MXPA97009746A - Homologo de receptor de tromb - Google Patents

Abstract

The present invention provides nucleotide and amino acid sequences that identify and encode a novel thrombin receptor homologue (TRH) expressed in the human liver. The present invention also provides antisense molecules to the nucleotide sequences, which encode TRH, diagnostic tests based on RTH-encoding nucleic acid molecules, expression drivers for the production of purified TRH, antibodies capable of binding to TRH, hybridization probes, or nucleotides for the detection of nucleotide sequences encoding TRH, genetically engineered host cells for the expression of TRH, and antagonists, antibodies and inhibitors, which bind to the T polypeptide

Description

HOMOLOGO DE RECEPTOR DE TROMBINA FIELD OF THE INVENTION The present invention is in the field of molecular biology; more particularly, the present invention describes the nucleic acid and amino acid sequences of a thrombin receptor homologue.

BACKGROUND OF THE INVENTION The receptor is a G protein coupled to the transmembrane-seven receptor (T7G), which is present in platelets, endothelial cells, fibroblasts, mesangial cells, neural cells, and smooth muscle cells. This receptor is activated by the irreversible splitting of the extracellular, amino terminal sequence between arg41 and ser42 through thrombin or another serine protease. In normal endothelial cells, the activation of the thrombin receptor stimulates the metabolism of phosphoinositide mediated by the Gq protein, intracellular, and inhibition of adenylate cyclase mediated by the Gi protein. In addition, endothelial cells secrete endothelin-derived relaxation factor (EDRF). Subsequently, EDRF stimulates the release of soluble guanylate cyclase followed by cyclic GMP formation and smooth muscle relaxation. Another function of EDRF is the inhibition of platelet adhesion and aggregation, which is beneficial for blood flow. Wilcox et al (1994, Circ Res. 75: 1029-38) reported that in the smooth muscle of the rat aorta, the expression of the thrombin receptor increases immediately after vascular damage and activates the proliferation of cells in the neointima . Blocking the site of cleavage of the thrombin receptor with the antibody stops cell proliferation induced by thrombin. After cleavage and activation, the thrombin receptors were desensitized, and internalized for recirculation. Recirculation of the receiver and replacement limit the duration of cases initiated by thrombin. The thrombin receptor has at least four N-glycosylation sites 35 62 259 Asn Asn Asn. 75 and Asn The presence of carbohydrate greatly affects the differences in predicted size and mass reported for thrombin receptors. In addition, glycosylation can represent as much as 30% of the mass of these receptors and apparently determines their distribution (Brass LF et al. (1992) J. Biol. Chem. 267: 13795-8). The thrombin receptor is classified with the receptors of T7G without neuquinin, which includes many glycoprotein receptors such as those for luteinizing hormone (LH) and follicle stimulating hormone (FSH). These have very long N-terms, bind to a structural motif of common ligand with low affinity to activate the receptor, and are based on the N term and extracellular loops to impart high affinity and specificity (Bolander FF (1994) Molecular Endrocrinology, Academic Press, San Diego, CA). They refer to the other T7Gs by their seven hydrophobic domains, which extend the plasma membrane and form a bundle of antiparallel helices. These transmembrane segments (TMS) are designated with Roman numerals l-VII and represent structural and functional aspects of the receiver. In most cases, the beam forms a joint cavity; however, when the binding site must adapt to larger molecules, the extracellular N-terminal segment or one or more of the three extracellular loops participate in the binding (Watson S and Arkinstall S (1994) The G-Protein Linked Receptor Facts Book, Academic Press, San Diego, CA) and in the subsequent induction of conformational change in intracellular portions of the receptor. The activated receptor, in turn, interacts with an intracellular protein G complex, which mediates additional intracellular signaling activities, generally the production of second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate or proteins of ion channel. Thrombin receptor homologs are expressed in tissues such as the liver, which is briefly described below. The basic functional unit of the human liver is the lobe, and each lobe consists of liver cell plates, which enclose a central vein. The small bile canaliculi divide the plates and empty into ducts found in septa between the lobes. The hepatic sinusoids associated with the septa are covered with endothelial and Kupffer cells. Bacteria and organic matter are removed from portal blood through Kupffer cells, which also express high levels of major histocompatibility complex proteins of class II and release nitric oxide, interleukin (IL) -1, IL -6 and TNF. Although they do not normally produce erythrocytes or divide into adults, the liver cells are basically embryonic. If a portion of the liver is destroyed or removed, the remaining parenchyma can regenerate the entire organ. Liver cells are characterized by large amounts of endoplasmic reticulum, which consists of metabolic, storage, endo- and exo-cytotic activities. The liver has three main functions: 1) vascular, for the storage and filtration of blood; 2) metabolic, for glucose regulation and storage of glycogen and fat; and 3) secretory / excretory, for the regulation of hormones and degradation of toxic substances. The normal blood volume of the liver is 450 ml, and an average flow of blood through the liver is 1450 ml / min. The liver can store an extra 500-1000 ml of blood or supply extra blood after trauma. Half of the lymph produced in the body comes from the liver via drainage through the Disse space (located below the liver cells).

High blood pressure within the liver is an important factor in edema or ascites. Liver cells have a very high rate of metabolism; and process, synthesize and degrade carbohydrates, fats and proteins, simultaneously. The mechanism of carbohydrate consists of the storage of glycogen, conversion of fructose and galactose to glucose, and gluconeogenesis. The liver regulates the amount of glucose in the blood in response to hormones. The cells of the liver have functions to metabolize fat, the oxidation of fatty acids and the production of lipoproteins, phospholipids and cholesterol. Liproteins transport phospholipids and cholesterol to other areas, where they are used to form cell membranes and other important substances in cell function. Most of the conversion of carbohydrates and proteins to fat occurs in the liver, although storage can be in adipose tissue, anywhere in the body. The liver also stores the fat-soluble vitamins and iron. The liver forms 90% of the proteins in the plasma, particularly albumin and several globulins, at a rate of 15-50 g / day. The liver also produces haptoglobin, ceruloplasmin and transferrin in response to cytokines. It manufactures non-essential amino acids; and in the presence of devitamin K, it synthesizes the accelerating globulin, erythropoietin, and the coagulation factors I, II, V, VII, and X. The lipid-soluble drugs are usually detoxified in the liver. The reactions of phase I involve the enzymatic modification of relative groups in drug molecules through oxidation, reduction, hydroxylation, sulfoxidation, deamination, dealkylation or methylation. These modifications deactivate barbiturates and benzodiazepines and activate cortisone or prednisone. The enzymes responsible for the phase I reactions are induced by ethanol and barbiturates and inhibited by chloramphenicol, cimetidine and ethanol. Phase II reactions involve the enzymatic conversion of substances to their acid or salt derivatives, for example, glucuronide, glycine or sulfate. When the liver is damaged, it reduces its processing of anticonvulsants (phenobarbital), anti-inflammatory agents (acetaminophen or glucocorticoids), tranquillizers (lidocaine, proprandol) or antibiotics (chloramphenicol, tetracycline or rifampin). In addition, the liver inactivates endogenous hormones through deamination, proteolysis or deiodination. Glucocorticoids and aldosterone are reduced to their tetrahydro derivatives and conjugated to glucuronic acid. Testosterone and estrogen are converted to ketosteroids and conjugated with sulfates or glucuronic acid. Abnormal physiological or pathological conditions of the liver include fatty liver, which is the excessive accumulation of lipids in hepatocytes in response to damage; jaundice, where large amounts of bilirubin are present in the extracellular fluid; hepatitis, which are viral infections caused by hepadnaviruses, but whose main pathology results from the host immune response; cirrhosis, which is caused by irreversible chronic damage of the hepatic parenchyma and progress to fibrosis; infiltrative diseases, such as granulomas and amylodosis; and adenomas and carcinomas. Doctors are currently evaluating bilirubin and urobilinogen levels to diagnose hemolytic liver disease and analyze the level of carcinoembryonic antigen to diagnose metastatic cancer. Depending on its functions, an assay for the thrombin receptor homolog can be a diagnostic tool for excessive fibrosis and results in liver damage. If the thrombin receptor homolog is expressed in response to mechanical or chemical damage, or is involved in unnecessary coagulation reactions or progressive fibrosis, then it may also represent an accessible therapeutic target for the control of inflammatory procedures in the liver. The anatomy, physiology and diseases of the liver are reviewed, among others, in Guyton, AC (1991) Textbook of Medical Physiology, WB Saunders Co., Philadelphia PA; Isselbacher, KJ et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York; and The Merck Manual of Diagnosis and Therapy (1992) Meck Researh Laboratories, Rahway NJ. The identification of thrombin receptor homologs described in this application provides the opportunity to diagnose or intervene in those cases of signal transduction associated with inflammation or disease, wherein said transmembrane-seven receptors are expressed or otherwise actively involved.

