8) and contains the cleavage εite for the restriction endonuclease BamHI and

18 nucleotides complementary to the 3' non-translated εequence of the G-protein coupled receptor gene. The amplified εequenceε are isolated from a 1% agarose gel using a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.). The fragment is then digested with the endonuclease BamHI and then isolated again on a 1% agarose gel. This fragment is designated F2.

The vector pRGl (modification of pVL94l vector, diεcuεsed below) is used for the expresεion of the G-protein coupled receptor protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods 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 restriction endonuclease BamHI. The polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an easy selection of recombinant viruseε the beta-galactosidase gene from E.coli iε inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co-tranεfected wild-type viral DNA. Many other baculoviruε vectors could be used in place of pRGl such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).

The plasmid is digested with the restriction enzymes BamHI and then dephosphorylated using calf intestinal phosphataεe by procedures known in the art. The DNA is then isolated from a 1% agarose gel as described above. This vector DNA iε designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase. E.coli HB101 cells are then

transformed and bacteria identified that contained the plasmid (pBacG-protein coupled receptor) with the G-protein coupled receptor gene using the enzyme BamHI . The sequence of the cloned fragment is confirmed by DNA sequencing.

5 μg of the plasmid pBacG-protein coupled receptor is co-transfected with 1.0 μg of a commercially available linearized baculovirus ("BaculoGold™ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)) . lμg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacG-protein coupled receptor are mixed in a εterile well of a microtiter plate containing 50 μl of εerum free Grace's medium (Life Technologies Inc., Gaithersburg, MD) . Afterwardε 10 μl Lipofectin pluε 90 μl Grace'ε medium are added, mixed and incubated for 15 minuteε at room temperature. Then the transfection mixture iε added dropwise to the Sf9 insect cells (ATCC CRL 1711) εeeded in a 35 mm tiεsue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27°C. After 5 hours the transfection εolution iε removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation continued at 27°C for four days.

After four days the supernatant is collected and a plaque assay performed similar as described by Summers and Smith (supra) . As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used which allows an easy iεolation of blue stained plaques. (A detailed description of a "plaque assay" can also be found in the user' ε guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9- 10) .

Four days after the serial dilution, the viruseε are added to the cellε and blue εtained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruseε is then resuεpended in an Eppendorf tube containing 200 μl of Grace'ε medium. The agar iε removed by a brief centrifugation and the εupernatant containing the recombinant baculoviruεes is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then stored at 4°C.

Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-G-protein coupled receptor at a multiplicity of infection (MOD of 2. Six hours later the medium iε removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc. , Gaithersburg) . 42 hours later 5 μCi of ³⁵ S-methionine and 5 μCi ³⁵ S cysteine (Amersham) are added. The cells are further incubated for 16 hourε before they are harveεted by centrifugation and the labelled proteinε viεualized by SDS- PAGE and autoradiography.

Example 4 Expression pattern of G-protein coupled receptor in human tissue

Northern blot analysiε iε carried out to examine the levelε of expreεεion of G-protein coupled receptor in human tiεεueε. Total cellular RNA samples are isolated with RNAzol™ B system (Biotecx Laboratories, Inc. Houston, TX) . About lOμg of total RNA isolated from each human tiεεue εpecified is separated on 1% agarose gel and blotted onto a nylon filter (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Press, (1989)). The labeling reaction is done according to the Stratagene Prime-It kit with 50ng DNA fragment. The labeled DNA is purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. Boulder, CO) .

The filter is then hybridized with radioactive labeled full length G-protein coupled receptor gene at 1,000,000 cpm/ml in 0.5 M NaP0 ₄ , pH 7.4 and 7% SDS overnight at 65'C. After being washed twice at room temperature and twice at 60"C with 0.5 x SSC, 0.1% SDS, the filter iε then expoεed at -70"C overnight with an intensifying screen. The mesεage RNA for G-protein coupled receptor iε abundant in peripheral lymphocyteε.

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.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: LI, ET AL.

(ii) TITLE OF INVENTION: Human G-protein coupled

Receptor

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,

CECCHI, STEWART & OLSTEIN

(B) STREET: 6 BECKER FARM ROAD

(C) CITY: ROSELAND

(D) STATE: NEW JERSEY

(E) COUNTRY: USA

(F) ZIP: 07068

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 INCH DISKETTE

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WORD PERFECT 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: Concurrently

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: FERRARO, GREGORY D.

