CN1157410C - Human G protein-coupled receptor (HETGQ23) - Google Patents

Abstract

The present invention discloses human G-protein coupled receptor polypeptide, a DNA(RNA) for encoding the polypeptide, and a method for generating the polypeptide by recombination technology. The present invention also discloses a method by which the polypeptide is used for identifying an antagonist and an excitant of the polypeptide, and a method by which the antagonist and the excitant are used for treating diseases corresponding to the low expression and the excess expression of the G-protein coupled receptor polypeptide. The present invention also discloses a diagnostic method for detecting the mutation in a G-protein coupled receptor nucleotide sequence, and a diagnostic method for detecting the change level of a soluble form of a receptor.

Description

Human G protein-coupled receptor (HETGQ23)

The present invention relates to newly identified polynucleotides, polypeptides encoded by these polynucleotides, uses of these polynucleotides and polypeptides, and methods of producing such polynucleotides and polypeptides. More specifically, the polypeptide of the invention is the human 7-transmembrane receptor. The invention also relates to methods of inhibiting the action of these polypeptides.

It is well established that many medically significant biological processes are mediated by proteins involved in signal transduction pathways involving G proteins and/or second messengers such as cAMP (Lefkowitz, Nature, 351:353 -354 (1991)). These proteins are referred to herein as proteins involved in pathways with G proteins or PPG proteins. Examples of these proteins include GPC receptors, such as those of adrenergic receptors and dopamine receptors (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 protein itself; effector proteins such as phospholipase C, adenylate cyclase and phosphodiesterase ; Stimulating proteins such as protein kinase A and protein kinase C (Simon, M.T., et al., Science, 252:802-8 (1991).

For example, in one form of signal transduction, hormone binding acts to activate an intracellular enzyme, adenylate cyclase. Enzyme activation by hormones is dependent on the presence of GTP nucleotides, and GTP also affects hormone binding. G proteins link hormone receptors to adenylyl cyclase, and when activated by hormone receptors, G proteins can be shown to convert bound GDP to GTP, the GTP-carrying form that then binds to activated adenylyl cyclase. Catalyzed by the G protein itself, GTP is hydrolyzed into GDP, and the G protein is restored to its ground state inactive form. In this way, the G protein has a dual role, as an intermediate to transmit the signal from the receptor to the effector, and as a clock to regulate the duration of the signal.

The membrane protein gene superfamily of G protein-coupled receptors is characterized by seven putative transmembrane domains. This region is believed to represent a transmembrane alpha-helix joined by extracellular or cytoplasmic loops. G protein-coupled receptors include many biologically active receptors, such as hormones, viruses, growth factors, and neural receptors.

G protein-coupled receptors are characterized by seven conserved hydrophobic segments comprising these approximately 20-30 amino acids, connected to at least eight divergently evolving hydrophilic loops. The G protein family of coupled receptors includes dopamine receptors that bind drugs used to treat psychotic and nervous disorders. Other members of this family include calcitonin, epinephrine, endothelin, cAMP, adenosine, muscarine, acetylcholine, serotonin, histamine, thrombin, cytokinins, follicle-stimulating hormone, opsin, endothelial differentiation Gene-1 receptor and rhodopsin, odorant, cytomegalovirus receptor, etc.

Most G protein-coupled receptors have a single conserved cysteine residue in each of the first two extracellular loops, which form disulfide bonds believed to stabilize functional protein structure. Seven transmembrane domains are labeled TM1, TM2, TM3, TM4, TM5, TM6 and TM7, and TM3 has been involved in signal transduction.

Phosphorylation and lipidation (hexadecylation or farnesylation) of cysteine residues can affect signal transduction by some G protein-coupled receptors. Most G protein-coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxyl terminus. Phosphorylation of some G protein-coupled receptors, such as β-adrenergic receptors, by protein kinase A and/or specific receptor kinases can mediate receptor desensitization.

For some receptors, the ligand-binding site of a G protein-coupled receptor is believed to consist of a hydrophilic cavity formed by several G protein-coupled receptor transmembrane Surrounded by hydrophobic residues. The hydrophilic side of each G protein-coupled receptor transmembrane helix faces inward and forms the polar ligand binding site. TM3 is included in some G protein-coupled receptors due to having a ligand binding site such as containing TM3 aspartic acid residues. In addition, TM5 serine, TM6 asparagine and TM6 or TM7 phenylalanine or tyrosine are also involved in ligand binding.

G protein-coupled receptors can be coupled intracellularly with various intracellular enzymes, ion channels, and transporters using heterotrimeric G proteins (see Johnson et al., Endoc., Rev., 10:317-331 (1989 )). Different G protein α-subunits preferentially stimulate specific effectors to regulate various biological functions within cells. Phosphorylation of cytoplasmic residues of G protein-coupled receptors has been identified as an important mechanism by which certain G protein-coupled receptors regulate G protein coupling. G protein-coupled receptors are found at numerous sites in mammalian hosts.

According to one aspect of the present invention, there are provided novel polypeptides and biologically active and diagnostically or therapeutically useful fragments and derivatives thereof. The polypeptides of the invention are of human origin.

According to another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNA and their antisense analogs and biologically active fragments useful in diagnosis or treatment .

According to yet another aspect of the present invention, there is provided a method for producing such a polypeptide by recombinant technology, the method comprising culturing a recombinant protein containing a nucleic acid sequence encoding a polypeptide of the present invention under conditions that promote expression of the polypeptide and subsequent recovery of the polypeptide. Prokaryotic and/or eukaryotic host cells.

According to yet another aspect of the invention, antibodies against such polypeptides are provided.

According to still another aspect of the present invention, there is provided a method for screening compounds and receptor ligands, the compound binds to and activates or inhibits the activation of the receptor polypeptide of the present invention.

According to another embodiment of the present invention, there is provided a method of using such an activating compound to stimulate a receptor polypeptide of the present invention to treat a condition associated with underexpression of a G protein coupled receptor.

According to another aspect of the invention, there are provided methods of using such inhibitory compounds to treat conditions associated with overexpression of G protein coupled receptors.

According to another aspect of the present invention, there is provided a non-naturally occurring synthetic, isolated and/or recombinant G protein-coupled receptor polypeptide that is a member of at least one transmembrane region of a G protein-coupled receptor of the present invention. Fragments, consensus fragments and/or sequences with conservative amino acid substitutions, and such receptors can bind G protein-coupled receptor ligands, or they can also quantitatively or qualitatively regulate G protein-coupled receptor ligand binding.

According to another aspect of the present invention, there are provided synthetic or recombinant G protein-coupled receptor polypeptides, conservative substitutions and derivatives thereof, antibodies, anti-idiotypic antibodies, compositions and methods, due to their expected biological properties, These substances can be used as potential modulators of G protein-coupled receptor function by binding to or modulating ligand binding, and thus can be used in diagnostic, therapeutic and/or research applications.

Yet another object of the present invention is to provide synthetic, isolated or recombinant polypeptides designed to inhibit or mimic various G protein-coupled receptors or fragments thereof according to receptor type and subtype.

According to another aspect of the present invention, there is provided a diagnostic probe comprising a nucleic acid molecule of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.

According to yet another aspect of the present invention, there is provided a diagnostic method for detecting a disease or susceptibility to a disease associated with a mutation in a nucleic acid sequence of the present invention.

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

The following drawings are intended to illustrate embodiments of the present invention, but are not intended to limit the scope of the invention covered by the claims.

Figure 1 depicts the cDNA sequence and corresponding deduced amino acid sequence of a G protein-coupled receptor of the invention. Standard single-letter abbreviations for amino acids are used. Sequencing was performed using 373 automatic DNA sequencer (Applied Biosystems.Inc.).

Figure 2 depicts the amino acid homology of polypeptides of the invention (top row) and human endothelial differentiation protein (edg-1) gene mRNA (bottom row).

Figure 3 is a schematic diagram of the secondary structure characteristics of G protein-coupled receptors. The first 7 schematic diagrams show the regions of the amino acid sequence of α-helix, β-sheet, turn region or coil region, and the boxed region represents the corresponding indicated area. Regions of the amino acid sequence that are exposed intracellularly, cytoplasmically, or transmembrane are shown in the second set of figures. The hydrophilic portion shows the hydrophobic regions of the protein sequence in the lipid bilayer of the membrane, and the hydrophilic regions outside the lipid bilayer membrane. The antigenicity index corresponds to the hydrophilicity graph, since the antigenic regions are the regions located outside the lipid bilayer membrane and capable of binding antigen. Surface probability maps further correspond to antigenicity index and hydrophilicity maps. The amphipathic map shows regions of 13 sequences that are polar and non-polar. The flexible regions correspond to the second set of schematic diagrams, ie the flexible regions represent extramembrane regions and the inflexible regions represent transmembrane regions.

According to one aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) encoding a mature polypeptide having the deduced amino acid sequence (SEQ ID No: 2) of FIG. The mature polypeptide encoded by the cDNA clone deposited with the deposit number ATCC NO.97130.