DESCRIPTION OF THE INVENTION The subject of the invention provides a unique nucleotide sequence, which encodes a novel human thrombin receptor homolog (TRH). The cDNA, here designated trh, was identified and cloned using Clone No. 86700 from a liver cDNA library. The invention also relates to the use of the nucleotide and amino acid sequences of TRH, or its variants, in the diagnosis and treatment of activated or inflamed cells and / or ejidos associated with their expression. Aspects of the invention include the antisense DNA of trh; cloning or expression vectors containing trh; host cells or organisms transformed with expression vectors containing trh; a method for the production and recovery of purified TRH from host cells; and purified protein, TRH, which can be used to identify antagonists, antibodies or inhibitors of the receptor for therapeutic use.

DESCRIPTION OF THE DRAWINGS Figures 1A and 1B show the nucleotide (SEQ ID NO: 1) and amino acid alignments (SEQ ID NO: 2) of the consensus sequence for TRH. The oligomers used to extend the nucleotide sequence to full length are XLR = TGCCTTCCGTTGCTGTATAGACCG and XLF = AAGGAGGGCATAATTCCA CAATGTG. Figure 2 presents the alignment of human TRH with a human thrombin receptor, HUMTHRR; the waste in the box is identical.

MODES FOR CARRYING OUT THE INVENTION As used herein, "TRH" in upper case, refers to a thrombin receptor homologue, whether natural or synthetic and active fragments thereof, which have the amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the TRH polypeptide is encoded by mRNA transcripts of the cDNA (trh in lower case) of SEQ ID NO: 1. The novel thrombin receptor homologue (trh), which is the subject of the present invention, was identified among the partial cDNAs derived from a liver collection. The clone Incyte 86700 is very similar to HUMTHRR, the human thrombin receptor (Dennington PM and MC Berndt (1994) Clin. Exp. Pharmacol. Physiol 21: 349-58). In addition, Incyte 86700 exhibits an amino acid sequence similarity to the platelet activating factor, residues 94-155. These residues cover most of TMS III, the second intracellular loop, and TMS IV of transmembrane-seven receptors. "Active" refers to those forms of, which retain the biological and / or immunological activities of any TRH of natural existence. "Naturally occurring TRH" refers to TRHs produced by human cells that have not been genetically engineered and specifically contemplates several TRHs that arise from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. "Derivative" refers to chemically modified TRHs through any techniques such as ubiquitination, labeling (eg, radionuclides, various enzymes, etc.), pegylation (derivatization with polyethylene glycol), and insertion or substitution by chemical synthesis of amino acids such as ornithine, which is not naturally present in human proteins. "Recombinant polypeptide variant" refers to any polypeptide that differs from naturally occurring TRHs through amino acid insertions, deletions and substitutions, created using recombinant DNA techniques. The guide for determining which amino acid residues can be replaced, added or deleted, without abolishing the activities of interest, such as signal transduction, can be found by comparing the particular TRH sequence with the particular TRH sequence with that of homologous peptides and reducing to the minimum the number of amino acid sequence changes made in highly conserved regions. Preferably, the amino acid "substitutions" are the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with an isoluezine or valine, an aspartate with glutamate, or a threonine with a serine, that is, conservative replacements. The "insertions" or "deletions" are typically on the scale of approximately 1 to 5 amino acids, the allowed variation can be experimentally determined by producing the peptide synthetically or by systematically making insertions, deletions, or substitutions of nucleotides in a trh molecule using techniques of DNA and analyzing the recombinant variants, expressed, for the activity. A "fragment," "portion," or "segment" of polypeptide is a stretch of amino acid residues of at least about 5 amino acids and as many as 20 amino acids, preferably about 9 to 13 amino acids. To be active, any TRH peptide must be of sufficient length to exhibit biological and / or immunological activity either alone or as part of a chimeric presentation molecule such as key limpet hemocyanin for the production of antibody or a chimeric test molecule that It consists mainly of a purinergic receptor for the evaluation activity. When desired, a "leader sequence" can direct the polypeptide through the membrane of a cell. Said sequence may be normally present on the polypeptides of the present invention or provided with heterologous sources through DNA techniques. "Probe" is an RNA or DNA of sufficient length to be used in molecular amplification or hybridization to detect complementary sequences. An "oligonucleotide" or "oligomer" is a stretch of nucleotide residues, which has a sufficient number of bases that will be used as an initiator or probe in a polymerase chain reaction (PCR) .These oligonucleotides are prepared based on the cDNA sequence, which was found in the Sequence List After an appropriate test to establish reaction conditions and to eliminate false positives, amplified, revealed or confirmed the presence of an identical or homologous DNA or its RNA transcribed in A particular cell or tissue The oligonucleotides or oligomers comprise portions of a DNA sequence having at least 10 nucleotides and as much as about 35 nucleotides, preferably about 25 nucleotides. "Degenerating oligonucleotide probes" are probes, in the which minor substitutions have been made based on the degeneracy of the genetic code (more than one codon can specify ficar the same amino acid). They are particularly useful in the amplification of a similar natural nucleic acid sequence of chromosomal DNA, as described by Walsh PS et al (1992, PCR Methods Appl 1: 2241-50). A "portion" or "fragment" of a polynucleotide or nucleic acid comprises all or any part of the nucleotide sequence having less nucleotide than about 6 kb, preferably less than about 1 kb, which can be labeled and used as probes. A probe can be labeled with a radioactive element, an enzyme, or a chromogenic or fluorogenic label. After effectively testing to establish the reaction conditions and to eliminate false positives, nucleic acid probes may be used in Southern blots or in situ to determine whether the DNA or RNA encoding a T7G or a T7G homologue is present in a type of cell, tissue or organ. Although the sequences for nucleic acid probes can be derived from natural, recombinant or computer consensus sequences, the physical probes can be chemically synthesized, completely or in part, and marked through nick translation, Klenow filling reaction , PC R or other methods known in the art. The probes of the present invention, their preparation and / or labeling are elaborated in Sambrook J. and others (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY; or Ausubel FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, both incorporated here by reference. Recombinant variants that encode T7Gs can be synthesized or selected by making use of "redundancy" in the genetic code. Several codon substitutions, such as silent charges, which produce specific restriction sites, can be introduced to optimize cloning to a plasmid or viral vector or to increase expression in a particular prokaryotic or eukaryotic system. Use-specific mutants or chimeras containing the added related peptide domains can also be introduced to test or modify the properties of any part of the polypeptide, particularly to change ligand binding affinities, interchain affinities, or degradation / change regime. The present invention provides a unique nucleotide sequence that identifies a novel homolog of the human purinergic receptor, which was first identified in a human liver cDNA library. The sequence for trh is shown in SEQ ID NO: 1 and is homologous, but significantly different from the GenBank sequence, HUMTHRR. Since TRH is specifically expressed in cells that respond to trauma or infection, the nucleic acid (trh), polypeptide (TRH) and antibodies to TRH are useful in investigations of and intervention in normal and abnormal physiological and pathological procedures, which regulate signaling, immunity, cell repair, etc. Therefore, an assay for the super-regulated expression of TRH can accelerate the diagnosis and appropriate treatment of conditions caused by abnormal signal transduction due to systemic and local infections, traumatic damage and other tissues, hereditary or environmental diseases associated with hypertension. , carcinomas, and other physiological or pathological problems. The nucleotide sequence encoding TRH (or its complement) has numerous other applications in techniques known to those skilled in the field of molecular biology. These techniques include the use as hybridization probes for Southern or Northern analysis, use as oligomers for PCR, use for chromosomal and gene mapping, use in the recombinant production of TRH, use in the generation of antisense DNA or RNA, its analogues and the like, and the use in the production of qimeric molecules to select agonists, inhibitors or antagonists for the design of domain-specific therapeutic molecules. The uses of the nucleotides encoding HRT, described herein, are illustrative of known techniques and are not intended to limit their use in any technique known to one skilled in the art. In addition, the nucleotide sequences described herein can be used in molecular biology techniques that have not been developed, provided that the new techniques are based on properties of the nucleotide sequences that are currently known, v. gr., the triplet genetic code, specific base pair interactions, etc. It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding TRH can be produced, some carrying minimal homology to the nucleotide sequence of any known and naturally occurring gene. The invention has specifically contemplated every possible variation of the