(B) REGISTRATION NUMBER: 36,134

(C) REFERENCE/DOCKET NUMBER: 325800-271

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 201-994-1700

(B) TELEFAX: 201-994-1744

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 2040 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: CDNA

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

CACGAGGAGA ACAGAAGAAG AGAAAGCTCA GCAAATTTTC TTGCCATACT TCATGACTTC 60

ACTGTGGCTA AGTGTGGGGA CCAGACAGGA CTCGTGGAGA CATCCAGGTG CTGAAGCCTT 120 CAGCTACTGT CTCAGTTTTT TGAAGTTTAG CAATGGCGTC TTTCTCTGCT GAGACCAATT 180

CAACTGACCT ACTCTCACAG CCATGGAATG AGCCCCCAGT AATTCTCTCC ATGGTCATTC 240

TCAGCCTTAC TTTTTTACTG GGATTGCCAG GCAATGGGCT GGTGCTGTGG GTGGCTGGCC 300

TGAAGATGCA GCGGACAGTG AACACAATTT GGTTCCTCCA CCTCACCTTG GCGGACCTCC 360

TCTGCTGCCT CTCCTTGCCC TTCTCGCTGG CTCACTTGGC TCTCCAGGGA CAGTGGCCCT 420

ACGGCAGGTT CCTATGCAAG CTCATCCCCT CCATCATTGT CCTCAACATG TTTGCCAGTG 480

TCTTCCTGCT TACTGCCATT AGCCTGGATC GCTGTCTTGT GGTATTCAAG CCAATCTGGT 540

GTCAGAATCA TCGCAATGTA GGGATGGCCT GCTCTATCTG TGGATGTATC TGGGTGGTGG 600

CTTGTGTGAT GTGCATTCCT GTGTTCGTGT ACCGGGAAAT CTTCACTACA GACAACCATA 660

ATAGATGTGG CTACAAATTT GGTCTCTCCA GCTCATTAGA TTATCCAGAC TTTTATGGAG 720

ATCCACTAGA AAACAGGTCT CTTGAAAACA TTGTTCAGCC GCCTGGAGAA ATGAATGATA 780

GGTTAGATCC TTCCTCTTTC CAAACAAATG ATCATCCTTG GACAGTCCCC ACTGTCTTCC 840

AACCTCAAAC ATTTCAAAGA CCTTCTGCAG ATTCACTCCC TAGGGGTTCT GCTAGGTTAA 900

CAAGTCAAAA TCTGTATTCT AATGTATTTA AACCTGCTGA TGTGGTCTCA CCTAAAATCC 960

CCAGTGGGTT TCCTATTGAA GATCACGAAA CCAGCCCACT GGATAACTCT GATGCTTTTC 1020

TCTCTACTCA TTTAAAGCTG TTCCCTAGCG CTTCTAGCAA TTCCTTCTAC GAGTCTGAGC 1080

TACCACAAGG TTTCCAGGAT TATTACAATT TAGGCCAATT CACAGATGAC GATCAAGTGC 1140

CAACACCCCT CGTGGCAATA ACGATCACTA GGCTAGTGGT GGGTTTCCTG CTGCCCTCTG 1200

TTATCATGAT AGCCTGTTAC AGCTTCATTG TCTTCCGAAT GCAAAGGGGC CGCTTCGCCA 1260

AGTCTCAGAG CAAAACCTTT CGAGTGGCCG TGGTGGTGGT GGCTGTCTTT CTTGTCTGCT 1320

GGACTCCATA CCACATTTTT GGAGTCCTGT CATTGCTTAC TGACCCAGAA ACTCCCTTGG 1380

GGAAAACTCT GATGTCCTGG GATCATGTAT GCATTGCTCT AGCATCTGCC AATAGTTGCT 1440

TTAATCCCTT CCTTTATGCC CTCTTGGGGA AAGATTTTAG GAAGAAAGCA AGGCAGTCCA 1500

TTCAGGGAAT TCTGGAGGCA GCCTTCAGTG AGGAGCTCAC ACGTTCCACC CACTGTCCCT 1560

CAAACAATGT CATTTCAGAA AGAAATAGTA CAACTGTGTG AAAATGTGGA GCAGCCAACA 1620

AGCAGGGGCT CTTAGGCAAT CACATAGTGA AAGTTTATAA GAGGATGAAG TGATATGGTG 1680

AGCAGCGGAC TTCAAAAACT GTCAAAGAAT CAATCCAGCG GTTCTCAAAC GGTACACAGA 1740

CTATTGACAT CAGCATCACC TAGAAACTTG TTAGAAATGC AAATTCTCAA GCCGCATCCC 1800

AGACTTGCTG AATCGGAATC TCTGGGGGTT GGGACCCAGC AAGGGCACTT AACAAACCCC 1860 CGTTTCTGAT TAATGCTAAA TGTAAGAATC ATTGTAAACA TTAGTTCTAT TTCTATCCCA 1920 AACTAAGCTA TGTGAAATAA GAGAAGCTAC TTTGTTTTTA AATGATGTTG AATATTTGTC 1980 GATATTTCCA TCATTAAATT TT CCTTAGC ATTGTCTAAG TCAAAAAAAA AAAAAAAAAA 2040

(2 ) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH : 482 AMINO ACIDS

(B) TYPE : AMINO ACID

(C) STRANDEDNESS :

(D) TOPOLOGY : LINEAR

(ii) MOLECULE TYPE: PROTEIN

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ala Ser Phe Ser Ala Glu Thr Asn Ser Thr Asp Leu Leu Ser

