WO1996005226A1

WO1996005226A1 - POLYNUCLEOTIDE ENCODING C5a RECEPTOR

Info

Publication number: WO1996005226A1
Application number: PCT/US1994/009234
Authority: WO
Inventors: Yi Li; Craig A. Rosen
Original assignee: Human Genome Sciences Inc
Current assignee: Human Genome Sciences Inc
Priority date: 1994-08-16
Filing date: 1994-08-16
Publication date: 1996-02-22
Anticipated expiration: 1997-02-16
Also published as: EP0787149A1; AU7566594A; US20060148708A1; JPH10504456A; EP0787149A4

Abstract

A human C5a receptor polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for identifying antagonists and agonists to such polypeptide. Antagonists and agonists may be used therapeutically to inhibit or stimulate the C5a receptor.

Description

'POLYNUCLEOTIDE ENCODING C5A RECEPTOR".

This invention relates to newly identified polynucleotideε, 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- tranεmembrane receptor. The transmembrane receptor is a G- protein coupled receptor. More particularly, the 7- transmembrane receptor has been putatively identified as an anaphylatoxin C5a receptor, sometimes hereinafter referred to as "C5a". 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 (Lefkowitz, 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.

A wide variety conditions, including infection by bacteria, viruses or fungi, infiltration by cancer cells, allergic or autoimmune disorders and physically or chemically-induced trauma causes an inflammatory response in humans. In all of these diseases and conditions in man and in most mammals, activation of the complement system (a set of proteins, regulatory factors and proteolytic enzymes) via either the classical or the alternative pathway results in the generation of biologically active peptides which serve to amplify and exacerbate the resulting inflammation.

The most active peptide, anaphylatoxin C5a, a 74-amino acid polypeptide, is 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, W. , Int. Arch. Allergy Appl. Immunol., 33:417- 428 (1967)), and stimulating pulmonary, cardiac, vascular and gastrointestinal tissues in vitro (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 basophils (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 responses elicited by C5a in polymorphonuclear leukocytes result from the winding of the anaphylatoxin to a high-affinity receptor on the plasma membrane (Chenoweth, D.E. and Hugli, T.E., Mol. Immunol., 17:151-161 (1980)). In these cells, it appears that the mechanism of signal transduction through the membrane involves one or more GTP- binding proteins (G proteins) as is the case with other chemotactic receptors. The receptor molecule for C5a on human neutrophils has been well characterized with respect to its kinetics and saturability and many of the structural requirements for its activity are known. Reports indicate that the neutrophil C5a receptor binds its ligand with a nanomolar affinity constant, is expressed in approximately 100,000 copies per cell, and the binding sub-unit has an apparent mass of approximately 52 kDa.

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 basophils (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 lysosomal 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. ., et al., J. Exp. Med., 155:1412 (1982)).

In accordance with one aspect of the present invention, there are provided novel polypeptides which have been putatively identified as a C5a receptor, as well as fragments, analogs 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 polynucleotides (DNA or RNA) which encode such polypeptides.

In accordance with a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques.

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 receptor to screen for receptor antagonists and/or agonists and/or receptor ligands.

In accordance with still another embodiment of the present invention there is provided a process of using such agonists for therapeutic purposes, for example, as a defense against bacterial infection, to stimulate the immunoregulatory effects of C5a, to treat cancers, immunodeficiency diseases and severe infections.

In accordance with another aspect of the present invention there is provided a process of using such antagonists for treating asthma, bronchial allergy, chronic inflammation, systemic lupus erythematosis, vasculitis, rheumatoid arthritis, osteoarthritis, gout, some auto- allergic diseases, transplant rejection, ulcerative colitis, in certain shock states, myocardial infarction, and post- viral encephalopathieε.

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 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 putative mature G-protein coupled receptor of the present invention. The standard one- letter abbreviation for amino acids is used.

Figure 2 is an illustration of the secondary structural features of the G-protein coupled receptor. The first 7 illustrations set forth the regions of the amino acid sequence which are alpha helices, beta helices, turn regions or coiled regions. The boxed areas are the areas which correspond to the region indicated. The second set of figures illustrate areas of the amino acid sequence which are exposed to intracellular, cytoplasmic or are membrane- spanning. The hydrophilicity plot illustrates areas of the protein sequence which are the lipid bilayer of the membrane and are, therefore, hydrophobic, and areas outside the lipid bilayer membrane which are hydrophilic. The antigenic index corresponds to the hydrophilicity plot, since antigenic areas are areas outside the lipid bilayer membrane and are capable of binding antigens. The surface probability plot further corresponds to the antigenic index and the hydrophilicity plot. The amphipathic plots show those regions of the protein sequences which are polar and non-polar. The flexible regions correspond to the second set of illustrations in the sense that flexible regions are those which are outside the membrane and inflexible regions are transmembrane regions.