nucleotide sequence that can be made by selecting combinations based on possible codon choices. These combinations are made according to the normal triplet genetic code as applied to the nucleotide sequence of the TRH of natural existence, and all these variations will be considered as being specifically described. Although the nucleotide sequences, which encode the TRH and its variants are preferably capable of hybridization to the nucleotide sequence of the naturally occurring TRH gene under severe conditions, it may be advantageous to produce the nucleotide sequences encoding TRH or its derivatives possessing a substantially different codon usage. The codons can be selected to increase the rate at which the expression of the peptide occurs in a prokaryotic or eukaryotic expression host according to the frequency with which the particular codons are used by the host. Other reasons for substantially altering the nucleotide sequence encoding TRH and its derivatives without altering the encoded amino acid sequence include the production of RNA transcripts that have more desirable properties, such as longer half-life, than the transcripts produced from the sequence of natural existence The nucleotide sequence encoding TRH can be linked to a variety of other nucleotide sequences through well-established recombinant DNA techniques (Sambrook et al., Supra). Useful nucleotide sequences for binding to trh include a determination of cloning vectors-plasmids, cosmids, lambda phage derivatives, phagemids, and the like, which are well known in the art and can be selected for such features as the size insert in which they can adapt, their usefulness, their fidelity, etc. Other vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, YAC and BAC mapping vectors, and the like. In general, these vectors may contain a functional origin of replication in at least one organism, conventional restriction endonuclease sensitive sites, and selectable markers for the host cell. Another aspect of the present invention is to provide trh-specific nucleic acid hybridization probes capable of hybridization with naturally occurring nucleotide sequences encoding TRH. Said probes can also be used for the detection of sequences encoding TRH and should preferably contain at least 50% of the nucleotides of any particular domain of interest from this sequence encoding trh. The hybridization probes of the present invention may be derived from the nucleotide sequence of SEQ ID NO: 1 or from the genomic sequence including promoters, enhancer elements and introns of the respective native trh. Hybridization probes can be labeled through a variety of reporter groups, including radionuclides such as 32P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin / biotin systems, and the like. PCR, as described in the patents of E.U.A. Nos. 4,683,195 and 4,965,188 provide additional uses for oligonucleotides based on the nucleotide sequences, which encode TRH. Said probes used in the PCR can be of recombinant origin, can be chemically synthesized, or can be a mixture of both, and comprise a discrete nucleotide sequence for diagnostic use or a deposit of degeneration of possible sequences for the identification of closely related sequences T7G The full-length genes can be cloned using a new method as described in Patent Application Serial No. 08 / 487,112, filed June 7, 1995, and incorporated herein by reference, which employs XL-PCR (Perkin- Elmer, Foster, CA) to amplify long pieces of DNA. This method was developed to allow a single researcher to process multiple genes (up to 20 or more) at a time and obtain an extended sequence (possibly full length) in 6-10 days. It replaces current methods that use labeled probes to classify collections and which allow a researcher to process only about 3-5 genes in 14-40 days. In the first step, which can be performed in approximately 2 days, are designated initiators and are synthesized based on a known partial sequence. In step 2, which takes approximately six to eight hours, the sequence is extended through PCR amplification of a selected collection. Steps 3 and 4, which take about a day, are the purification of amplified c-DNA and its ligation to an appropriate vector. Step 5, which takes about a day, involves the transformation and growth of host bacteria. In step 6, which takes approximately five hours, PCR is used to classify bacterial clones for an extended sequence. The final steps, which take about a day, involve the preparation and sequencing of the selected clones. If the full-length cDNA has not been obtained, the entire procedure is repeated using either the original collection or some other preferred collection. The preferred collection may be one that has been selected by size to include only larger AD Nc or may consist of individual or combined commercially available collections, eg, lung, liver, heart and brain from Gibco / BR L (Gaithersburg MD) . The cDNA library may have been prepared with oligo d (T) or random primers. The advantage of using collections with random primers is that they will have more sequences containing 5 'ends of genes. A collection with random primer can be particularly useful if an oligo d (T) collection does not produce a complete gene. Obviously, the larger the protein, the less likely that the entire gene is found in an individual plasmid. Other means for producing hybridization probes for closely related sequences include the cloning of nucleic acid sequences encoding TRH or its derivatives to vectors for the production of mRNA probes. Such vectors are known in the art and are commercially available and can be used to synthesize RNA probes in vitro through the addition of the appropriate RNA polymerase such as T7 or SP6 RNA polymerase and appropriate labeled nucleotides. It is now possible to produce a DNA sequence, or portions thereof, that encodes TRH and / or its derivatives completely by synthetic chemistry. Said molecules can be inserted into any of the many available vectors using reagents and methods that are known in the art at the time of filing this application. In addition, synthetic chemistry can be used to introduce mutations to the trh sequences or any portion thereof. The nucleotide sequence can be used to develop an assay to detect activation, inflammation or disease associated with abnormal levels of TRH expression. The nucleotide sequence can be labeled by methods known in the art and added to a sample of fluid or tissue from a patient. After an incubation period sufficient to effect hybridization, the sample was washed with a compatible fluid, which contains a visible marker, a dye or other appropriate molecule (s), if the nucleotide has been labeled with an enzyme. After the compatible fluid is rinsed, the dye is quantified and compared to a normal one. If the amount of dye is significantly elevated (or reduced, as the case may be), the nucleotide sequence has hybridized to the sample, and the test indicates an abnormal condition such as nflammation or disease. The nucleotide sequence for trh can be used to construct hybridization probes for gene mapping. The nucleotide sequence provided herein can be mapped to a chromosome and specific regions of a chromosome using well known genetic and / or chromosomal mapping techniques. These techniques include in situ hybridization, analysis of binding against known chromosomal markers, classification of hybridization with collections or chromosomal preparations classified by flow, specific to known chromosomes, and the like. The technique of fluorescent in situ hybridization of chromosomes has been described extensively, among other places, in Verma et al. (1988) Human Ch romosomes: A Manual of Basic Techniques, Pergamon Press, New York. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data. Examples of map data can be found in 1994 Genome Issue of Science (265: 1981f). The correlation between the location of trh on a physical chromosomal map and a specific disease (or predisposition to a specific disease) can help to narrow the region of DNA associated with that genetic disease. The nucleotide sequence of the present invention can be used to detect differences in the gene sequence between normal individuals and carriers or affected individuals. The nucleotide sequence encoding TRH can be used to produce purified TRH using well known methods of recombinant DNA technology. Among the many publications that teach methods for gene expression after they have been isolated is Goeddel (1990) Gene Expression Technology, Methods and Enzymology, Vol. 185, Academic Press, San Diego CA. TRH can be expressed in a variety of host cells, either prokaryotic or eukaryotic. The host cells can be of the same species in which the nucleotide sequences of trh are endogenous or of a different species. The advantages of producing TRH through recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures. Cells transformed with DNA encoding TRH can be cultured under conditions suitable for the expression of TRH and recovery of the protein from the cell culture. The TRH produced through a recombinant cell can be secreted or it can be contained intracellularly depending on the particular genetic construct. In general, it is more convenient to prepare recombinant proteins in secreted form. The purification steps vary with the production process and the particular protein produced. Various methods for the isolation of the TRH polypeptide can be achieved through procedures well known in the art. For example, said polypeptide can be purified by immunoaffinity chromatography using the antibodies provided by the present invention. Various other protein purification methods, well known in the art, include those described in Deutscher M (1990) Methods in Enzymology, Vol. 182, Academic Press, San Diego CA; and in Scopes R (1982) Protein Purification: Principles and Practice, Springer-Verlag, New York, both incorporated herein by reference. In addition to recombinant production, fragments of TRH can be produced through direct peptide synthesis using solid phase techniques (Stewart et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco CA; Merrifield J. (1963 J.