The polynucleotide encoding the polypeptide of the present invention can be isolated from a cDNA library derived from human endometrial tumor tissue, which is structurally related to the G protein-coupled receptor family, which comprises a protein encoding 364 amino acid residues open reading frame. The protein showed the highest degree of homology with human EDG-1 protein, with 36% identity and 61% similarity over a stretch of 364 amino acid sequences. Potential ligands for receptor polypeptides of the invention include, but are not limited to, anandamide, serotonin, epinephrine and norepinephrine, platelet activating factor, thrombin, C5a and bradykinin, chemokines and platelet activating factor.

The polynucleotides of the present invention may be in the form of RNA or DNA, where DNA includes cDNA, genomic DNA and synthetic DNA. This DNA can be double-stranded or single-stranded, and if single-stranded, it can be the coding strand or the non-coding (antisense) strand. The coding sequence of this coding mature polypeptide can be identical with the coding sequence shown in Figure 1 (SEQ ID NO: 1) or the coding sequence of the clone of deposit; Due to the redundancy or degeneracy of the genetic code, this coding sequence also It may be a different coding sequence that encodes the same mature polypeptide as the DNA of Figure 1 (SEQ ID NO: 1) or the deposited cDNA.

The polynucleotide encoding the mature polypeptide of Figure 1 (SEQ ID NO: 2) or encoding the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence encoding the mature polypeptide; the coding sequence encoding the mature polypeptide (and optionally Additional coding sequences) and non-coding sequences, such as introns or 5' and/or 3' non-coding sequences of the mature polypeptide coding sequence.

Thus, the term "polynucleotide encoding a polypeptide" includes polynucleotides that contain only coding sequences for a polypeptide as well as polynucleotides that also contain additional coding and/or non-coding sequences.

The present invention also relates to polynucleotide variants described above that encode a polypeptide having the deduced amino acid sequence (SEQ ID NO: 2) of Figure 1 or a fragment of a polypeptide encoded by a deposited cDNA clone, analogs and derivatives. Such nucleotide variants may be naturally occurring allelic variants of the polynucleotide or non-naturally occurring polynucleotide variants.

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 deposited cDNA clone, as well as encoding the mature polypeptide as shown in Figure 1 or encoded by A polynucleotide variant that is a fragment, derivative or analogue of the mature polypeptide encoded by the deposited cDNA clone. These nucleotide variants include deletion variants, substitution variants, addition or insertion variants.

As indicated above, the polynucleotide may have a coding sequence that is a naturally occurring allele of the coding sequence shown in Figure 1 (SEQ ID NO: 1) or the coding sequence of the deposited clone Variants. It is known in the art that an allelic variant is another form of a polynucleotide sequence that may have a substitution, deletion or addition of one or more nucleotides without substantially altering the function of the encoded polypeptide.

The polynucleotide may also encode a soluble form of the receptor polypeptide of the invention, which is the extracellular portion of the polypeptide cleaved from the TM and intracellular regions of the full-length polypeptide of the invention.

A polynucleotide of the invention may also have a coding sequence fused in frame to a marker sequence that allows purification of the polynucleotide of the invention. In the case of bacterial hosts, the marker sequence can be the hexahistidine tag provided by the pQE-9 vector, which is used to purify the mature polypeptide fused with the marker, or, for example, when using mammalian cells (such as COS-7 cells). ), the tag sequence may be a hemagglutinin (HA) tag. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term "gene" refers to the segment of DNA involved in the production of a polypeptide chain: it includes the regions preceding and following the coding region (leader and trailer) and intervening between individual coding segments (exons) sequence (intron).

Fragments of the full-length gene of the present invention can be used as hybridization probes for cDNA libraries to separate the full-length gene from other genes that have a high sequence similarity or similar biological activity to the gene. Probes of this type preferably have at least 20 or 30 bases, and may contain, for example, 50 or more bases. Said probes can also be used to identify cDNA clones corresponding to full-length transcripts and genomic clones or clones containing the complete gene of the invention including regulatory and promoter regions, exons and introns. Examples of screening include isolating the coding region of a gene by synthesizing oligonucleotide probes using known DNA sequences. Labeled oligonucleotides having a sequence complementary to the gene sequence of the invention can be used to screen human cDNA, genomic DNA or mRNA libraries to determine which library members the probe hybridizes to.

The present invention also relates to polynucleotides that hybridize to the sequences described above (provided that there is at least 70%, preferably at least 90%, more preferably at least 95% identity between the two sequences). The present invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described above. As used herein, the term "stringent conditions" means that hybridization can only occur if there is at least 95%, preferably at least 97%, identity between the sequences. In a preferred embodiment, the polynucleotide hybridized with the polynucleotide described above encodes a polypeptide that substantially remains identical to the cDNA of Figure 1 (SEQ ID NO: 1) or the deposited cDNA(S) The same biological function or activity of the encoded mature polypeptide.

In addition, the polynucleotide may have at least 20 bases, preferably at least 30 bases, more preferably at least 50 bases, which hybridize to the polynucleotide of the present invention and have the same identity as described above , may or may not retain activity. For example, such polynucleotides can be used as probes for the polynucleotide of SEQ ID NO: 1, for example for the recovery of polynucleotides or as diagnostic probes or as PCR primers.

Thus, the present invention relates to polynucleotides having at least 70% identity, preferably at least 90% identity and more preferably 95% identity to a polynucleotide encoding a polypeptide of SEQ ID NO: 2, and fragments thereof (such fragments have at least 20 or 30 bases, preferably at least 50 bases) and the polypeptides encoded by these polynucleotides.

The deposits referred to herein will be maintained in accordance with the provisions of the Budapest Treaty on the Internationally Recognized Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits are provided merely as a convenience to those skilled in the art and are not required deposits under 35 U.S.C. section 112. The polynucleotide sequences contained in said deposited materials and the amino acid sequences encoded thereby are incorporated herein by reference and are used to resolve any inconsistencies in the sequence descriptions herein. Any manufacture, use, or sale of the deposited materials is subject to license, and no such license is hereby granted.

The present invention also relates to a G protein-coupled receptor polypeptide having the deduced amino acid sequence (SEQ ID NO: 2) of Figure 1 or a G protein-coupled receptor polypeptide having an amino acid sequence encoded by a deposited cDNA and fragments thereof, similar substances and derivatives.

The terms "fragment", "derivative" and "analogue" when referring to the polypeptide of Figure 1 (SEQ ID NO: 2) or the polypeptide encoded by the deposited cDNA refer to a polypeptide that substantially retains the same biological function or activity as such polypeptide A polypeptide that functions as a G protein-coupled receptor, or retains the ability to bind to a receptor ligand even though the polypeptide does not have the function of a G protein-coupled receptor, such as a soluble form of the receptor. Analogs include proproteins that can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or synthetic polypeptides. Preferred are recombinant polypeptides.

Said fragment, derivative or analog of the polypeptide (SEQ ID NO: 2) of Figure 1 or the polypeptide encoded by the deposited cDNA may be: (i) one in which one or more amino acid residues are conserved or a non-conservative amino acid residue substitution (preferably a conservative amino acid residue substitution), and the substituted amino acid residue may or may not be an amino acid residue encoded by a selective codon, or (ii) one in which one or multiple amino acid residues comprising substituents, or (iii) one in which the mature polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide (e.g. polyethylene glycol), or (iv) One in which additional amino acids are fused to the mature polypeptide, which can be used to purify the mature polypeptide, or (v) one in which fragments of the polypeptide are soluble, i.e. non-membrane bound but still associated with membrane bound receptors Ligand binding. Such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art from the teachings herein.

The invention preferably provides polypeptides and polynucleotides in isolated form, and preferably the polypeptides and polynucleotides are purified to homogeneity.

The term "isolated" means that the material in question is removed from its original environment (eg, the natural environment, if it occurs naturally). 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. The polynucleotide may be part of a vector and/or the polynucleotide or polypeptide may be part of a composition and still be isolated in that such vector or composition is not part of its natural environment.

The polypeptides of the present invention include polypeptides of SEQ ID NO: 2 (especially mature polypeptides) and have at least 70% similarity (preferably 70% identity) with the polypeptide of SEQ ID NO: 2, more preferably 90% Similarity (preferably 90% identity), most preferably 95% similarity (preferably 95% identity) polypeptides, also includes portions of these polypeptides, such portions of polypeptides usually comprising at least 30 amino acids , and preferably at least 50 amino acids.

As is well known in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequences and conservative amino acid substitutions of one polypeptide to another.

Fragments or portions of polypeptides of the invention can be used by peptide synthesis to generate the corresponding full-length polypeptides; thus the fragments can be used as intermediates in the production of full-length polypeptides. Fragments or portions of polynucleotides of the invention can be used to synthesize full-length polynucleotides of the invention.

The present invention also relates to vectors containing the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors of the present invention, and methods for producing polypeptides of the present invention through recombinant techniques.

The host cell is produced by genetic engineering (transduction, transformation or transfection) with the vector of the present invention, and the vector can be a cloning vector or an expression vector. The vector may be in the form of, for example, a plasmid, a virus particle, and a bacteriophage. Engineered host cells can be cultured in modified conventional nutrient media suitable for activation of promoters, selection of transformants, or amplification of G protein-coupled receptor genes. Culture conditions, such as temperature and pH, etc., are those previously used for expression of the host cell of choice and will be apparent to those of ordinary skill.