Figure 3 illustrates an amino acid alignment of the G- protein coupled receptor of the present invention and C5a receptors from various species of animals. Faded areas are those areas which match with the other amino acid sequences in the figure.

It should be pointed out that sequencing inaccuracies are a common problem which occurs in polynucleotide sequences. Accordingly, the sequence of the drawing is based on several sequencing runs and the sequencing accuracy is considered to be at least 97%.

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. 75821 on June 24, 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 human osteoclastoma stromal cells. It is structurally related to the G protein- coupled receptor family. It contains an open reading frame encoding a protein of 355 amino acid residues. The protein exhibits the highest degree of homology to a human C5a receptor with 27 % identity and 54 % similarity over the entire amino acid sequence.

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 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 or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of Figure 1 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 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 ' 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 as a polynucleotide which includes 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 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 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 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 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 nucleotides, which does not substantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host 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 is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains. 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 corresponds 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 is at least 50% and preferably 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . As herein used, the term "stringent conditions" means 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 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 soluble 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 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 contained 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 G-protein coupled receptor polypeptide which has the deduced amino acid sequence of Figure 1 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 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 or that encoded by the deposited cDNA 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 such 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 group, or (iii) one in which the mature polypeptide is fused 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 polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

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 animal 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 environment.

The present invention 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 of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the C5a receptor genes. 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 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; 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 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 sequence in the expression 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 expresεion 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 expresεion vectorε preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductaεe or neomycin reεiεtance for eukaryotic cell culture, or εuch as tetracycline or ampicillin resiεtance in E. coli.

The vector containing the appropriate DNA εequence aε hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

As repreεentative exampleε of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli. Strepto yces. Salmonella typhimurium: fungal cellε, εuch aε yeast; insect cells such as Drosophila and Sf9; animal cells such as CHO, COS or Boweε melanoma; plant cellε, etc. The εelection of an appropriate hoεt iε deemed to be within the εcope of those skilled in the art from the teachingε herein.

More particularly, the present invention also includes recombinant constructs comprising 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 haε been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises 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), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbskε, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may 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 transferaεe) vectorε or other vectorε with εelectable 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 promoters include CMV immediate early, HSV thymidine kinase, 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 host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such aε 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 constructε in hoεt 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 synthesizers.

Mature proteins can be expressed in mammalian cellε, yeaεt, bacteria, or other cellε under the control of appropriate promoterε. Cell-free tranεlation systems can also be employed to produce such proteins using RNAs derived from the DNA constructε of the preεent invention. Appropriate cloning and expresεion vectorε for uεe 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 eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually 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 enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinaεe (PGK), α-factor, acid phoεphataεe, or heat εhock proteinε, among otherε. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination εequenceε, and preferably, a leader εequence capable of directing εecretion of translated protein into the periplasmic εpace or extracellular medium. Optionally, the heterologouε εequence can encode a fuεion protein including an N-terminal identification peptide imparting deεired characteriεtics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expresεion vectorε for bacterial uεe are constructed by inserting a structural DNA sequence encoding a deεired protein together with suitable translation initiation and termination signalε in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of 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 tγphi urium and various species within the genera Pseudomonaε, 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 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 GEM1 (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 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 resulting crude extract retained for further purification.

Microbial cellε employed in expreεεion of proteinε can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use 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 expresε recombinant protein. Examples of mammalian expresεion εyεtemε include the COS-7 lines of monkey kidney fibroblaεtε, deεcribed by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expresεing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expresεion 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 siteε, tranεcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation εiteε may be uεed to provide the required nontranεcribed genetic elementε.

The G-protein coupled receptor polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocelluloεe 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 present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue. The G-protein coupled receptor of the present invention may be employed in a process for screening for antagonists and/or agonists for the receptor.

In general, such screening procedureε involve providing appropriate cells which expresε 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 G-protein coupled receptor. Such transfection may be accomplished by procedures as hereinabove described.

One such screening procedure involves the use of the melanophores which are transfected to express the G-protein coupled 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 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 indicateε 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 determining whether such compound generates a signal, i.e., activates the receptor.