Am. Chem. Soc. 85: 2149-2154). In vitro protein synthesis can be performed using manual techniques or by automation. Automatic synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (ABI, Foster, California) according to the instructions provided by the manufacturer. Several fragments of TRH can be chemically synthesized separately and combined using chemical methods to produce the full-length molecule. The TR H for the induction of antibody does not require biological activity; however, the protein must be antigenic. The peptides used to induce specific antibodies can have an amino acid sequence consisting * of at least five amino acids, preferably at least 10 amino acids. These must resemble a structural portion or epitope of the polypeptide amino acid sequence and may contain the entire amino acid sequence of an individual domain of TRH. The short stretches of TRH amino acids can be fused with those of another protein such as a key limitation hemocyanin, and an antibody produced against the fusion protein. Antibodies specific for TR H can be produced by inoculating an appropriate animal with the polypeptide or an antigenic fragment. An antibody is specific for TRH if it is produced against an epitope of the polypeptide and binds to at least part of the natural or recombinant protein. The production of antibody includes not only the stimulation of an immune response by injection to animals, but also analogous steps in the production of synthetic antibodies or other specific binding molecules such as the classification of recombinant immunoglobulin collections (Orlandí R. et al. others (1989) PNAS 86: 3833-37, or Huse WD et al (1989) Science 256: 1275-81) or in vitro stimulation of lymphocyte populations. Current technology (Winter G and Milstein C (1991) Nature 349: 293-99) provides a number of highly specific binding reagents based on the principles of antibody formation. These techniques can be adapted to produce molecules specifically by binding particular domains of TRH. A further embodiment of the present invention is the use of TRH-specific antibodies or the like as bioactive agents to treat abnormal signal transduction associated with systemic and local infections, traumatic or tissue damage, hereditary or environmental diseases associated with hypertension, carcinomas, and other pathological problems. Bioactive compositions comprising TRH agonists, antagonists or inhibitors can be administered in a suitable therapeutic dose determined by any of several methodologies including clinical studies in mammalian species to determine the maximum tolerable dose and in normal human subjects to determine the dose safe. In addition, the bioactive agent can be formed as a complex with a variety of well-established compounds or compositions, which improve stability or pharmacological properties such as half-life. It is contemplated that a therapeutic, bioactive composition is delivered through intravenous infusion to the bloodstream or any other effective means that can be used for treatment. The examples presented below are provided to describe the present invention. These examples are provided by way of illustration and are not included for the purpose of limiting the invention.

INDUSTRIAL APPLICABILITY I. Isolation of mRNA and Construction of the cDNA Collection The TRH sequence of this application was first identified in clone Incyte No. 96700 among the sequences comprising the human liver collection. The human cells used for this collection come from a 49-year-old man (Catalog # 937224; Stratagene, LaJolla CA). The cDNA synthesis for the cDNA collection was started with oligo d (T), and synthetic oligonucleotide adapters were ligated onto cDNA ends that allow their insertion into the Uni-ZAP ™ vector system (Stratagene). This allowed a high efficiency of the unidirectional lambda collection construction (sense orientation) and the convenience of a plasmid system with blue / white color selection to detect clones with cDNA inserts. The cDNA library was sorted using probes from ADNo antibody probes, and then, phagemid pBluescript® (Stratagene) was cut rapidly in vivo. The phagemid allows the use of a plasmid system to facilitate characterization of the insert, sequencing, site-directed mutagenesis, creation of unidiretional deletions and expression of fusion polypeptides. The collection phage particles constructed as usual were infected in XL1-Bue® of the host strain of E. coli (Stratagene), which has a high transformation efficiency, increasing the probability of obtaining rare clones, sub-represented in the cDNA collection. Alternative unidirectional vectors may include, but are not limited to, cDNAp (Invitrogen, San Diego, CA) and pSHIox-1 (Novagen, Madison Wl).

II. Isolation of cDNA Clones The phagemid forms of individual cDNA clones were obtained through the in-cut procedure. vivo, in which the host bacterial strain was co-infected both with the collection phage and with an auxiliary phage f1. The polypeptides or enzymes derived from both the phage containing the collection and the auxiliary phage grooved to the DNA, initiated the new DNA synthesis of the sequences delineated on the target DNA, and created a circular phagemid DNA molecule with an individual chain structure. , smaller, which included all the pBluescript phagemid DNA sequences and the cDNA insert. The phagemid DNA was secreted from the cells and purified and used to reinfect fresh host cells where phagemid DNA of double-stranded structure was produced. Since the phagemid carries the gene for β-lactamase, newly transformed bacteria were selected in a medium containing ampicillin. The phagemid DNA was purified using the MAGIC MINPREPS ™ DNA Purification System (Catalog # A7100; Promega Corp. Madison, Wl). This small-scale procedure provides a simple and reliable method for lysing bacterial cells and rapidly isolating purified phagemid DNA using a proprietary DNA-binding resin. The phagemid DNA was also purified using the QIAWELL-8 Plasmid Purification System (QIAGEN Inc. Chatsworth CA). This product line provides a convenient, fast and reliable high production method for lysing bacterial cells and isolating highly purified phagemid DNA using anion exchange resin particles, QIAGEN. The DNA was eluted from the purification resin and prepared for DNA sequencing and other analytical manipulations.