The polynucleotides of the invention can be used to produce polypeptides via recombinant techniques. Thus, for example, polynucleotides may be contained in any of a variety of expression vectors for expressing polypeptides. Such vectors include chromosomal DNA sequences, non-chromosomal DNA sequences, synthetic DNA sequences such as SV 40 derivatives; bacterial plasmids; phage DNA; baculoviruses; yeast plasmids; vectors derived from combinations of plasmid and phage DNA, viral DNA such as vaccinia virus, adenovirus, fowl pox virus and pseudorabies virus. However, other vectors can also be used as long as they are replicable and stable in the host.

Inserting the appropriate DNA sequence into the vector can be done in a variety of ways. The DNA sequence is generally inserted into appropriate restriction endonuclease sites by methods known in the art. Such methods and others are considered to be within the knowledge of those skilled in the art.

The DNA sequence in the expression vector is operably linked to an appropriate expression control sequence (promoter) that directs mRNA synthesis. Representative examples of such promoters may be mentioned: the LTR or SV 40 promoter, the lac or trp of Escherichia coli, the bacteriophage lambda PL promoter, and viruses known to control gene expression in prokaryotic or eukaryotic cells or their other promoters. Expression vectors also contain ribosome binding sites for translation initiation and translation termination, which may also include appropriate sequences for amplified expression.

In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic characteristic for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance in eukaryotic cell culture, or Examples include tetracycline or ampicillin resistance in E. coli.

A vector comprising the appropriate DNA sequence described above together with an appropriate promoter or control sequence can be used to transform an appropriate host to enable expression of the protein.

As representative examples of suitable hosts there may be mentioned: bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium; fungal cells such as yeast cells; insect cells Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenovirus; plant cells, etc. Selection of an appropriate host is within the purview of those skilled in the art from the teachings herein.

More specifically, the invention also includes recombinant constructs comprising one or more of the sequences broadly described above. The construct comprises a vector, such as a plasmid or viral vector, into which the sequence of the invention has been inserted in forward or reverse direction. In a more desirable aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, promoters, operably linked to said sequences. Large numbers of suitable vectors and promoters are known to those skilled in the art and are commercially available. Examples include the following vectors: Bacterial vectors: pQE 70, pQE 60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX 174, pbluescriptSK, pbsks, pNH 8A, pNH 16a, pNH 18A, pNH46A (Stratagene), ptrc 99a , pKK 223-3, pKK233-3, pDR540, pRIT 5 (Pharmacia); eukaryotic vectors: pWLNEO, pSV2CAT, pOG 44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmids or vectors can be used as long as they are replicable and stable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two suitable vectors are pKK232-8 and pCM7. Bacterial promoters of particular mention include lacI, lacZ, T3, T7, gpt, _λPR , _PL , and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV 40, _LTRs from retroviruses and mouse metallothionein-I. Selection of appropriate vectors and promoters is within the level of ordinary skill in the art.

In another embodiment, the present invention relates to a host cell comprising the above construct. The host cell may be a higher eukaryotic cell (such as a mammalian cell), or a lower eukaryotic cell (such as a yeast cell), or the host cell may be a prokaryotic cell (bacterial cell). Constructs can be efficiently introduced into host cells by calcium phosphate transfection, DEAE-dextran-mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology , (1986)).

The construct in the host cell can be used to produce the gene product encoded by the recombinant sequence in a conventional manner. In addition, the polypeptides of the present invention can be synthesized by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria or other cells under the control of appropriate promoters. Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the invention. Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y., (1989) (incorporated herein by reference) describes suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts.

Transcription in higher eukaryotes of DNA encoding a polypeptide of the present invention is enhanced by an enhancer sequence inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, which act on the promoter to increase its transcription. Examples include the SV 40 enhancer on the late side of the replication origin 100-270 bp, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

In general, recombinant expression vectors include an origin of replication and selectable markers that allow transformation of host cells (such as the ampicillin resistance gene in Escherichia coli and the TRP 1 gene in S. Promoter. Such promoters may be from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), alpha-factor acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in an appropriate manner with translation initiation and termination sequences, preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium. The heterologous sequence may or may not encode a fusion protein including an N-terminal identifying peptide that confers desired characteristics, such as stabilization of expressed recombinant products or simplified purification steps.

Useful expression vectors for use in bacteria are constructed by inserting a structural DNA sequence encoding the desired protein and appropriate translation initiation and termination signals in operative reading together with a functional promoter. The vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector in the host and to provide amplification when required. Prokaryotic hosts suitable for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, Pseudomonas, Streptomyces, and Staphylococcus species (although others may alternatively be used).

As a representative and non-limiting example, useful expression vectors for use in bacteria may contain a selectable marker and a bacterial origin of replication derived from a commercially available plasmid comprising genetic elements of the well-known cloning vector pBR322 (ATCC37017). Such commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, WI, USA). These pBR 322 "backbone" fragments are combined with the appropriate promoter and structural sequence to be expressed.

After transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by a suitable method (eg temperature shift or chemical induction) and the cells are cultured for an additional period of time.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude product retained for further purification.

Microbial cells used for protein expression may be disrupted by any conventional method, including freeze-thaw cycles, sonication, mechanical disruption, or use of cell lysing agents, which are well known to those skilled in the art.

Various mammalian cell culture systems can also be used to express recombinant proteins. Examples of mammalian expression systems include the monkey kidney fibroblast COS-7 cell line described by Gluzman (Cell, 23:175 (1981)) and other cell lines capable of expressing compatible vectors such as C127, 3T3, CHO, Hela and BHK cell lines. Mammalian expression vectors contain an origin of replication, a suitable promoter and enhancer, and may also contain any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences and 5' flanking non-transcribed sequences. DNA sequences from the SV 40 splice and adenylation sites can be used to provide the desired non-transcribed genetic elements.

The G protein-coupled receptor polypeptides of the present invention can be recovered and purified from recombinant cell cultures by various methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, Hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be used, if desired, in completing the conformation of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used as a final purification step.

The polypeptides of the present invention may be natural purified products, or products of chemical synthesis methods, or produced from prokaryotic or eukaryotic hosts (such as bacteria, yeast, higher plants, cultured insect and mammalian cells) through recombinant techniques. Depending on the host used in the recombinant production method, the polypeptides of the invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine residue.

The G protein-coupled receptors of the invention are useful in methods of screening for compounds that activate (agonists) or inhibit (antagonists) the receptor polypeptides of the invention.

Generally, this screening step involves providing suitable cells which express the receptor polypeptide of the invention on their surface. Such cells include cells from mammals, yeast, Drosophila or E. coli. In particular, polynucleotides encoding receptors of the invention can be used to transfect cells to express G protein-coupled receptors. The expressed receptor is then contacted with a test compound to observe binding, whether to stimulate or inhibit a functional response.

One such screening step involves the use of melanocytes transfected to express a G protein-coupled receptor of the invention. This screening technique is described in PCT WO 92/01810 (published February 6, 1992).

Thus, for example, this method can be used to screen for compounds that inhibit the activation of receptor polypeptides of the invention by contacting receptor-encoding melanocytes with a receptor ligand and the compound to be screened. Inhibition of ligand-generated signaling indicates that the compound is a potential antagonist of the receptor, ie, inhibits its activation.

The screen can be used to identify compounds that activate the receptor by contacting the cells with the compound to be screened and testing whether the compound produces a signal (ie, activates the receptor).

Other screening techniques include the use of proteins expressing G protein-coupled receptors in a system for measuring changes in extracellular pH resulting from receptor activation, as described in Science, Vol. 246, pp. 181-296 (October 1989). Cells such as transfected CHO cells. For example, the compound is bound to a cell expressing a receptor polypeptide of the invention, and a second messenger response (eg, signal transduction or pH change) is assayed to detect whether the potential compound activates or inhibits the receptor.

Another screening technique involves introducing RNA encoding G protein-coupled receptors into Xenopus oocytes to transiently express the receptors. The recipient oocyte is then contacted with the receptor ligand and the compound to be screened, followed by detection of inhibition or activation of calcium signaling if the screening compound is believed to inhibit receptor activation.

Another screening technique involves expressing a G protein-coupled receptor wherein the receptor is linked to phospholipase C or D. As representative examples of such cells there may be mentioned endothelial cells, smooth muscle cells, embryonic kidney cells and the like. The above screening can be accomplished by detecting activation of the receptor or inhibition of activation of the receptor from a phospholipase secondary signal.

Another method involving screening for compounds that inhibit the activation of receptor polypeptides of the invention is by assaying for the inhibition of binding of labeled ligands to cells having the receptor on their surface. The method involves transfecting a eukaryotic cell with DNA encoding a G protein-coupled receptor such that the cell expresses the receptor on its surface, contacting the cell with a compound in the presence of a labeled form of a known ligand that can be detected by For example radiolabelling. The amount of labeled ligand bound to the receptor can be determined, for example, by measuring the radioactivity of the receptor. If a decrease in the labeled ligand binding to the receptor is detected, it indicates that the compound binds to the receptor, and the binding of the labeled ligand to the receptor is inhibited.

G protein-coupled receptors are ubiquitous in mammalian hosts and are responsible for many biological functions, including some pathological states. Accordingly, it is necessary to find compounds and drugs that can stimulate G protein-coupled receptors on the one hand and inhibit G protein-coupled receptors on the other hand.