Other screening techniques include the use of cellε which expreεs the G-protein coupled receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as deεcribed in Science, volume 246, pageε 181-296 (October 1989). For example, potential agoniεtε or antagoniεts may be contacted with a cell which expresses the G-protein coupled receptor and a second meεεenger response, e.g. .signal transduction or pH changes, may be measured to determine whether the potential agonist or antagonist is effective.

Another such screening technique involves introducing RNA encoding the G-protein coupled receptor into xenopuε oocytes to tranεiently express 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 expresεing the G- protein coupled receptor in which the receptor iε linked to a phospholipase C or D. Aε representative examples of εuch 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 involves 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 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 amount of labeled ligand bound to the receptors iε meaεured, 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.

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

In general, antagonistε 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, 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 osteoporoεis.

A potential antagonist is an antibody, or in some caεeε an oligonucleotide, which binds to the G-protein coupled receptor but does not elicit a second mesεenger response such that the activity of the G-protein coupled receptor is prevented. Potential antagonists also include proteins which are closely related to the ligand of the G-protein coupled receptor, i.e. a fragment of the ligand, which have lost biological function and when binding to the G-protein coupled receptor, elicit no response.

A potential antagonist alεo includeε 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 binding 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 antisenεe RNA oligonucleotide of from about 10 to 40 baεe 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 receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the G-protein coupled receptors (antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisenεe Inhibitorε of Gene Expreεεion, CRC Preεs, Boca Raton, FL (1988)). The oligonucleotides deεcribed above can also be delivered to cells such that the antisenεe RNA or DNA may be expreεsed in vivo to inhibit production of G-protein coupled receptors.

Another potential antagoniεt is a small molecule which binds to the G-protein coupled receptor, making it inaccesεible to ligands such that normal biological activity is prevented. Examples of 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 membrane bound G-protein coupled receptors.

The G-protein coupled receptor of the present invention haε been putatively identified aε a C5a receptor. Thiε identification haε been made aε a result of amino acid sequence homology.

The antagonists may be used to treat all pathological conditions which result from anaphylaxis εtimulated by the C5a polypeptide and mediated by the C5a receptor. These pathological conditions include asthma, bronchial allergy, chronic inflammation, systemic lupus erythematosus, vaεculitis, serum sickness, angioedema, rheumatoid arthritis, osteoarthritis, gout, bullouε εkin diseases, hypersensivity, pneumonitis, idiopathic pulmonary fibrosis, immune complex- mediated glomerulonephritis, psoriaεis, allergic rhinitis, adult respiratory distress syndrome, acute pulmonary disorders, endotoxin shock, hepatic cirrhosis, pancreatitis, inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), thermal injury, gram-negative sepsis, necrosis in yocardial infarction, leukophoresiε, expoεure to medical devices (including, but not limited to, hemodialyzer membranes and extracorpeal blood circulation equipment), chronic hepatitis, transplant rejection, post-viral encephalopathies, and/or ischemia induced myocardial or brain injury. These antagonist may also be used as prophylactics for such conditions as shock accompanying Deng Urea fever. The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.

The agonistε identified by the εcreening method aε described above, may be employed to enhance the C5a reactions mediated through the C5a receptor, which include defense against bacterial infection, stimulation of the immunoregulatory effects of C5a, treatment of cancers, immunodeficiency diseases and severe infections.

The C5a receptor and antagonists or agonistε may be employed in combination 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 is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit 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 compositionε of the invention. Associated with such containers) can be a notice in the form prescribed 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 polypeptides of the present invention may be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositionε may be administered in a convenient manner such as by the topical, intravenous, intraperitoneal, intramuscular, subcutaneouε, intranaεal or intradermal routeε. The pharmaceutical compoεitionε are adminiεtered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, the pharmaceutical compositionε will be adminiεtered in an amount of at leaεt about 10 μg/kg body weight and in moεt caεes they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most caseε, the doεage is from about 10 /xg/kg to about 1 mg/kg body weight daily, taking into account the routeε of adminiεtration, symptoms, etc.

The C5a receptor polypeptides and antagonists or agonists which are polypeptides, may alεo be employed in accordance with the present invention by expresεion of εuch polypeptideε in 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 cellε then being provided to a patient to be treated with the polypeptide. Such methodε are well-known in the art. For example, cells may be engineered by procedures known in the art by use 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 adminiεtered to a patient for engineering cellε in vivo and expresεion of the polypeptide in vivo . These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expreεεion vehicle for engineering cells may be other than a retroviruε, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

The sequenceε of the preεent invention are alεo valuable for chromoεome identification. The εequence iε specifically 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 chromoεomeε according to the present invention is an important first step in correlating those sequences with genes associated with disease.