III. Sequencing of cDNA Clones The cDNA inserts of the randomized isolates from the placental collection were partially sequenced. Methods for sequencing DNA are well known in the art. Conventional enzymatic methods employed Klenow fragments of DNA polymerase, SEQUENASE® (US Biochemical Corp. Cleveland OH) or Taq polymerase to extend the DNA strands of a reinforced oligonucleotide primer to the DNA template of interest. Methods for the use of templates of both single and double chain structure have been developed. The chain termination reaction products were electrophoresed on urea-acrylamide gels and detected either by autoradiography (for radionuclide-labeled precursors) or by fluorescence (or fluorescently labeled precursors). Recent improvements in mechanized reaction preparation, sequencing and analysis using the fluorescent detection method have allowed the expansion in the number of sequences that can be determined per day using machines such as Catalyst 800 and Applied Biosystems 377 or 373 DNA sequencers .

IV. Homology Search of cDNA Clones and Deduced Proteins Each sequence thus obtained was compared with sequences in GenBank, using a search algorithm developed by Applied Biosystems and incorporated in the INHERIT ™ 670 Sequence Analysis System. In this algorithm, an algorithm was used. Pattern Specification Language (Pattern Specification Language, developed by TRW Inc., Los Angeles CA) to determine regions of homology. The three parameters determine how the sequence comparisons performed the window size, window deviation, and error tolerance. Using a combination of these three parameters, the DNA database was searched for sequences containing regions of homology to the sequence in question, and the appropriate sequences were classified with an initial value. Subsequently, these homologous regions were examined using dot matrix homology graphs to distinguish regions of homology from chance comparisons. Smith-Waterman alignments were used to show the results of the homology search.

Peptide and protein sequence homologies were ascertained using the INHERIT ™ 670 Sequence Analysis System in a manner similar to that used in DNA sequence homologies. The Pattern Specification Language and the parameter windows were used to search the protein databases for sequences containing regions of homology, which were classified with an initial value. The dot matrix homology plots were examined to distinguish regions of significant homology from the opportunity comparisons. Alternatively, BLAST, which represents the Basic Local Aligment Search Tool, was used to search for local sequence alignments (Altschul SF (1993) J. Mol. Evol. 36: 290-300; Altschul, SF et al. (1990) J. Mol. Biol. 215: 403-10). BLAST produces alignments of both nucleotide and amino acid sequences to determine the similarity of the sequence. Due to the local nature of the alignments, BLAST is especially useful for determining exact comparisons or for identifying homologs. While it is ideal for combinations with hollows, it is inappropriate to perform the motif style search. The fundamental unit of production of the BLAST algorithm is the High Classification Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths, whose alignment is locally external and for which the classification of the alignment satisfies or exceeds a threshold or cut classification set by the user. The BLAST aspect is to search for HSPs between a sequence in question and a database sequence, to evaluate the statistical significance of any comparisons found, and to report only those comparisons that satisfy the threshold of significance selected by the user. Parameter E establishes the statistically significant threshold to report the comparisons of the database sequence. E is interpreted as the superior union of the expected frequency of occurrence of opportunity of an HSP (or group of HSPs) within the context of the entire database search. Any database sequence, whose comparison satisfies E is reported in the program production.

V. Identification, Full Length Cloning, Sequencing and Translation The INHERIT ™ analysis results from the randomly collected and sequenced portions of clones from the human liver collection identified as Incyte 86700 as a homologue of the romybin receptor, HUMTHRR. The cDNA insert comprising the Incyte 86700 was fully sequenced and used as the basis for the cloning of the full-length cDNA. The Incyte 86700 cDNA was extended to a full length using a modified XL-PCR method (Perkin Elmer) as described in Serial Patent Application No. 08 / 487,112, by Guegler et al., Filed June 7, 1995 , and incorporated here by reference. The primers were designed based on a known sequence; an initiator was synthesized to initiate the extension in the antisense direction (XLR = TGCCCTCCGTTGCTGTATAAGACCG) and the other to extend the sequence in the direction of direction (XLF = AAGGAGGGCATAATTCCACAATGTG). The primers allowed the sequence to be extended "outward" by generating amplicons containing a new and unknown nucleotide sequence for the genes of interest. The primers were designed using Oligo 4.0 (National Biosciences Inc. Plymouth MN) to have a length of 22-30 nucleotides, have a GC content of 50% or more, and to reinforce the target sequence at temperatures of approximately 68-72 °. C. Any stretching of nucleotides, which could result in hairpin structures and inator-initiator dimerizations were avoided. The liver cDNA collection was used as a template, and the XLR and XLS primers were used to amplify the 86700 sequence. Following the instructions of the XL-PCR equipment and through a good mixing and reaction mixture, a high fidelity amplification was obtained. Starting with 25 pMoles of each primer and the recommended concentrations of all equipment components, PCR was performed using the MJ PTC200 thermocycler (MJ Research, Watertown MA) and the following parameters: Step 1 94 ° C for 60 sec. (initial denaturation) Step 2 94 ° C for 15 sec. Step 3 65 ° C for 1 min. Step 4 68 ° C for 7 min. Step 5 Repeat steps 2-4 15 additional times Step 6 94 ° C for 15 sec. Step 7 65 ° C for 1 min. Step 8 68 ° C for 7 min + 15 sec. / cycle Step 9 Repeat steps 6-8 11 additional times Step 10 72 ° C for 8 min. Step 11 4 ° C (and maintain) At the end of the 28 cycles, 50 μl of the reaction mixture was removed; and the remaining reaction mixture was operated for 10 additional cycles as noted above: Step 1 94 ° C for 15 sec. Step 2 65 ° C for 1 min. Step 3 68 ° C for (10 min. + 15 sec.) / Cycle Step 4 Repeat steps 1-39 additional times Step 5 72 ° C for 10 min. An aliquot of 5-10 μl of the reaction mixture was analyzed by electrophoresis at a low concentration (about 0.6-0.8%) on a mini gel to determine successful reactions in the extension. Although all extensions potentially contained the full-length gene, some of the larger products were selected and separated from the gel. The additional purification involved using a QIAQuick ™ gel extraction method (QIAGEN Ine, Chatsworth CA). After DNA recovery, the Klenow enzyme was used to convert the final protrusions into shaved ends to facilitate religation and cloning of the products. After precipitation with ethanol, the products were redissolved in 13 μl of ligation buffer. Then, 1 μl of T4-DNA ligase (15 units) and 1 μl of T4 polynucleotide kinase were added, and the mixture was incubated at room temperature for 2-3 hours or overnight at 16 ° C. The cells of J. Competent coli (in 40 μl of appropriate media) were transformed with 3 μl of ligation mixture and cultured in 80 μl of SOC medium (Sambrook J. et al, supra). After incubation for one hour at 37 ° C, the complete transformation mixture was plated on Luria Bertani (LB) agar (Sambrook J. et al, supra) containing carbenicillin at 25 mg / l. The next day, 12 colonies were picked randomly from each plate and cultured in 150 μl of an LB / carbenicillin medium placed in a single cavity of a sterile, commercially available 96-well microtiter plate. The next day, 5 μl of each culture was transferred overnight to a 96-well non-sterile plate and after dilution with 1:10 with water, 5 μl of each sample was transferred to a PCR setup. For PCR amplification, 15 μl of the PCR mixture (1.33x concentrate containing 0.75 Taq polymerase units, a vector primer and one or both gene-specific primers used for the extension reaction) were added to each well. The amplification was performed using the following conditions: Step 1 94 ° C for 60 min. Step 2 94 ° C for 20 sec. Step 3 55 ° C for 30 sec. Step 4 72 ° C for 90 sec. Step 5 Repeat steps 2-4 for an additional 29 times Step 6 72 ° C for 180 sec. Step 7 4 ° C (and maintain). The aliquots of these PCR reactions were performed on agarose gels together with molecular weight markers. The sizes of the PCR products were compared to the original, partial cDNAs, and appropriate clones were selected, ligated to the plasmid and sequenced. The cDNA (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequences for human TRH are shown in Figure 1. When the translation of the sequence was searched against protein databases such as SwissProt and PIR, no exact comparisons were found. Figure 2 shows the comparison of the trh sequence and HUMTHRR sequences.