5 10 15

Gin Pro Trp Asn Glu Pro Pro Val lie Leu Ser Met Val lie Leu

20 25 30

Ser Leu Thr Phe Leu Leu Gly Leu Pro Gly Aεn Gly Leu Val Leu

35 40 45

Trp Val Ala Gly Leu Lyε Met Gin Arg Thr Val Aεn Thr lie Trp

50 55 60

Phe Leu Hiε Leu Thr Leu Ala Aεp Leu Leu Cyε Cys Leu Ser Leu

65 70 75

Pro Phe Ser Leu Ala Hiε Leu Ala Leu Gin Gly Gin Trp Pro Tyr

80 85 90

Gly Arg Phe Leu Cys Lys Leu He Pro Ser He He Val Leu Asn

95 100 105

Met Phe Ala Ser Val Phe Leu Leu Thr Ala He Ser Leu Asp Arg

110 115 120

Cyε Leu Val Val Phe Lyε Pro He Trp Cyε Gin Aεn His Arg Asn

125 130 135

Val Gly Met Ala Cys Ser He Cys Gly Cys He Trp Val Val Ala

140 145 150

Cys Val Met Cys He Pro Val Phe Val Tyr Arg Glu He Phe Thr

155 160 165

Thr Asp Asn His Asn Arg Cys Gly Tyr Lyε Phe Gly Leu Ser Ser

170 175 180

Ser Leu Aεp Tyr Pro Aεp Phe Tyr Gly Aεp Pro Leu Glu Aεn Arg

185 190 195

Ser Leu Glu Aεn He Val Gin Pro Pro Gly Glu Met Asn Asp Arg

200 205 210

Leu Asp Pro Ser Ser Phe Gin Thr Asn Asp His Pro Trp Thr Val

215 220 225

Pro Thr Val Phe Gin Pro Gin Thr Phe Gin Arg Pro Ser Ala Asp

230 235 240

Ser Leu Pro Arg Gly Ser Ala Arg Leu Thr Ser Gin Asn Leu Tyr

245 250 255

Ser Asn Val Phe Lys Pro Ala Asp Val Val Ser Pro Lys He Pro

260 265 270 Ser Gly Phe Pro He Glu Asp Hiε Glu Thr Ser Pro Leu Aεp Aεn

275 280 285

Ser Asp Ala Phe Leu Ser Thr Hiε Leu Lyε Leu Phe Pro Ser Ala

290 295 300

Ser Ser Aεn Ser Phe Tyr Glu Ser Glu Leu Pro Gin Gly Phe Gin

305 310 315

Asp Tyr Tyr Asn Leu Gly Gin Phe Thr Asp Asp Asp Gin Val Pro

320 325 330

Thr Pro Leu Val Ala He Thr He Thr Arg Leu Val Val Gly Phe

335 340 345

Leu Leu Pro Ser Val He Met He Ala Cys Tyr Ser Phe He Val

350 355 360

Phe Arg Met Gin Arg Gly Arg Phe Ala Lys Ser Gin Ser Lys Thr

365 370 375

Phe Arg Val Ala Val Val Val Val Ala Val Phe Leu Val Cys Trp

380 385 390

Thr Pro Tyr His He Phe Gly Val Leu Ser Leu Leu Thr Asp Pro

395 400 405

Glu Thr Pro Leu Gly Lys Thr Leu Met Ser Trp Asp Hiε Val Cyε

410 415 420

He Ala Leu Ala Ser Ala Asn Ser Cys Phe Asn Pro Phe Leu Tyr

425 430 435

Ala Leu Leu Gly Lys Asp Phe Arg Lys Lyε Ala Arg Gin Ser He

440 445 450

Gin Gly He Leu Glu Ala Ala Phe Ser Glu Glu Leu Thr Arg Ser

455 460 465

Thr His Cys Pro Ser Aεn Aεn Val He Ser Glu Arg Aεn Ser Thr

470 475 480

Thr Val

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 32 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GACTAAAGCT TATGGCGTCT TTCTCTGCTG AG 32

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 32 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GAACTTCTAG ACTTCACACA GTTGTAOTAT TT 32

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 33 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GTCCAAGCTT GCCACCATGG GTCTTTCTCT GCT 33

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 58 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

CTAGCTCGAG TCAAGCGTAG TCTGGGACGT CGTATGGGTA GCACACAGTT GTACTATT 58

(2 ) INFORMATION FOR SEQ ID NO : 7 :

( i ) SEQUENCE CHARACTERISTICS

(A) LENGTH : 30 BASE PAIRS

(B) TYPE : NUCLEIC ACID

(C) STRANDEDNESS : SINGLE

(D) TOPOLOGY : LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

CGGGATCCCT CCATGGCGTC TTTCTCTGCT 30

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 29 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

CGGGATCCCG CTCACACAGT TGTACTATT 29