Briefly, εequenceε can be mapped to chromoεomeε by preparing PCR primerε (preferably 15-25 bp) from the cDNA. Computer analyεiε of the cDNA iε used to rapidly select primerε that do not εpan more than one exon in the genomic DNA, thuε complicating the amplification proceεε. These primers are then used for PCR screening of εomatic cell hybridε containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment. PCR mapping of εomatic 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 panelε 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 situ 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 chromoεomal εpread can be uεed to provide a preciεe chromoεomal location in one εtep. This technique can be used with cDNA as short aε 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 iε probably not neceεεary to get good reεultε a reasonable percentage of the time. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence haε been mapped to a preciεe 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. McKuεick, 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 chromoεomal region are then identified through linkage analyεiε (coinheritance of physically adjacent genes).

Next, it iε neceεεary to determine the differenceε 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 phyεical mapping and genetic mapping techniqueε, a cDNA preciεely localized to a chromoεomal region aεεociated with the diεeaεe could be one of between 50 and 500 potential causative genes. (This assumeε 1 megabaεe mapping resolution and one gene per 20 kb) .

The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expresεing them can be uεed as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized 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 antibodies and fragments.

Antibodies generated against the polypeptides corresponding to a sequence of the preεent 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 εo obtained will then bind the polypeptideε itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expresεing that polypeptide.

For preparation of monoclonal antibodies, 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 antibodieε (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 antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain 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 is not limited to such 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.

"Plasmidε" are deεignated by a lower case p preceded and/or followed by capital letterε and/or numberε. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids 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 restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used aε would be known to the ordinarily εkilled artisan. For analytical purposeε, typically 1 μg of plaεmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragmentε for plaεmid conεtruction, typically 5 to 50 μg of DNA are digeεted with 20 to 250 unitε 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's instructionε. After digeεtion the reaction iε electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size εeparation of the cleaved fragmentε iε performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980).

"Oligonucleotides" referε 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 adding a phosphate with an ATP in the preεence of a kinaεe. A εynthetic oligonucleotide will ligate to a fragment that haε not been dephosphorylated.

"Ligation" refers to the proceεε of forming phoεphodieεter bondε between two double εtranded nucleic acid fragmentε (Maniatiε, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditionε with 10 unitε to T4 DNA ligaεe ("ligaεe") per 0.5 μg of approximately equimolar amountε of the DNA fragmentε to be ligated.

Unless otherwise stated, transformation was 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 C5a Receptor The DNA sequence encoding the C5a receptor, ATCC # 75821, is initially amplified using PCR oligonucleotide primerε corresponding to the 5' end sequences of the processed C5a receptor protein (minus the signal peptide sequence) and the vector sequences 3' to the gene. Additional nucleotides corresponding to the C5a receptor were added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence 5 ' GACTAAAGCTTAATGGAAGATTTGGAGGAA 3 ' contains a Hindlll restriction enzyme site followed by 19 nucleotides of C5a receptor coding sequence starting from the presumed terminal amino acid of the proceεεed protein codon. The 3' sequence 5 ' GAACTTCTAGACCGTTATTGAGCTGTTTCCAGGAG 3 ' contains complementary sequences to an Xbal site and is followed by 18 nucleotides of the gene. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311). pQE-9 encodeε antibiotic reεiεtance (Amp^r), a bacterial origin of replication (ori), an IPTG- regulatable promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites. pQE-9 was then digested with Hindlll and Xbal. The amplified sequences were ligated into pQE-9 and were inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture waε then uεed to tranεform E. coli εtrain available from Qiagen under the trademark M15/rep 4 by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Presε, (1989). M15/rep4 containε multiple copieε of the plaεmid pREP4, which expresses the la repressor and also confers kana ycin resiεtance (Kan^r) . Tranεformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA waε iεolated and confirmed by restriction analysiε. Cloneε containing the deεired conεtructε were grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ l) 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 were grown to an optical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG ( "Isopropyl-B-D-thiogalacto pyranoside") was then added to a final concentration of 1 mM. IPTG induces by inactivating the la repressor, clearing the P/O leading to increased gene expression. Cells were grown an extra 3 to 4 hours. Cells were then harvested by centrifugation. The cell pellet waε solubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, solubilized C5a receptor was 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 C5a receptor was eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpoεe of renaturation adjuεted to 3 molar guanidine HCl, lOOmM εodium 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.