SAW. Antisense Analysis Knowledge of the complete, correct cDNA sequence of trh allows its use as a tool for antisense technology in the investigation of the function of the gene. Oligonucleotides, cDNAs or genomic fragments comprising the antisense strand structure of trh are used either in vivo or in vitro to inhibit mRNA expression. Such technology is now well known in the art, and antisense molecules are designated at various locations along the nucleotide sequences. By treating the test cells or animals complete with said antisense sequences, the gene of interest can be effectively deactivated. Frequently, the function of the gene was ascertained by observing the behavior at an intracellular level, cellular, tissue or organism (eg, lethal capacity, loss of differentiated function, changes in morphology, etc.). In addition to using the sequences constructed to interrupt the transcription of a particular open reading frame, modifications of gene expression are obtained by designing antisense sequences to intron regions, promoters / enhancer elements, or even trans-regulating regulation genes. Similarly, inhibition is achieved using the Hogeboom base pair methodology, also known as "triple helix" base pairs.

Vile. Expression of TRH Expression of trh is achieved by subcloning the cDNAs to appropriate expression vectors and transfecting the vectors to analogous expression hosts. In this particular case, the cloning vector previously used for the generation of the cDNA library, pBluescript, also provides direct expression of the trh sequences in EL coli. Upstream of the cloning site, this vector contains a promoter for β-galactosidase, followed by the sequence containing the amino-terminal Met and the 7 subsequent residues of β-galactosidase. Immediately after these eight residues is a gene-engineered promoter-useful bacteriophage promoter for artificial primers and transcription and a number of unique restriction sites, including Eco RI, for cloning. The induction of the transfected bacterial strain, isolated with IPTG using normal methods, produces a fusion protein corresponding to the first seven residues of β-galactosidase, approximately 15 residues of "linker", and the peptide encoded within the cDNA. cDNA clone inserts are generated by an essentially random procedure, there is an opportunity in three that the included cDNA lies in the correct frame for the appropriate translation If the cDNA is not in the proper reading frame, it is obtained by eliminating or insertion of the appropriate number of bases by well-known methods, including in vitro mutagenesis, exonuclease III digestion or "mung bean" nuclease, or the inclusion of an oligonucleotide linker of appropriate length Alternatively, the trh cDNA is released on other vectors which are known to be useful for the expression of the protein in specific hosts. Adores of or gonucleotides containing cloning sites as well as a DNA segment (approximately 25 bases) sufficient for the hybridization of the stretches at both ends of the target cDNA are chemically synthesized by normal methods. These primers are then used to amplify the gene segment. desired by PCR The resulting gene segment is digested with appropriate restriction enzymes under normal conditions and isolated through gel electrophoresis. Alternatively, similar gene segments are produced by digestion of the cDNA with appropriate restriction enzymes. Using appropriate primers, the segments to encode the sequence of more than one gene are ligated together and cloned into appropriate vectors. It is possible to optimize the expression by constructing said chimeric sequences. Suitable expression hosts for said chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9 cells, yeast cells such as Saccharomvces. cerevisiae, and bacteria such as E. coli For each of these cell systems, a useful expression vector includes an origin of replication that allows propagation in bacteria and a selectable marker such as the β-lactamase antibiotic resistance gene to allow selection of the plasmid in the bacterium. In addition, the vector includes a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells. Vectors for use in eukaryotic expression hosts usually require A RN processing elements such as the 3 'polyadenylation sequences, if they are not part of the AD Nc of interest. In addition, the vector contains promoters or enhancers, which increase the expression of the gene. Said promoters are specific hosts and include MMTV, SV40 and metalotionin promoters for C HO cells; trp, lac, tac and T7 promoters for bacterial hosts; and an alpha factor, alcohol oxidase and PG H promoters for yeast. Transcription enhancers, such as the rous sarcoma virus enhancer, are used in mammalian host cells. Once homogenous cultures of recombinant cells are obtained through normal culture methods, large quantities of recombinantly produced TRH were recovered from the conditioned medium and analyzed using chromatographic methods known in the art.

VIII. Isolation of Recombinant TRH TRH is expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate the purification of protein. Said purification facilitating domains includes, but is not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains allow purification on immobilized immunoglobulin, and the domain used in the FLAGS affinity extension / purification system (Immunex Corp. Seattle WA). The inclusion of a cleavage link sequence such as Factor XA or enterokinase Invitrogen, San Diego CA) between the purification domain and the trh sequence is useful to facilitate the expression of TRH.