For example, compounds that activate G protein-coupled receptors are useful for therapeutic purposes, such as the treatment of asthma, Parkinson's disease, acute heart failure, hypotension, urinary retention, and osteoporosis, among others.

In general, compounds that inhibit the activation of G protein-coupled receptors are useful for many therapeutic purposes, such as the treatment of hypertension, angina, myocardial infarction, ulcers, asthma, allergies, benign prostatic hyperplasia, psychiatric and nervous disorders (including schizophrenia) , manic irritation, depression, delirium, dementia, mental retardation) and movement disorders such as Huntington's disease or Gillesdila Tourett's syndrome. Compounds that inhibit G protein-coupled receptors are also useful in reversing endogenous anorexia and controlling hyperphagia.

An antibody may antagonize a G protein-coupled receptor of the invention, or in some cases an oligopeptide that binds a G protein-coupled receptor but does not elicit a second messenger response, thus allowing Prevents activation of G protein-coupled receptors. Antibodies include anti-idiotypic antibodies, which recognize a single determinant normally associated with the antigen-binding site of an antibody. Potential antagonist compounds also include proteins that are closely related to the ligand of the G protein-coupled receptor, ie, fragments of the ligand that have lost biological function and do not elicit a response when bound to the G protein-coupled receptor.

Antisense constructs prepared by antisense technology can control gene expression through triple helix formation or antisense DNA or RNA, both of which are based on the combination of polynucleotides and DNA or RNA. For example, the 5' coding portion of a polynucleotide sequence encoding a mature polypeptide of the invention can be used to design antisense RNA oligonucleotides about 10-40 base pairs in length. A DNA oligonucleotide is designed to be complementary to the region of the gene involved in transcription (triple helix - see Lee et al., Nucleic Acids Res., 6:3073 (1979); Cooney et al., Science, 241:456, (1988); and Dervan etc., Science, 251: B 60 (1991)), thereby preventing the transcription and production of G protein-coupled receptors. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block the translation of mRNA molecules into G protein-coupled receptors (antisense-Okano, J. Neurochem, 56:560 (1991); oligodeoxynucleotides as gene Expression of antisense inhibitors (CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells so that antisense RNA and DNA can be expressed in vivo to inhibit G protein-coupled receptors. body production.

The activation of the receptor polypeptides of the invention can also be inhibited by small molecules that bind to G protein coupled receptors and block their binding to ligands, thereby blocking normal biological activity.

Soluble forms of G protein-coupled receptors, such as fragments of the receptor, can be used to inhibit activation of the receptor and prevent interaction of the ligand with the membrane-bound G protein-coupled receptor by binding to the ligand of the polypeptide of the invention.

The present invention also provides a method for treating abnormal states associated with excess activity of G protein-coupled receptors, comprising administering the above-mentioned inhibitory compound to an individual together with a pharmaceutically acceptable carrier in an amount sufficient to block Binding of the body to G protein-coupled receptors or inhibiting activation by inhibiting second messengers abolishes the abnormal state.

The present invention also provides a method for treating the abnormal state associated with the overexpression of G protein-coupled receptor activity, comprising administering to the individual a therapeutically effective amount of the above-mentioned compound that activates the receptor polypeptide of the present invention and a pharmaceutically acceptable carrier, thereby eliminating the abnormal state.

Soluble forms of G protein-coupled receptors and compounds that activate or inhibit such receptors may be used in combination with suitable pharmaceutical carriers. Such compositions comprise a therapeutically effective amount of a polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Its formulation should suit the mode of administration.

The invention also provides pharmaceutical packs or kits comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Such containers may carry a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of drugs and biological products, which notice reflects the approval of the governmental agency for the manufacture, use, or sale of a product for human use. In addition, the polypeptides or compositions of the invention may be used in combination with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenient manner, eg, topically, intravenously, intraperitoneally, intramuscularly, subcutaneously, intranasally or intradermally. Said pharmaceutical composition is administered in an effective amount for treating and/or preventing a particular disease. Generally, they are administered in an amount of at least about 10 micrograms/kg body weight, and in most cases they are administered in amounts not to exceed about 8 mg/kg body weight per day. In most cases, the dose ranges from approximately 10 μg/kg to 1 mg/kg body weight per day, taking into account factors such as the route of administration and the condition.

G protein-coupled receptor polypeptides and activating or inhibiting compounds in the form of polypeptides can be utilized in accordance with the present invention by expressing such polypeptides in vivo, which is often referred to as "gene therapy".

Thus, for example, polynucleotides (DNA or RNA) encoding polypeptides can be genetically engineered in vitro on patient cells, and the engineered cells can provide said polypeptides to the treated patients. Such methods are well known in the art and will be apparent from the description herein. For example, cells can be engineered with retroviral particles comprising RNA encoding a polypeptide of the invention.

Likewise, cells can be engineered in vivo to express a polypeptide in vivo, eg, by methods known in the art. For example, producer cells producing retroviral particles containing RNA encoding a polypeptide of the invention can be administered to a patient in order to genetically engineer the cells in vivo and express the polypeptide in vivo. These and other methods of administering the polypeptides of the invention in this manner will be apparent to those skilled in the art from the description herein. For example, the expression vector used to engineer cells may not be a retrovirus, but such as an adenovirus, which can be used to engineer cells in vivo after being combined with a suitable delivery vector.

Retroviruses from which the retroviral plasmid vectors described above can be derived include, but are not limited to: Moloney murine leukemia virus, spleen necrosis virus, retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus Virus, Gibbon Orangutan Leukemia Virus, Human Immunodeficiency Virus, Adenovirus, Myeloproliferative Sarcoma Virus, and Mammary Tumor Virus. In one embodiment, the retroviral plasmid vector is from Moloney murine leukemia virus.

The vector contains one or more promoters. Suitable promoters that may be used include, but are not limited to: the retroviral LTR; the SV 40 promoter; and the human cytomegalovirus (CMV) promoter (Miller, et al., Biotechnology, Vol. 7, No. 9, 980 -990 (1989) description); or any other promoters (such as eukaryotic cell promoters, such as including but not limited to histone, pol III and β-actin promoters). Other viral promoters used include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. Selection of a suitable promoter is within the knowledge of those skilled in the art from the description herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters that can be used include, but are not limited to: adenoviral promoters (such as the adenovirus major late promoter); or heterologous promoters (such as the cytomegalovirus (CMV) promoter); respiratory syncytial virus (RSV ) promoters; inducible promoters (such as MMT promoter, metallothionein promoter); heat shock promoters; albumin promoters; ApoAI promoters; human globin promoters; viral thymidine kinase promoters (such as herpes simplex thymidine kinase promoter); retroviral LTRs (including the modified retroviral LTRs described above); the beta-actin promoter; and the human growth hormone promoter. The promoter may also be the native promoter controlling the gene encoding the polypeptide.

Retroviral plasmid vectors are used to transduce packaging cell lines to form production cell lines. Examples of packaging cells that can be transfected include, but are not limited to: PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψ-CRE, -ψ-CRIP, GP +E-86, GP+envAm12 and DAN cell lines (described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), the entire contents of which are incorporated herein by reference). Packaging cells can be transduced with the vector by any method known in the art. These methods include, but are not limited to: electroporation, use of liposomes, and _CaPO4 precipitation. Alternatively, retroviral plasmid vectors can be encapsulated in liposomes, or conjugated to lipids, and administered to the host.

The producer cell line produces infectious retroviral vector particles comprising the nucleic acid sequence encoding the polypeptide. These retroviral vector particles can then be used to transduce eukaryotic cells in vivo or in vitro. Transduced eukaryotic cells will express the nucleic acid sequence encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, and hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

The present invention also provides a method for detecting whether a ligand that is unknown to be able to bind to a G protein-coupled receptor can bind to the receptor, which comprises combining a mammalian cell expressing a G protein-coupled receptor with the ligand in a state where the ligand is allowed to bind. The body is contacted under the condition that the G protein-coupled receptor binds, and the presence of the receptor-bound ligand is detected to determine whether the ligand binds to the G protein-coupled receptor.

The present invention also provides a method of screening drugs to identify a drug that specifically interacts with and binds to a human G protein-coupled receptor on the surface of a cell, comprising sucking a mammalian drug comprising an isolated DNA molecule encoding a G protein-coupled receptor Animal cells are exposed to a series of drugs, and the drugs bound to the mammalian cells are determined, thereby identifying drugs that specifically interact with and bind to the human G protein-coupled receptors of the invention. This drug can then be used to therapeutically activate the receptors of the invention or to inhibit the activation of the receptors of the invention.

The present invention also provides a method for detecting the expression of G protein-coupled receptors on the cell surface by detecting the presence of mRNA encoding G protein-coupled receptors, which comprises obtaining total mRNA from cells, and combining the mRNA with Can contact with the nucleic acid probe of the present invention that specifically hybridizes under hybridization conditions with the sequence contained in the nucleic acid molecular sequence encoding human G protein-coupled receptors, and detect the hybridization of mRNA and the probe, thereby detecting G protein-coupled receptors expression by cells.

The invention also relates to the use of the G protein-coupled receptor gene as part of a diagnostic test to detect diseases or susceptibility to diseases associated with mutations present in the nucleic acid sequences encoding the receptor polypeptides of the invention, such as those associated with Related to cellular transformation, such as tumors and cancers.