Example 2 Expression of Recombinant C5a Receptor in COS cells

The expresεion of plaεmid, pC5a HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 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 pC5a protein and a HA tag fused in rame 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 hemagglutinin protein as previously deεcribed (I. Wilson, H. Niman, R. Heighten, A Cherenson, 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 aε followε: The DNA sequence encoding for the C5a receptor, ATCC # 75821, was constructed by PCR on the full-length gene cloned using two primers: the 5' primer 5' GTCCGAAGCTTGCCACCATGGAA GATTTGGAGGAA 3' contains a Hindlll εite followed by 18 nucleotides of C5a receptor coding sequence starting from the initiation codon; the 3' sequence 5' CTAGCTCGAGTCAAGCGTAGTCTG GGACGTCGTATGGGTAGCATTGAGCTGTTTCCAGGAG 3 ' contains complementary sequences to an Xhol site, translation stop codon, HA tag and the last 18 nucleotides of the C5a receptor coding sequence (not including the stop codon). Therefore, the PCR product contains a Hindlll site, C5a receptor coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xhol site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digeεted with Hindlll and Xhol reεtriction enzyme and ligated. The ligation mixture waε tranεformed into E. coli strain SURE (available from Stratagene Cloning Systemε, 11099 North Torrey Pineε Road, La Jolla, CA 92037) the tranεformed culture waε plated on ampicillin media plateε and reεiεtant colonieε were selected. Plasmid DNA was isolated from tranεformantε and examined by restriction analysiε for the presence of the correct fragment. For expression of the recombinant C5a receptor, COS cells were transfected with the expreεsion vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Preεs, (1989)). The expresεion of the C5a receptor HA protein was detected by radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with ^3SS-cysteine two dayε post tranεfection. Culture media were then collected and cellε were lysed 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 lyεate and culture media were precipitated with a HA specific monoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.

Example 3 Cloninσ and expression of C5a receptor uεinσ the baculoviruε expreεsion system

The DNA sequence encoding the full length C5a receptor protein, ATCC # 75821, was amplified using PCR oligonucleotide primers corresponding to the 5 ' and 3 ' sequences of the gene:

The 5 ' primer has the sequence 5' GCCGGATCCGCCA CCATGGAAGATTTGGAGGAA 3 ' and contains a BamHI restriction enzyme site (in bold) followed by 6 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (J. Mol. Biol. 1987, ϋό, 947-950, Kozak, M.), and is just behind the first 18 nucleotides of the gene (the initiation codon for translation "ATG" is underlined).

The 3 ' primer haε the εequence 5' GCCGGATCCGT TATTGAGCTGTTTCCAG 3 ' and contains the cleavage site for the restriction endonucleaεe BamHI and 18 nucleotideε complementary to the 3' non-tranεlated εequence of the C5a receptor gene. The amplified sequences were isolated from a 1% agarose gel using a commercially available kit ( "Geneclean, " BIO 101 Inc., La Jolla, Ca. ) . The fragment was then digested with the endonucleases BamHI and then isolated again on a 1% agarose gel. This fragment is designated F2.

The vector pRGl (modification of pVL941 vector, discussed below) is used for the expreεεion of the C5a receptor protein uεing the baculoviruε expresεion 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 polyhedroεiε viruε (AcMNPV) followed by the recognition εiteε 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-galactoεidase gene from E.coli is 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 sequenceε for the cell-mediated homologouε recombination of co-tranεfected wild-type viral DNA. Many other baculoviruε vectorε could be uεed in place of pRGl εuch aε pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31- 39).

The plasmid was digeεted with the reεtriction enzymeε BamHI and then dephoεphorylated using calf intestinal phosphataεe by procedures known in the art. The DNA was then isolated from a 1% agarose gel as described above. This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plaεmid V2 were ligated with T4 DNA ligaεe. E.coli HB101 cellε were then tranεformed and bacteria identified that contained the plasmid (pBacC5a) with the C5a receptor gene using the enzyme BamHI. The sequence of the cloned fragment was confirmed by DNA sequencing.

5 μg of the plasmid pBacC5a was co-transfected with 1.0 μg of a commercially available linearized baculoviruε ( "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 pBacC5a were mixed in a εterile well of a microtiter plate containing 50 μl of εeru free Grace'ε medium (Life Technologieε Inc., Gaitherεburg, MD). Afterwardε 10 μl Lipofectin pluε 90 μl Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture was added dropwise to the Sf9 inεect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1ml Grace' medium without εerum. The plate waε rocked back and forth 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's 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.