IX: Chimeric T7Gs Test The functional chimeric T7Gs were constructed by combining the extracellular receptive sequences of a new isoform with the transmembrane and intracellular segments of a known isoform. Said chimeric molecules are useful for testing purposes. This concept was demonstrated by Kobilka et al. (1988, Science 240 1310-1316) who created a series of chimeric α2-β2 adrenergic receptors (AR) by inserting progressively larger amounts of the a2-AR adrenergic sequence into β2-AR activity. binding of known agonists changed as the molecule shifted from having a conformation of more than a2 to that of ß2, and the intermediate constructs showed specific mixed character The specific character for binding antagonists, however, was correlated with the source domain The importance of the T7G Vil cominium for ligand recognition was also found in chimeras that use two a-factor receptors of two yeasts and is important since the yeast receptors are classified as diverse receptors. Thus, the functional role of the specific domains seem to be preserved across the entire T7G family without considering the category In a In parallel, the internal segments or cytoplasmic domains of an isoform are exchanged with the analogous domains of a known T7G and are used to identify the structural determinants responsible for the coupling of receptors to Gp proteins (Dohlman et al., 1991). Rev Biochem 60653-88) A chimeric receptor in which the domains V, VI, and the intracellular connection loop of ß2-AR are substituted to a2-AR, are shown to bind hgandos with a specific character of a2-AR, but stimulate adenylate cyclase in the form of ß2-AR. This shows that for the adrenergic type receptors, the recognition of the G protein is present in the V and VI domains and their connection loop. The opposite situation was predicted and observed for a chimera where the loop V? VI from α2-AR replaced the corresponding domain in ß2-AR and the resulting receptor binding ligands with the specific character of ß2-AR and activated phosphatidylinositol mediated by G protein in the a2AR form. Finally, the chimaeras constructed from muscarinic receptors also showed that the V-> loop. VI is the main determinant for the specific character of G protein activity (Bolander, supra). The chimeric or modified T7Gs, which contain substitutions in the extracellular and transmembrane regions, have shown that both portions determine the specific character of the ligand binding. For example, two residues were conserved. Ser in the V domain of all adrenergic receptors and D-catecholamine are necessary for potent agonist activity. These serines are thought to be in the binding site of T7G and to form hydrogen bonds with the catechol portion of the agonists. Similarly, an Asp residue present in the I I I domain of all T7Gs, which bind biogenic amines is believed to be in the T7G binding site and form an ion pair with the amine ligand group. The cloned, functional T7Gs are expressed in heterologous expression systems and their activity was analyzed (Marullo et al. (1988) Proc. Nati. Acad. Sci, 85: 7551 -55; King et al. (1990) Science 250 12-23) A heterologous system introduces genes for a mammalian T7G and a mammalian G protein towards yeast cells The T7G had an appropriate ligand-specific character and affinity and activates the activation-restriction of appropriate biological growth, and morphological changes, of the yeast cells The novel sequences for T7G domains were tested in a similar way X. Production of TRH-specific Antibodies Two aspects were used to give antibodies to TRH, and each aspect is useful to generate both polyclonal and monoclonal antibodies. In one aspect, the denatured protein of the reversed-phase HPLC separation is obtained in amounts up to 75 mg Denatured protein is used to immunize mice or rabbits using normal protocols approximately 100 micrograms are suitable for immunization of a mouse, while up to 1 mg can be used to immunize a rabbit. To identify mouse hibpdomas, the denatured protein It is radioiodinated and used to classify potential mupno B cell hibpomains for those that produce the antibody. This procedure requires only small amounts of protein, so that 20 mg could be enough to label and classify several thousand clones. In a second aspect, the amino acid sequences of a T domain Appropriate RH, as deduced from the translation of the cDNA, was analyzed to determine regions of high immunogenicity Oligopeptides comprising appropriate hydrophobic regions, as illustrated in Figure 3, are synthesized and used in suitable immunization protocols to produce antibodies. for selecting suitable epitopes is described by Ausuble FM et al. (Supra) Optimal amino acid sequences for immunization are usually in the C-terminus, the N-terminus and those hydrophilic, intervening regions of the polypeptide, which are likely to be exposed to the external environment when the protein is in natural conformation Typically, the selected peptides, approximately 15 residues in length, are synthesized using an Applied Biosystems Peptide Synthesizer Model 431A using fmoc chemistry and coupled to the keyhole modem hemocyanin (KLH, Sigma, St Louis MO) by reaction with M-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS, cf Ausubel FM et al., supra) If necessary, a cysteine can be introduced at the N-terminus of the peptide to allow coupling to KLH Rabbits can be immunized with the complex of peptide-KLH in a complete Freund's assistant The resulting antisera can be tested for antipeptide activity by binding the peptide to the plastic, blocking with 1% bovine serum albumin, reacting with antisera, washing and reacting with anti-IgG. specific goat rabbit, purified by affinity, marked (radioactive or fluorescent). Hybridomas can be prepared and classified using normal techniques. Hybridomas of interest can be detected by classifying with labeled TRH to identify those fusions that produce the monoclonal antibody with the desired specific character. In a typical protocol, the plate cavities (FAST, Becton-Dickinson, Palo Alto, CA) are coated, during incubation, with specific rabbit-anti-mouse antibodies (or suitable antispecies Ig) at 10 mg / ml. The coated cavities are blocked with 1% BSA, washed and incubated with supernatants of the hybridomas. After washing, the cavities are incubated with TRH labeled at 1 mg / ml. Supernatants with specific antibodies bind more marked TRH than the detectable above the background. Then the specific antibodies that produce clones can be expanded and subjected to 2 cycles of cloning at a limiting dilution. Cloned hybridomas are injected into mice treated with pristane to produce ascites, and the monoclonal antibody is purified from mouse ascitic fluid by affinity chromatography using Protein A. Monoclonal antibodies with affinities of at least 10e8 Me "1, preferably from 10e9 to 10e10 or stronger, will typically be produced through normal procedures as described in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York, and in Goding (1986) Monoclonal Antibodies: Principies and Practice, Academic Press, New York, both incorporated here by reference.

XI. Diagnostic Test Using TRH Specific Antibodies Particular TRH antibodies are useful for investigating signal transduction and diagnosis of infectious or hereditary conditions, which are characterized by differences in the amount or distribution of TRH or products downstream of a cascade of active signaling. Since TRH was found in a collection of human liver, it appears to be over-regulated in cell types primarily involved in immune protection or defense. Diagnostic tests for TRH include methods that use the antibody and a label to detect HRT in fluids of the human body, membranes, cells, tissues or extracts of said tissues. The polypeptides and antibodies of the present invention are used with or without modification. Frequently, polypeptides and antibodies will be labeled by joining them, either covalently or non-covalently, with a substance, which provides a detectable signal. A wide variety of brands and conjugation techniques are known and have been reported extensively in both scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescent agents, magnetic particles and the like. The patents that teach the use of said marks include the patents of E.U.A. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins can be produced as shown in the patent of E.U.A. No. 4,816,567, incorporated herein by reference. A variety of protocols for measuring soluble TRH or membrane binding, using polyclonal or monoclonal antibodies specific for the protein, is known in the art. Examples include the enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site monoclonal-based immunoassay is preferred using monoclonal antibodies reactive to two epitopes without interference in TRH, but a competitive binding assay may be employed. These assays are described, inter alia, in Maddox, DE et al. (1983, J. Exp. Med. 158: 1211).

XII. Purification of Native TRH Using Specific Antibodies Native or recombinant TRH was purified by immunoaffinity chromatography using antibodies specific for TRH. In general, an immunoaffinity column is constructed by covalently coupling the anti-TRH antibody to an activated chromatographic resin. Polyclonal immunoglobulins will be prepared from immune serum either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, NJ). Also, monoclonal antibodies are prepared from mouse ascites fluid by precipitation of ammonium sulfate or chromatography on immobilized Protein A. The partially purified immunoglobulin is covalently linked to a chromatographic resin such as activated CnBr-Sepharose (Pharmacia Piscataway, NJ). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions. Said immunoaffinity columns are used in the purification of TRH by the preparation of a fraction of cells containing TRH in a soluble form. This preparation is derived by solubilization of the whole cell or of a sub-cellular fraction obtained via differential centrifugation (with the addition of detergent or not) or by other methods well known in the art. Alternatively, the TRH containing a signal sequence is secreted in a useful amount to the medium in which the cells grow. A preparation containing soluble TRH is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of TRH (eg, pH regulators of high ionic strength in the presence of detergent). The column is then eluted under conditions that break the binding of the antibody / TRH (eg, a pH regulator with a pH of 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and TRH is collected.

XII. Drug Classification This invention is particularly useful for classifying therapeutic compounds using TRH or its binding fragments in any variety of drug classification techniques. The polypeptide or fragment employed in said test is either free in solution, fixed to a solid support, exits a cell surface, or is located intracellularly. A method for classifying drugs utilizes eukaryotic or prokaryotic host cells, which are stably transformed with recombinant nucleic acids that express the polypeptide, fragment or chimera as discussed above. The drugs are classified against said transformed cells in competitive binding assays. Said cells, either in a viable or fixed form, can be used for normal binding assays. The formation of complexes between TRH and the agent being tested is measured. Alternatively, one can examine the decrease in complex formation between TRH and a receptor caused by the agent being treated. Thus, the present invention provides methods for classifying drugs or any other agents that can affect signal transduction. These methods, well known in the art, comprise contacting said agent with a TRH polypeptide or fragment thereof and analyzing, (i) the presence of a complex between the agent and the TRH polypeptide or fragment, or (ii) the presence of a complex between the TRH polypeptide or fragment and the cell. In such competitive binding assays, the TRH polypeptide or fragment is typically labeled. After a suitable incubation, the TRH polypeptide or fragment was separated from that present as a binding, and the amount of free or complex labeling is a measure of the ability of the particular agent to bind to TRH or to interfere with formation. of the TRH complex and agent complex. Another technique for classifying the drug provides a high throughput classification for compounds that have adequate binding affinity to the TRH polypeptide and is described in detail in European Patent Application 84/03564, published on September 13, 1984, incorporated herein by reference. In summary, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with the TRH polypeptide and washed. The bound TRH polypeptide is then detected by methods well known in the art. Alternatively, the purified TRH can also be directly coated onto plates for use in the aforementioned drug classification techniques. In addition, antibodies without neutralization can be used to capture the peptide and immobilize it on the solid support. This invention also contemplates the use of competitive drug classification assays wherein neutralizing antibodies capable of binding TRH, specifically compete with a test compound to bind TRH polypeptides or fragments thereof. In this way, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with TRH.