Individuals with mutations in the human G protein-coupled receptor genes of the present invention can be detected at the DNA level using various techniques. Nucleic acids for diagnosis can be obtained from patient cells such as blood, urine, saliva, tissue biopsy and autopsy material. Genomic DNA can be used directly for detection, or it can be amplified enzymatically by PCR before analysis (Saiki et al., Nature, 324:163-166 (1986)). RNA or cDNA can also be used for the same purpose. As an example, PCR primers complementary to a nucleic acid encoding a G protein-coupled receptor can be used to identify and analyze mutations in the G protein-coupled receptor gene. For example, deletions or insertions can be detected by changes in the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridization of amplified DNA to radiolabeled GPCR RNA or radiolabeled GPCR antisense DNA sequences. Digestion with RNase A, or the difference in melting point temperature to distinguish between perfectly paired sequences and mismatched duplexes.

Sequence differences between the reference gene and the gene with the mutation are revealed by direct DNA sequencing methods. In addition, cloned DNA segments can be used as probes to detect specific DNA segments. When combined with PCR, the sensitivity of this method is greatly improved. For example, sequencing primers are used with double-stranded PCR products or single-stranded template molecules generated by modified PCR methods. Sequences are determined by conventional radiolabeled nucleotide methods or automated sequencing methods with fluorescent labels.

Genetic testing based on DNA sequence differences can be accomplished by detecting changes in the electrophoretic mobility of DNA fragments on gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high-resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels, in which the migration of different DNA fragments stops at different locations on the gel according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Sci. , 230:1242 (1985)).

Sequence changes at specific positions can also be revealed by nuclear protection assays, such as RNase and S1 protection, or by chemical cleavage methods (eg, Cotton et al., PNAS, USA, 85:4397-4401, 1985).

Thus, specific DNA sequences can be detected by methods such as hybridization, ribonuclease protection, chemical cleavage, direct DNA sequencing, or the use of restriction enzymes (e.g., restriction fragment length polymorphism (RFLP)) and analysis of genomic DNA. Southern blotting.

In addition to more routine gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

Additionally, certain diseases are caused by or are characterized by altered gene expression, which can be detected by changes in mRNA. In addition, the genes of the present invention can be used as a reference in identifying individuals with underexpressed functions associated with this type of receptor.

The invention also relates to diagnostic assays for detecting altered levels of soluble forms of receptor polypeptides of the invention in various tissues. Analytical methods for detecting soluble receptor polypeptide levels in host samples are well known to those skilled in the art and include: radioimmunoassays, competitive binding assays, Western blot analysis, preferably ELISA assays.

The ELISA assay initially involves the preparation of antibodies, preferably monoclonal antibodies, specific for the antigen of the receptor polypeptide. In addition, a reporter antibody for the monoclonal antibody is prepared. A detectable reagent, such as radioactive, fluorescent or in this example horseradish peroxidase, is conjugated to the reporter antibody. A sample is obtained from the host and incubated on a solid support such as a polystyrene dish that binds the proteins in the sample. Any free protein binding sites in the dish will be covered by incubation with a non-specific protein such as bovine serum albumin. The monoclonal antibody is then incubated in the dish, during which time the monoclonal antibody binds to any coupled receptor protein bound to the polystyrene dish. Wash away any unbound mAb with buffer. At this point, a reporter antibody conjugated to horseradish peroxidase is placed in the dish, resulting in binding of the reporter antibody to any monoclonal antibody bound to the coupled receptor protein. Unbound reporter antibody is then washed away. The peroxidase substrate is then added to the dish and the amount of color produced within a given time is the amount of coupled receptor protein present in a given volume of patient sample compared to a standard curve.

The sequences of the invention are also valuable for chromosome identification. The sequence specifically targets and hybridizes to a particular location on a single human chromosome. Furthermore, there is now a need to identify specific loci on chromosomes. Currently, there are only a few chromosomal labeling reagents based on actual sequence data (repeated polymorphisms) that can be used to mark chromosomal locations. The DNA chromosome mapping of the present invention is an important first step in correlating these sequences with disease-associated genes.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from cDNA. In silico analysis of cDNA allows rapid selection of primers that should not span more than one exon on the genomic DNA or complicate the amplification method. These primers were 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 produce amplified fragments.

PCR mapping of somatic cell hybrids is a rapid procedure for mapping a specific DNA to a specific chromosome. Using the same oligonucleotide primers according to the invention, sublocalization can be achieved in a similar manner with a set of fragments from a specific chromosome or from a collection of large genome clones. Other mapping strategies that can similarly be used to map chromosomes include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome-specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of cDNA clones and metaphase chromosome smears can be used to achieve accurate chromosome mapping in one step. The technique can be used with cDNAs as short as 50 or 60 bases in length. For a review of this technique see Verma et al., The Human Chromosome: A Handbook of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to an accurate chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data can be found, for example, in V. McKusick, Mendelian Inheritance in Man (available online through the Johns Hopkins University Welch Medical Library). Linkage analysis (co-inheritance of physically adjacent genes) is then used to identify relationships between genes and diseases that have mapped to the same chromosomal region.

Differences in cDNA or genome sequences between affected and unaffected individuals then need to be determined. If a mutation is observed in some or all affected individuals, but not in any normal individual, then the mutation may be the cause of the disease.

Based on the current resolution of physical and genetic mapping techniques, a cDNA pinpointed to a disease-associated chromosomal region could be one of 50-500 potentially causative genes (of which 1 is assumed to be causative). Megabase mapping resolution and every 20kb is a gene).

The polypeptide, its fragments or derivatives or analogs, or cells expressing the above substances can be used as immunogens for producing antibodies thereof. These antibodies may be, for example, polyclonal or monoclonal antibodies. The invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments or products of Fab expression libraries. Various methods known in the art can be used to produce these antibodies and fragments.

Antibodies raised against polypeptides corresponding to the sequences of the present invention can be obtained by directly injecting the polypeptides into animals or by administering the polypeptides to animals, wherein the animals are preferably non-human. The antibody thus obtained will then bind to the polypeptide. In this way, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies that bind the entire native polypeptide. The antibody can then be used to isolate the polypeptide from tissue expressing the polypeptide.

For the preparation of monoclonal antibodies, any technique for producing antibodies by continuous cell line culture can be used. Examples include hybridoma technology (Kohler and Milstein, 1975, Nature, 256:495-497), trisomy hybridoma technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immunology Today, 4:72) and production of human EBV-hybridoma technology for monoclonal antibodies (Cole, et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques for the production of single chain antibodies (US Patent 4,946,778) can be adapted to produce single chain antibodies directed against the immunogenic polypeptide products of the invention. Transgenic mice can also be used to express humanized antibodies raised against the immunogenic polypeptides of the invention.

The present invention will be further illustrated with reference to the following examples; however, it should be understood that the invention is not limited to these examples. All parts or amounts are by weight unless otherwise stated.

In order to facilitate the understanding of the following embodiments, some frequently occurring methods and/or terms are now described.

"Plasmids" are designated by a preceding lowercase p and/or followed by several uppercase letters and/or numbers. The starting plasmids herein are either commercially available or publicly available on an unrestricted basis, or can be constructed from available plasmids according to published methods. Furthermore, equivalent plasmids to those described are known in the art and will be apparent to those of ordinary skill in the art.

"Digestion" of DNA refers to the catalytic cleavage of DNA with a restriction enzyme that acts only on certain sequences on the DNA. A variety of restriction enzymes employed herein are commercially available, and the reaction conditions, cofactors, and other requirements for their use are known to those of ordinary skill in the art. For analytical purposes, typically 1 µg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 µl of buffer solution. To isolate DNA fragments for plasmid construction, 5 to 50 μg of DNA is usually digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffer solutions and amounts of substrate for a particular restriction enzyme are specified by the manufacturer. An incubation time of about 1 hour at 37 degrees Celsius is typically employed, but this time may vary according to the product supplier's instructions. After digestion, the reaction mixture was run directly on a polyacrylamide gel to isolate the desired fragments.

Size separation of cleaved fragments was performed using an 8% polyacrylamide gel as described by Goeddel, D. et al., Nucleic Acids Res. 8:4057 (1980).

"Oligonucleotide" refers to either a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which can be chemically synthesized. These synthetic oligonucleotides do not have a 5' phosphate, so unless a phosphate is added with ATP in the presence of a kinase, the oligonucleotide will not ligate to another oligonucleotide. Synthetic oligonucleotides will be ligated to the non-dephosphorylated fragments.

"Ligation" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments (Maniatis, T., et al., supra, p. 146). Unless otherwise provided, ligation was accomplished using known buffers and conditions with approximately equimolar amounts of 10 units of T4 DNA ligase ("ligase") per 0.5 μg of the DNA fragments to be ligated.