After four days the supernatant was collected and a plaque asεay performed similar as described by Summers and Smith (supra). Aε a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) waε uεed which allows an easy isolation of blue stained plaques. (A detailed description of a "plaque assay" can also 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 containing the recombinant viruεes was then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar waε removed by a brief centrifugation and the supernatant containing the recombinant baculoviruseε was used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes were harvested and then stored at 4°C.

Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells were infected with the recombinant baculovirus V-C5a at a multiplicity of infection (MOI) of 2. Six hourε later the medium waε removed and replaced with SF900 II medium minuε methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 μCi of ^3SS-methionine and 5 μCi ³⁵S cyεteine (Amerεham) were added. The cellε were further incubated for 16 hours before they were harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.

Example 4 Expresεion pattern of C5a receptor in human tissue

Northern blot analysis was carried out to examine the levels of expression of C5a receptor in human tisεueε. Total cellular RNA samples were isolated with RNAzol™ B system (Biotecx Laboratorieε, Inc. 6023 South Loop Eaεt, Houεton, TX 77033). About 10/xg of total RNA iεolated from each human tissue specified was separated on 1% agarose gel and blotted onto a nylon filter. (Sambrook, Fritsch, and Maniatiε, Molecular Cloning, Cold Spring Harbor Preεs, (1989)). The labeling reaction was done according to the Stratagene Prime- It kit with 50ng DNA fragment. 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 full length C5a receptor gene at 1,000,000 cpm/ml in 0.5 M NaP0₄, 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 waε then expoεed at -70^*C overnight with an intenεifying εcreen. The message RNA for C5a receptor is abundant in peripheral lymphocytes.

Numerous modifications and variations of the present invention are poεεible in light of the above teachingε 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: C5a Receptor

(iii) NUMBER OF SEQUENCES: 2

(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:

(Vϋ) 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: 325800-195

(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: 2024 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: cDNA

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

CGGCAAAGCA GGCATGGACA ATAGCTTCTC TCCTCACAGA AATTTAACTG ATTTCTTCAT 60

TCTCCATTTA GCAAGGTCAT GGAAGATTTG GAGGAAACAT TATTTGAAGA ATTTGAAAAC 120

TATTCCTATG ACCTAGACTA TTACTCTCTG GAGTCTGATT TGGAGGAGAA AGTCCAGCTG 180

GGAGTTGTTC ACTGGGTCTC CCTGGTGTTA TATTGTTTGG CTTTTGTTCT GGGAATTCCA 240

GGGAAATGCC TCTATCATTT GGTTCACGGG GTTCAAGTGG AAGAAGACAG TCACACTCTG 300

TGGTTCCTCA ATCTAGCCAT TGCGGATTTC ATTTTTCTTC TCTTTCTGCC CCTGTACATC 360

TCCTATGTGG CCATGAATTT CCACTGGCCC TTTGGCATCT GGCTGTGCAA AGCCAATTCC 420

TTCACTGCCC AGTTGAACAT GTTTGCCAGT GTTTTTTTCC TGACAGTGAT CAGCCTGGAC 480

CACTATATCC ACTTGATCCA TCCTGTCTTA TCTCATCGGC ATCGAACCCT CAAGAACTCT 540

CTGATTGTCA TTATATTCAT CTGGCTTGTG GCTTCTCTAA TTGGCGGTCC TGCCCTGTAC 600

TTCCGGGATA CTGTGGAGTT CAATAATCAT ACTCTTTGGT ATAACAATTT TCAGAAGCAT 660

GATCCTGACC TCACTTGGAT CAGGCACCAT GTTCTGACTT GGGTGAAATT TATCATTGGT 720

TATCTCTTCC CTTTGCTAAC AATGAGTATT CGGTACTTGT GTCTCATCTT CAAGGTGAAG 780

AAGCGAAGCA TCCTGATCTC CAGTAGGCAT TTCTGGACAA TTCTGGTTGT GGTTGTGGCC 840

TTTGTGGTTT GGTGGACTCC TTATCACCTG TTTAGCATTG GGGAGCTCAC CATTCACCAC 900

AATAGCTATT CCCACCATGT GATGCAGGCT GGAATCCCCC TCTCCACTGG TTTGGCATTC 960

CTCAATAGTT GCTTGAACCC CATCCTTTAT GTCCTAGTTA GTAAGAAGTT CCAAGCTCGC 1020 TTCCGGTCCT CAGTTGCTGA GATACTCAAG TACACACTGT GGGAAGTCAG CTGTTCTGGC 1080