Unless otherwise stated, transformations were performed as described by Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

Example 1

  Bacterial expression and purification of G protein-coupled receptor (GPRC) polypeptides

Amplification of the GPRC-encoding DNA sequence (ATCC No. 97130) was initiated using PCR oligonucleotide primers corresponding to the 5' and 3' end sequences of the processed GPRC nucleotide sequence. Additional nucleotides corresponding to the GPRC nucleotide sequence were added to the 5' and 3' sequences, respectively. The 5' oligonucleotide primer has the sequence 5'CACAGGATCCC GTGGCTGCCATCTCTACTTC3 ' (SEQ ID NO: 3), which primer contains a BamHT restriction endonuclease site followed by a putative second The 17 nucleotides of the GPRC coding sequence starting with amino acids. The 3' sequence 5'TCTCAGGTACCGTTCTCTAAACCACAGAGTGGTCA (SEQ ID NO: 4) contains the sequence complementary to the ASP718 site, followed by 19 nucleotides of the GPRC coding sequence. The restriction enzyme sites correspond to the restriction enzyme sites of the bacterial expression vector pQE-31 (Qiagen, Chatsworth Corporation, CA, 91311). pQE-31 encodes antibiotic resistance (Ampr), bacterial origin of replication (ori), IPTG regulatable promoter operon (P/O), ribosome binding site (RBS), 6-histidine tag and restriction endonuclease Dicer site. pQE-31 was then digested with BamHT and ASP718. The amplified sequence was ligated into pQE-31 and inserted in frame with the sequence encoding the histidine tag and RBS. The ligation mixture was then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the method described by Sambrook et al. (A Laboratory Manual of Molecular Cloning, Cold Spring Harbor Laboratory Press, (1989)). M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor while conferring kanamycin resistance (Kanr). Transformants were identified by their ability to grow on LB plates, and ampicillin/kanamycin resistant colonies were screened. Plasmid DNA was isolated and confirmed by restriction analysis.

Colonies containing the desired construct were grown overnight (O/N) in LB broth supplemented with Amp (100 μg/ml) and Kan (25 μg/ml). O/N cultures were used to inoculate bulk cultures at a ratio of 1:100 to 1:250. When the cells had grown to an optical density 600 (OD ⁶⁰⁰ ) between 0.4 and 0.6, IPTG (isopropyl-BD-thiogalactopyranoside) was added to a final concentration of 1 mM. IPTG clears P/O by inactivating the lacI repressor, causing an increase in gene expression. Cells were incubated for an additional 3 to 4 hours. Cells were harvested by centrifugation. The cell pellet was dissolved in 6M guanidine hydrochloride chaotrope. After clarification, the solubilized GPRC was purified from solution by chromatography on a nickel-chelate column under conditions that allow tight binding of the protein containing the 6-histidine tag (Hochuli, E. et al., Journal of Chromatography 411 : 177-184 (1984)). The GPRC was eluted from the column with 6M guanidine hydrochloride (pH 5.0), and adjusted to 3 mM guanidine hydrochloride, 100 mM sodium phosphate, 10 mM glutathione (reduced), 2 mM glutathione (oxidized) for renaturation. Type). After 12 hours of incubation in solution, the protein was dialyzed against 10 mM sodium phosphate.

Example 2

    Expression of recombinant GPRC in COS7 cells

The expression plasmid GPRC HA is derived from the vector pcDNA3/Amp (Invitrogen), which contains: 1) SV 40 origin of replication, 2) ampicillin resistance gene, 3) Escherichia coli origin of replication, 4) CMV promoter, followed by a polylinker region, SV 40 intron and polyadenylation site. A DNA fragment encoding the complete GPRC precursor with an HA tag fused in frame to its 3' end was cloned into the polylinker region of the vector so that expression of the recombinant protein was under the control of the CMV promoter. The HA tag corresponds to an epitope from the influenza hemagglutinin protein, which has been described previously (I.Wilson, H.Niman, R.Heighten, A.Cherenson, M.Connolly, and R.Lerner, 1984, Cell 37 :767, (1984)). The fusion of HA to the target protein allows easy detection of the recombinant protein with antibodies recognizing HA epitopes.

The plasmid construction strategy is described as follows:

Two kinds of primers were used to construct the DNA sequence (ATCC No.97130) encoding GPRC by PCR method, said primers were: 5' primer 5'CAACCACAGGGATCCC ATG GCTGCCATCTCTACTTCCATCCCTGTA3' (SEQ ID NO: 5) contained a BamHI site (bold body), This is followed by 27 nucleotides of the GPRC coding sequence beginning with the start codon: 3' primer 5' CCCCTCGAGCTAAACCACAGAGTGGTCATTGCTGTGAACTCCAGCC 3' (SEQ ID NO: 6), which contains a site complementary to XhoI, a translation stop codon, an HA marker and Sequence of the last 24 nucleotides (excluding the stop codon) of the GPRC coding sequence. The PCR product thus contained a HindIII site, the GPRC coding sequence followed by an HA tag incorporated in frame, a translation stop codon after the HA tag and an XhoI site. The PCR-amplified DNA fragment and the vector pcDNA3/Amp were digested and ligated with HindIII and XhoI restriction enzymes. The ligation mixture was transformed into E. coli strain DH5α (, the transformed culture was plated on ampicillin medium plates, and resistant colonies were screened. Plasmid DNA was isolated from the transformants and checked for the presence of the correct fragment by restriction analysis. For To express recombinant GPRC, COS7 cells were transfected with expression vectors by the DEAE-DEXTRAN method (J.Sambrook, E.Fritsch, T.Maniatis, Molecular Cloning Experiment Manual, published by Cold Spring Harbor Laboratory, (1989)).By radiolabeling and immunization Precipitation method was used to detect the expression of GPRC HA protein (E.Harlow, D.Lane, Antibody Experiment Manual, published by Cold Spring Harbor Laboratory (1988). Cells were labeled with ¹⁵ S-cysteine for 8 hours two days after transfection The culture was then harvested and cells were lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984). Cell lysates and media were precipitated with HA-specific monoclonal antibodies. Protein precipitates were analyzed on 15% SDS-PAGE gels.

Example 3

  Cloning and expressing GPRC using baculovirus expression system

Utilize PCR oligonucleotide primers to amplify the DNA sequence (ATCC No.97130) that encodes the full-length GPRC protein, and said primers correspond to the 5' and 3' sequences of the gene:

5' Primer: 5'TTCACCACCTACCTGGATCCACAGAGCTGTC ATG GCTGCC 3' (SEQ ID NO: 7), containing a BamHI restriction endonuclease site (bold), followed by 11 nucleotides representing an efficient translation initiation signal in eukaryotic cells (J. Mol. Biol. 1987, 196, 947-950, Kozak, M), followed by the first 9 nucleotides of the GPRC gene (the translation start and stop codons ATG are underlined).

The 3' primer sequence is 5' CCTCATCTCA GGTACC GTTCTAAAACCAGAGTGG3' (SEQ ID NO: 8), which includes a restriction endonuclease ASP718 cleavage site, and 10 nucleotides complementary to the 3' untranslated sequence of the GPRC gene acid. The amplified sequence was separated from a 1% agarose gel using a commercially available kit ("Geneclean", BIO101 Company, La Jolla, Ca.). The fragment was digested with the endonuclease BamHI and purified again on a 1% agarose gel. This fragment is designated F2.

The GPRC protein was expressed by the baculovirus expression system using the vector pA2 (modified from the pVL941 vector, discussed below) (for review see: Summer, M.D. and Smith, G.E. 1987, Baculovirus Vectors and Methods Manual for Insect Cell Culture Procedures, Experiment Station Bulletin of Texas Agriculture 1555). This expression vector contains the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcMNPV), followed by a recognition site for the restriction endonuclease BamHI. The polyadenylation site of Simian Virus (SV) 40 is used for efficient polyadenylation. For easy selection of recombinant viruses, the E. coli β-galactosidase gene was inserted in the same orientation as the polyhedrin promoter, followed by the polyadenylation signal for the polyhedrin gene. The polyhedrin sequence is flanked by viral sequences for cell-mediated homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectors can be used in place of pA2, such as pAc373, pVL941, pRG1 and pAcIM1 (Luckow, V.A. and Summer, M.D., Virology, 170:31-39).

The plasmid was digested with restriction enzymes ASP718 and BamHT, and then dephosphorylated by calf intestinal phosphatase by methods known in the art. DNA was isolated from a 1% agarose gel using a commercially available kit ("Geneclean", BIO101 Company, La Jolla, Ca.) as described above. This vector DNA is called V2.

The F2 fragment and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. E. coli DH5α cells were then transformed and bacteria containing the plasmid (pBacGPRC) carrying the GPRC gene were identified using the enzyme BamHI. The sequence of the cloned fragment was confirmed by DNA sequencing.

5 μg of plasmid pBacGPRC was mixed with 1.0 μg of commercially available linear baculovirus ("BaculoGold ^™ baculovirus DNA", Pharmingen, San Diego, CA.) co-transfection.

1 μg of BaculoGold ^™ viral DNA and 5 μg of plasmid pBacGPRC were mixed in sterile wells of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies, Inc., Gaithersburg, MD). After adding 10 microliters of lipofection reagent and 90 microliters of Grace's medium, mix and incubate at room temperature for 15 minutes. The transfection mixture was then added dropwise to Sf9 insect cells (ATCC CRL 1711 ) plated on 35 mm tissue culture plates with 1 ml of serum-free Grace's medium. Shake the plate back and forth to mix the newly added solution. Plates were then incubated at 27°C for 5 hours. After 5 hours, the transfection solution was removed from the plate and 1 microliter of Grace's insect medium supplemented with 10% fetal bovine serum was added. The plate was returned to the incubator and incubated at 27°C for 4 consecutive days.