ACAGTGAGTG AACAGCTCAG GAACTCAGAA ACCAAGAATC TGTGTCTCCT GGAAACAGCT 1140

CAATAAGTTA TTACTTTTCC ACAAATCAGT ATATGGCTTT TTATGTGGGT CCTCTGACTG 1200

ATGCTTTCAG ATTAAAATTG TTTCCAAGAT AGAGAGCCGA CTCCACTTTC ATAGTTATTG 1260

TTTCTGGTCA CTATATAGGC ATCACATTTT TGTGTGGATA TGAAACTTAG GAAGGATCCT 1320

CTTGACTCCT TGTGATGTGG CAATAAATTT TTTTTAAAAA ACTGAAAATA CTTAGGAAGG 1380

ATCCGCATAA TTTTTTTCTG CAACTTAAAT GAAATGCATC ATTCTTGTTA ATCATACCAT 1440

GGTGAATTAA TCACTTTTGA AGCAATATCA GTTATTTTTT GAATAATAAC TTTTCTAAAG 1500

CCTTAAGTCT TAATATTAAA TATATGATTA GCCAGGCCCG GTGGCTGACA CCTGTAATCC 1560

CAGCACTTTG GGAGGCCAAG GTGGGGGGAT TACCCGAGGT CAGGAATTCG AGACCAGCCT 1620

GACCAACATG GAGAAACCCC GTCTCTACTA AAAATCCAAA ATTAGCCGGT CATGGTGGTG 1680

CATGTCTGCA AACCCAGCTA CTCGGGAGGC TGAAGCAGGA GAATCC CTT GAACCTGGGA 1740

GGCAGAGGTT GTGGTGAGCC AACATCACAC CATTGCACTC CAGCCTGGGC CACAAGAGTA 1800

AAACTCTGTC TCAAAAATAA ATAAATAAAA TAGATAAATA AATATATGAT TAACTAATTT 1860

TAAAAATGTT AAAATGTATT CTTAAATTCA TTTTAATTTT GTACAATAAC CTGCTAGACA 1920

CATTTTTAAA ATGCAACATG TGTACTTAAT TTCTTTATGT AATCTATGTA TATACATTTA 1980

TGAATTAAAG TAATTGTTGG TTATCTTAAA AAAAAAAAAA AAAA 2024

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 355 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) STRANDEDNESS:

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN

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

Met Glu Asp Leu Glu Glu Thr Leu Phe Glu Glu Phe Glu Asn Tyr

5 10 15

Ser Tyr Asp Leu Asp Tyr Tyr Ser Leu Glu Ser Asp Leu Glu Glu

20 25 30

Lys Val Gin Leu Gly Val Val His Trp Val Ser Leu Val Leu Tyr

35 40 45

Cys Leu Ala Phe Val Leu Gly lie Pro Gly Lyε Cyε Leu Asp His

50 55 60

Leu Val His Gly Val Gin Val Glu Glu Asp Ser His Thr Leu Trp 65 70 75

Phe Leu Asn Leu Ala lie Ala Asp Phe He Phe Leu Leu Phe Leu

80 85 90 Pro Leu Tyr lie Ser Tyr Val Ala Met Asn Phe His Trp Pro Phe

95 100 105 Gly lie Trp Leu Cys Lys Ala Asn Ser Phe Thr Ala Gin Leu Asn

110 115 120 Met Phe Ala Ser Val Phe Phe Leu Thr Val He Ser Leu Asp His

125 130 135 Tyr lie His Leu lie His Pro Val Leu Ser His Arg His Arg Thr

140 145 150 Leu Lys Asn Ser Leu lie Val lie lie Phe He Trp Leu Val Ala

155 160 165 Ser Leu lie Gly Gly Pro Ala Leu Tyr Phe Arg Asp Thr Val Glu

170 175 180 Phe Aεn Aεn His Thr Leu Trp Tyr Asn Asn Phe Gin Lys His Asp

185 190 195 Pro Asp Leu Thr Trp lie Arg His His Val Leu Thr Trp Val Lys

200 205 210 Phe lie lie Gly Tyr Leu Phe Pro Leu Leu Thr Met Ser He Arg

215 220 225 Tyr Leu Cys Leu lie Phe Lyε Val Lyε Lys Arg Ser He Leu He

230 235 240 Ser Ser Arg His Phe Trp Thr lie Leu Val Val Val Val Ala Phe

245 250 255 Val Val Trp Trp Thy Pro Tyr Hiε Leu Phe Ser He Gly Glu Leu

260 265 270 Thr lie His His Asn Ser Tyr Ser Hiε Hiε Val Met Gin Ala Gly

275 280 285 lie Pro Leu Ser Thr Gly Leu Ala Phe Leu Aεn Ser Cys Leu Aεn

290 295 300 Pro lie Leu Tyr Val Leu Val Ser Lys Lyε Phe Gin Ala Arg Phe

305 310 315 Arg Ser Ser Val Ala Glu lie Leu Lys Tyr Thr Leu Trp Glu Val

320 325 330 Ser Cys Ser Gly Thr Val Ser Glu Gin Leu Arg Asn Ser Gly Thr

335 340 345

Lys Asn Leu Cys Leu Leu Glu Thr Ala Gin

350 355

Claims

WHAT IS CLAIMED IS:

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

(a) a polynucleotide encoding the G-protein coupled receptor polypeptide having the deduced amino acid εequence of Figure 1 or a fragment, analog or derivative of εaid polypeptide;

(b) a polynucleotide encoding the G-protein coupled receptor polypeptide having the amino acid εequence encoded by the cDNA contained in ATCC Depoεit No. 75821 or a fragment, analog or derivative of εaid polypeptide.

2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.

3. The polynucleotide of Claim 1 wherein the polynucleotide iε RNA.

4. The polynucleotide of Claim 1 wherein the polynucleotide iε genomic DNA.

5. The polynucleotide of Claim 2 wherein εaid polynucleotide encodeε G-protein coupled receptor having the deduced amino acid εequence of Figure 1.

6. The polynucleotide of Claim 2 wherein εaid polynucleotide encodes the G-protein coupled receptor polypeptide encoded by the cDNA of ATCC Deposit No. 75821.

7. The polynucleotide of Claim 1 having the coding sequence of G-protein coupled receptor as shown in Figure 1.

8. The polynucleotide of Claim 2 having the coding sequence of G-protein coupled receptor deposited as ATCC Deposit No. 75821.

9. A vector containing the DNA of Claim 2.

10. A host cell genetically engineered with the vector of Claim 9.

11. A proceεε for producing a polypeptide comprising: expressing from the host cell of Claim 10 the polypeptide encoded by said DNA.

12. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 9.

13. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having G-protein coupled receptor activity.

14. A polypeptide selected from the group consiεting of (i) a G-protein coupled receptor polypeptide having the deduced amino acid εequence of Figure 1 and fragmentε, analogε and derivativeε thereof and (ii) a G-protein coupled receptor polypeptide encoded by the cDNA of ATCC Depoεit No. 75821 and fragments, analogs and derivatives of said polypeptide.

15. The polypeptide of Claim 14 wherein the polypeptide is a G-protein coupled receptor having the deduced amino acid sequence of Figure 1.

16. An antibody against the polypeptide of claim 14.

17. A compound which activates the G-protein coupled receptor polypeptide of claim 14.

18. A compound which inhibits activation of the G-protein coupled receptor polypeptide of claim 14.

19. A method for the treatment of a patient having need of activation of a G-protein coupled receptor polypeptide of claim 14 comprising: administering to the patient a therapeutically effective amount of a compound Claim 17.

20. A method for the treatment of a patient having need to inhibit activation of the G-protein coupled receptor of claim 14 comprising: administering to the patient a therapeutically effective amount of a compound of Claim 18.

21. The polypeptide of Claim 14 wherein the polypeptide is a soluble fragment of the G-protein coupled receptor and is capable of binding a ligand for the receptor.

22. A process for identifying antagonistε and agonists to the G-protein coupled receptor compriεing: expreεεing the G-protein coupled receptor on the εurface of a cell; contacting the cell with a receptor ligand and compound to be εcreened; determining whether a second signal is generated from the interaction of the ligand and the receptor; and identifying if the compound to be screened is an agonist or antagonist.

23. A proceεε for determining whether a ligand not known to be capable of binding to a G-protein coupled receptor can bind thereto compriεing: contacting a mammalian cell which expresses the G- protein coupled receptor with a potential ligand; detecting the presence of the ligand which bindε to the receptor; and determining whether the ligand binds to the G- protein coupled receptor.