After 4 days, supernatants were collected and subjected to plaque assays similar to that described by Summers and Smith (supra). A variation is the use of agarose gel with "Blue Gal" (Life Technologies, Gaithersburg), which allows easy separation of blue-stained plaques. (A thorough description of the "plaque assay" can also be found on pages 9-10 of the Insect Cell Culture and Baculovirology User's Guide published by Life Technologies, Inc. (Gaithersburg).

Four days later, serially diluted virus was added to the cells and blue-stained plaques were picked with an Eppendorf pipette tip. The agar containing the recombinant virus was then suspended in an Eppendorf tube containing 200 microliters of Grace's medium. The agar was removed by brief centrifugation, and the supernatant containing the recombinant baculovirus was used to infect Sf9 cells seeded in 35 mm dishes. After 4 days, the supernatants from these dishes were harvested and stored at 4°C.

Sf9 cells were grown in Grace' medium supplemented with 10% heat-inactivated FBS. Cells were infected with recombinant baculovirus V-GPRC at a multiplicity of infection (MOI) of 2. After 6 hours, the medium was removed and replaced with SF900II medium (minus methionine and cysteine) (Life Technologies, Gaithersburg). After 42 hours, 5 μCi ³⁵ S methionine and 5 μCi ³⁵ S cysteine (Amersham) were added. The cells were further cultured for 72 hours, the cells were collected, and then the cells were lysed in hypotonic phosphate buffer, the cell membranes were collected by centrifugation, and the labeled proteins were displayed by SDS-PAGE and autoradiography.

Example 4

Expression via gene therapy

Fibroblasts were obtained from a study subject by skin biopsy. The resulting tissue was placed on tissue culture medium and dissected into small pieces. Place small tissue pieces on the wet surface of tissue culture flasks, with approximately 10 pieces of tissue in each bottle. The bottle was placed upside down, capped tightly and left overnight at room temperature. Place at room temperature for 24 hours and then invert the bottle, the tissue pieces are still fixed at the bottom of the bottle, add fresh medium (such as Ham's F12 medium containing 10% FBS, penicillin and streptomycin), and then incubate at 37°C Incubate for approximately 1 week. Fresh medium was added at this point, followed by medium changes every few days. After an additional 2 weeks of culture, a monolayer of fibroblasts appeared. Cell monolayers were trypsinized and placed in larger flasks.

pMV-7 flanked by Moloney murine sarcoma virus long terminal repeats (Kirschmeier, P.T. et al., DNA, 7:219-25 (1988)) was digested with EcoRI and Hind III, followed by treatment with calf intestinal phosphatase. Linear vectors were fractionated on agarose gels and purified using glass beads.

The cDNA encoding the polypeptide of the present invention is amplified using PCR primers corresponding to the 5' and 3' terminal sequences, respectively. The 5' primer contains an EcoRI site and the 3' primer contains a Hind III site. Equal amounts of the linearized backbone of Moloney murine sarcoma virus were added to the amplified EcoRI and Hind III fragments in the presence of T4 DNA ligase, and the resulting mixture was maintained under conditions suitable for ligation of the two fragments. The ligation mixture was used to transform bacteria HB101, which were then plated on agar plates containing kanamycin in order to confirm that the vector had inserted the correct gene of interest.

Amphotropic (amphotropic) pA317 or GP+am12 packaging cells were cultured on tissue culture plates in Dulbecco's Modified Eagles Medium (DMEM) containing 10% calf serum (CS), penicillin, and streptomycin until to achieve a paved density. A vector containing the gene is then added to the culture medium and packaging cells are transduced with the vector. The packaging cells then produce infectious virus particles containing the gene (packaging cells are now called producer cells).

Fresh medium was added to the transduced producer cells, followed by harvesting the medium from a 10 cm plate confluent with the producer cells. The medium containing infectious viral particles is filtered through a millipore filter to remove detached producer cells, and then used to infect fibroblasts. Medium was removed from sub-confluent plates of fibroblasts and quickly replaced with medium from producer cells. The medium was removed and replaced with fresh medium. If the titer of virus is high, then virtually all fibroblasts are infected and no selection is required. If the titer is very low, retroviruses with selectable markers such as neo or his may be needed.

The engineered fibroblasts are then injected into the host, either alone or after they have been grown to confluence on a cytodex3 microcarrier bead. At this point the fibroblasts produce protein products.

Many modifications and variations of the present invention are possible in light of the above teaching, so that the invention may be otherwise embodied within the scope of the appended claims.

Sequence Listing

(1) General information:

(i) Applicant: Li Yi et al.

(ii) Invention name: human G protein-coupled receptor

(iii) Number of sequences: 8

(iv) Contact address:

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

         CECCHI, STEWART & OLSTEIN

(B) Street: 6 BECKER FARM ROAD

(C) City: ROSELAND

(D) State: New Jersey

(E) Country: United States

(F) Zip code: 07068

(v) In computer readable form:

(A) Media type: 3.5-inch floppy disk

(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:

(C) Classification:

(vii) Prior application data:

(A) Application number:

(B) Filing date:

(viii) Lawyer/Representative Information:

(A) Name: FERRARO, GREGORY D.

(B) Registration No.: 36,134

(C) Case number/document number: 325800-358

(ix) Telecommunications information:

(A) Tel: 201-994-1700

(B) Fax: 201-994-1744

(2) SEQ ID NO: 1 information:

(i) Sequence features:

(A) Length: 2456 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 1:

GGAACCGCCC CACCGTGGTG GCGGCCGCCC AGAACTAGTG GATCCCCCGG GCTGCAGGAA 60

TTCGGCACGA GCAGACACAC TTGCTTTGGT TTACAGATCC AGTGAAGTGA AAAATCAGAA 120

CTAGAAACGT ATGCACCTTC CTAGCAGCAA AGCCGCTTCT GCGTTCTTCG CAGCCTCCAG 180

TGCAGGGCGG CGCTGGGAGA AACTTTGCGC CTTCTGGAAA GTTTAGAAAG TGAGCCACGA 240

AAGAGAGGCC ACATTTCCGG GGTTTTGCGG GCCCCGCGAT GTTTTCCAGA GCTTTTCGAG 300

TGGGAAGAGG AGAGCGACAA CGTGAAAATG CCCCGTGCCG GGGCGTCCAC CGGAGTCCTG 360

CCAGCTGTCC GGCGCTGGGG TGGACGTCTG ATTTATGAAG CTCCCCATCC ACCTATCTGA 420

GTACCTGACT TCTCAGGACT GACACCTACA GCATCAGGTA CACAGCTTCT CCTAGCATGA 480

CTTCGATCTG ATCAGCAAAC AAGAAAATTT GTCTCCCGTA GTTCTGGGGC GTGTTCACCA 540

CCTACAACCA CAGAGCTGTC ATGGCTGCCA TTCCTACTTC CATCCCTGTA ATTTCACAGC 600

CCCAGTTCAC AGCCATGAAT GAACCACAGT GCTTCTACAAA CGAGTCCATT GCCTTCTTTT 660

ATAACCGAAG TGGAAAGCAT CTTGCCACAG AATGGAACAC AGTCAGCAAG CTGGTGATGG 720

GACTTGGAAT CACTGTTTGT ATCTTCATCA TGTTGGCCAA CCTATTGGTC ATGGTGGCAA 780

TCTATGTCAA CCGCCGCTTC CATTTTCCTA TTTATTACCT AATGGCTAAT CTGGCTGCTG 840

CAGACTTCTT TGCTGGGTTG GCCTACTTCT ATCTCATGTT CAACACAGGA CCCAATACTC 900

GGAGACTGAC TGTTAGCACA TGGCTCCTTC GTCAGGGCCT CATTGACACC AGCCTGACGG 960

CATCTGTGGC CAACTTACTG GCTATTGCAA TCGAGAGGCA CATTACGGTT TTCCGCATGC 1020

AGCTCCACAC ACGGATGAGC AACCGGCGGG TAGTGGTGGT CATTGTGGTC ATCTGGACTA 1080

TGGCCATCGT TATGGGTGCT ATACCCAGTG TGGGCTGGAA CTGTATCTGT GATATTGAAA 1140

ATTGTTCCAA CATGGCACCC CTCTACAGTG ACTCTTACTT AGTCTTCTGG GCCATTTTCA 1200

ACTTGGTGAC CTTTGTGGTA ATGGTGGTTC TCTATGCTCA CATCTTTGGC TATGTTCGCC 1260

AGAGGACTAT GAGAATGTCT CGGCATAGTT CTGGACCCCG GCGGAATCGG GATACCATGA 1320

TGAGTCTTCT GAAGACTGTG GTCATTGTGC TTGGGGCCTT TATCATCTGC TGGACTCCTG 1380

GATTGGTTTT GTTACTTCTA GACGTGTGCT GTCCACAGTG CGACGTGCTG GCCTATGAGA 1440

AATTCTTCCT TCTCCTTGCT GAATTCAACT CTGCCATGAA CCCCATCATT TACTCCTACC 1500

GCGACAAAGA AATGAGCGCC ACCTTTAGGC AGATCCTCTG CTGCCAGCGC AGTGAGAACC 1560

CCACCGGCCC CACAGAAGGC TCAGACCGCT CGGCTTCCTC CCTCAACCAC ACCATCTTGG 1620

CTGGAGTTCA CAGCAATGAC CACTCTGTGG TTTAGAACGG AAACTGAGAT GAGGAACCAG 1680

CCGTCCTCTC TTGTAGGATA AACAGCCTCC CCCTACCCAA TTGCCAGGGC AAGGTGGGGT 1740

GTGAGAGAGG AGAAAAGTCA ACTCATGTAC TTAAACACTA ACCAATGACA GTATTTGTTC 1800

CTGGACCCCCA CAAGACTTGA TATATATTGA AAATTAGCTT ATGTGACAAC CCTCATCTTG 1860

ATCCCCATCC CTTCTGAAAG TAGGAAGTTG GAGCTCTTGC AATGGAATTC AAGAACAGAC 1920

TCTGGAGTGT CCATTTAGAC TACACTAACT AGACTTTTAA AAGATTGTGT GTGGTTTGGT 1980

GCAAGTCAGA ATAAATTCTG GCTAGTTGAA TCCACAACTT CATTTATATA CAGGCTTCCC 2040

TTTTTTATTT TTAAAGGATA CGTTTCACTT AATAAACACG TTTATGCCTA TCAGCATGTT 2100

TGTGATGGAT GAGACTATGG ACTGCTTTTA AACTACCATA ATTCCATTTT TTCCCTTACA 2160

TAGGAAAACT GTAAGTTGGA ATTATCTTTT GGTTAGAAAG CATGCATGTA ATGTATGTAT 2220

GCAGCATGCC TTACTTAAAA AGATTAAAAG GATACTAATG TTAAATCTTC TAGGAAATAG 2280

AACCTAGACT TCAAAGCCAG TATTTGTTTA GGTCATGAAG CAAACAATGC TCTAATCACA 2340

ATATTAACTG TTTAATTAAA ATGTTGTAAC AAGTATAAAA CAGGGAATGT AAGTTTTATTA 2400

CCAAAGTGAT ATGTATTCCA AAAAAGGTCA TAGAAGATGA AGCAACTATA ATATTG 2456

(2) SEQ ID NO: 2 information:

(i) Sequence features:

(A) Length: 364 amino acids

(B) type: amino acid

(C) chain type:

(D) Topology: Linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 2:

Met Ala Ala Ile Ser Thr Ser Ile Pro Val Ile Ser Gln Pro Gln

5 10 15

Phe Thr Ala Met Asn Glu Pro Gln Cys Phe Tyr Asn Glu Ser Ile

20 25 30

Ala Phe Phe Tyr Asn Arg Ser Gly Lys His Leu Ala Thr Glu Trp

35 40 45

Asn Thr Val Ser Lys Leu Val Met Gly Leu Gly Ile Thr Val Cys

50 55 60

Ile Phe Ile Met Leu Ala Asn Leu Leu Val Met Val Ala Ile Tyr

65 70 75

Val Asn Arg Arg Phe His Phe Pro Ile Tyr Tyr Leu Met Ala Asn

80 85 90

Leu Ala Ala Ala Asp Phe Phe Ala Gly Leu Ala Tyr Phe Tyr Leu

95 100 105

Met Phe Asn Thr Gly Pro Asn Thr Arg Arg Leu Thr Val Ser Thr

110 115 120

Trp Leu Leu Arg Gln Gly Leu Ile Asp Thr Ser Leu Thr Ala Ser

125 130 135

Val Ala Asn Leu Leu Ala Ile Ala Ile Glu Arg His Ile Thr Val

140 145 150

Phe Arg Met Gln Leu His Thr Arg Met Ser Asn Arg Arg Val Val

155 160 165

Val Val Ile Val Val Ile Trp Thr Met Ala Ile Val Met Gly Ala

170 175 180

Ile Pro Ser Val Gly Trp Asn Cys Ile Cys Asp Ile Glu Asn Cys

185 190 195

Ser Asn Met Ala Pro Leu Tyr Ser Asp Ser Tyr Leu Val Phe Trp

200 205 210

Ala Ile Phe Asn Leu Val Thr Phe Val Val Met Val Val Leu Tyr

215 220 225

Ala His Ile Phe Gly Tyr Val Arg Gln Arg Thr Met Arg Met Ser

230 235 240

Arg His Ser Ser Gly Pro Arg Arg Asn Arg Asp Thr Met Met Ser

245 250 255

Leu Leu Lys Thr Val Val Ile Val Leu Gly Ala Phe Ile Ile Cys

260 265 270

Trp Thr Pro Gly Leu Val Leu Leu Leu Leu Asp Val Cys Cys Pro

275 280 285

Gln Cys Asp Val Leu Ala Tyr Glu Lys Phe Phe Leu Leu Leu Ala

290 295 300

Glu Phe Asn Ser Ala Met Asn Pro Ile Ile Tyr Ser Tyr Arg Asp

305 310 315

Lys Glu Met Ser Ala Thr Phe Arg Gln Ile Leu Cys Cys Gln Arg

320 325 330

Ser Glu Asn Pro Thr Gly Pro Thr Glu Gly Ser Asp Arg Ser Ala

335 340 345

Ser Ser Leu Asn His Thr Ile Leu Ala Gly Val His Ser Asn Asp

350 355 360

His Ser Val Val

(2) SEQ ID NO: 3 information:

(i) Sequence features:

(A) Length: base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 3:

(2) SEQ ID NO: 4 information:

(i) Sequence features:

(A) Length: base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 4:

(2) SEQ ID NO: 5 information:

(i) Sequence features:

(A) Length: 46 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 5:

CAACCACAGG GATCCCATGG CTGCCATCTC TACTTCCATC CCTGTA 46

(2) SEQ ID NO: 6 information:

(i) Sequence features:

(A) Length: 46 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 6:

CCCCTCGAGC TAAACCACAG AGTGGTCATT GCTGTGAACT CCAGCC 46

(2) SEQ ID NO: 7 information:

(i) Sequence features:

(A) Length: 40 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 7:

TTCACCACCT ACCTGGATCC ACAGAGCTGT CATGGCTGCC 40

(2) SEQ ID NO: 8 information:

(i) Sequence features:

(A) Length: 35 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(xi) Sequence description: SEQ ID NO: 8:

CCTCATCTCA GGTACCGTTC TAAACCACAG AGTGG 35

Claims (13)

1. An isolated polynucleotide comprising a member selected from the group consisting of: (a) a polynucleotide encoding the polypeptide shown in SEQ ID NO: 2; (b) a polynucleotide encoding a polypeptide expressed from the DNA contained in ATCC Accession No. 97130; (c) a polynucleotide capable of hybridizing under stringent conditions to and having at least 70% identity to the polynucleotide of (a) or (b); (d) A fragment of the polynucleotide of (a) or (b) or (c). 2. The polynucleotide of claim 1, encoding a polypeptide comprising amino acids 1 to 364 shown in SEQ ID NO:2. 3. A vector comprising the polynucleotide of claim 1. 4. A host cell genetically engineered with the vector of claim 3. 5. A method for producing a polypeptide, comprising: expressing the polypeptide encoded by the polynucleotide of claim 1 in the host cell of claim 4. 6. A method of producing cells capable of expressing a polypeptide comprising genetically engineering the cells with the vector of claim 3. 7. A polypeptide comprising a member selected from the group consisting of: (i) a polypeptide having a deduced amino acid sequence shown in SEQ ID NO: 2 and fragments thereof, analogs and derivatives thereof, said fragments, analogs and derivatives having the activity of the polypeptide of SEQ ID NO: 2; (ii) polypeptides encoded by the cDNA of ATCC deposit number 97130 and fragments, analogs and derivatives thereof, said fragments, analogs and derivatives having the activity of the polypeptide of SEQ ID NO:2. 8. The polypeptide of claim 7, wherein the polypeptide has the deduced amino acid sequence of SEQ ID NO:2. 9. An antibody to the polypeptide of claim 7. 10. A method of identifying a compound that binds to and activates the polypeptide of claim 7, comprising: contacting a cell expressing a polypeptide of claim 7 on its surface with a compound under conditions sufficient to bind the compound to the polypeptide, said polypeptide and a second component capable of providing a detectable signal responsive to binding of the compound to said polypeptide associated; Compounds capable of binding to the polypeptide are identified by detecting the presence of a signal produced by said second component. 11. A method of identifying a compound that binds to the polypeptide of claim 7 and inhibits activation of the polypeptide, comprising: Contacting an analytically detectable ligand known to bind to the receptor polypeptide of claim 7 and a compound with a cell expressing the polypeptide of claim 7 on its surface under conditions that allow binding to the polypeptide that provides the response compound is associated with the second component of the detectable signal of binding of the polypeptide; Whether the ligand is bound to the polypeptide is determined by detecting the absence of a signal resulting from the interaction of the ligand and the polypeptide. 12. An in vitro method for diagnosing a patient with a disease or susceptibility to disease associated with low expression of the polypeptide of claim 7, comprising: detecting a mutation in a nucleic acid sequence encoding said polypeptide in a sample from the patient. 13. An in vitro diagnostic method comprising: analyzing a sample derived from a host for the presence or absence of the polypeptide of claim 7.

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