XIV. Rational Drug Design The objective of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, for example, agonists, antagonists, or inhibitors. Any of these examples can be used to design drugs, which are more active or stable forms of the polypeptide or which improve or interfere with the function of a polypeptide in vivo (Hodgson (1991) Bio / Technology 9: 19-21, incorporated herein by reference). In one aspect, the three-dimensional structure of a protein of interest, or of a protein inhibitor complex, it is determined through x-ray crystallography, through computer modeling or, more typically, through a combination of the two aspects. Both the form and charges of the polypeptide must be rtained to see the structure and to determine the active sites of the molecule. Less frequently, useful information regarding the structure of a polypeptide can be gained through modeling based on the structure of homologous proteins. In both cases, the relevant structural information is used to design efficient inhibitors. Useful examples of rational drug design include molecules that have improved activity or stability as shown by Braxton S and Wells JA (1992 Biochemistry 31 7796-7801) or which act as inhibitors, agonists or antagonists of native peptides as shown by Athauda SB et al. (1993 J Biochem 113742-746), incorporated herein by reference. It is also possible to isolate a specific antibody on the target, selected by functional assay, as described above, and then solve its crystal structure. This aspect, in principle , produces a farmanucleus on which the subsequent drug design can be based. It is possible to derive protein crystallography by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the The anti-ids binding site could be expected to be an analogue of the original receptor. The anti-id can then be be used to identify and isolate peptides from chemically or biologically produced peptide libraries. The isolated peptides could then act as the farmanucleus. By virtue of the present invention, a sufficient amount can be made available to perform such analytical studies as X-ray crystallography. the knowledge of the TRH amino acid sequence provided herein will provide guidance for those who employ computer modeling techniques in place of or in addition to X-ray crystallography.

XV Identification of other Members of the Signal Transduction Complex The purified TRH is a research tool for the identification, characterization and purification of interaction G proteins, phospholipase C, adenylate cyclase, or other signal transduction pathway proteins. The radioactive labels are incorporated into a selected TRH domain through various methods known in the art and used in vitro to capture interaction molecules. A preferred method involves labeling the primary amino groups in TRH with the Bolton-Hunter 125l reagent (Bolton, AE and Hunter, WM (1973) Biochem J. 133: 529). This reagent has been used to label molecules without concomitant loss of biological activity (Herbert CA et al. (1991) J. Biol. Chem. 266: 18989; McColl S. et al. (1993) J. Immunol. 150: 4550-4555) . The labeled TRH is useful as a reagent for the purification of molecules with which it interacts. In an affinity purification mode, the TRH bound to the membrane is covalently coupled to a chromatography column. The cell-free extract derived from collection or target cells is passed over the column, and the molecules with the appropriate affinity binding for TRH. The TRH complex is recovered from the column, dissociated and the recovered molecule is subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for the cloning of the relevant gene from an appropriate DNA library. In an alternative method, antibodies are developed against TRH, specifically monoclonal antibodies, as described above. The monoclonal antibodies are classified to identify those that inhibit the binding between ligands and TRH. These monoclonal antibodies are then used therapeutically.

XVI. Use and Administration of Antibodies, Inhibitors or Antagonists Antibodies, inhibitors or antagonists of TRH (or other treatments to limit signal transduction, LST), provide different effects when administered therapeutically. The LSTs are formulated in an aqueous, non-toxic, inert, pharmaceutically acceptable carrier medium, preferably at a pH of about 5 to 8, most preferably 6 to 8, although the pH varies according to the characteristics of the antibody, inhibitor or antagonist that is formulated, and the condition that will be treated. The characteristics of the LSTs include the molecule's solubility, half-life and antigenicity / immunogenicity; these and other characteristics help define an effective vehicle. Native human proteins are preferred as LSTs, but organic or synthetic molecules resulting from drug classifications are equally effective in particular situations. LSTs are delivered through administration routes that include, but are not limited to, topical creams and gels; spray and transmucosal spray; patches and transdermal bandages; injectable, intravenous and washing formulations; and orally administered fluids and pills formulated to resist stomach acid and enzymes. The particular formulation, the exact dose, and the route of administration are determined by the attending physician, and vary according to each specific situation. These determinations are made considering multiple variables such as the condition that will be treated, the LST that will be administered, and the pharmacokinetic profile of the particular LST. Additional factors, which are taken into account, include the state of the disease (eg, severity) of the patient, age, weight, gender, diet, time and frequency of administration, combination of the drug, reaction sensitivities and tolerance / response to therapy. Long-acting LST formulations are administered every 3 to 4 days, every week, or once every two weeks, depending on the half-life and clear regimen of the particular LST. Normal amounts of doses vary from 0.1 to 100,000 micrograms, up to a total dose of approximately 1 g, depending on the route of administration. The guide of particular doses and methods of supply is provided in the literature. See patents of E.U.A. Nos. 4,657,760; 5,206,344; or 5,225,212. Those skilled in the art will employ different formulations for different LSTs. Administration to cells such as nerve cells requires different methods of delivery of those to other cells such as vascular endothelial cells. It is contemplated that abnormal signal transduction in those conditions or diseases, which attack the cell-priming activity, cause damage that can be treated with LSTs. Said conditions, particularly anaphylactic or hypersensitive responses are treated as discussed above. LST is also used to treat other systemic and local infections, traumatic tissue damage, hereditary or environmental diseases associated with allergies, hypertension, carcinomas, cystic fibrosis, and other physiological or pathological problems associated with abnormal signal transduction. All publications and patents mentioned in the specification are incorporated herein by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described along with the preferred specific embodiments, it should be understood that the invention as claimed should not be unduly limited to said specific embodiments. In fact, several modifications of the modes described above for carrying out the invention, which are obvious to those skilled in the field of molecular biology or related fields, are within the scope of the claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: INCYTE PHARMACEUTICALS, INC. (ii) TITLE OF THE INVENTION: Thrombin Receptor Homologue (iii) SEQUENCE NUMBER: 3 (iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT: INCYTE PHARMACEUTICALS, INC. (B) STREET: 3330 Hillview Avenue (C) CITY: Palo Alto (D) STATE: CA (E) COUNTRY: USA (F) CODE: 94304 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: will be assigned (B) DATE OF SUBMISSION: submitted with the same (vii) DATA FROM THE PREVIOUS REQUEST: (A) SERIES NO. OF APPLICATION: US 08 / 467,125 (B) DATE OF SUBMISSION: June 6, 1995 (viii) POWDER / AGENT INFORMATION: (A) NAME: Luther, Barbara J. (B) REG. NUMBER. : 33954 (C) NO. REF. / PERMANENT: PF-0041 PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 415-855-0555 (B) TELEFAX: 415-852-0195 (2) INFORMATION FOR SEC ID NO: 1: (i) FEATURES OF SEQUENCE: (A) LENGTH: 1143 base pairs (B) TYPE: nucleic acid (C) STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) IMMEDIATE SOURCE: (A) COLLECTION: Liver (B) CLON: 86700 (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATGAGAAGTC ATACCATAAC AATGACGACA ACTTCAGTCA GCAGCTGGCC TTACTCCTCC 60 CACAGAATGC GCTTTATAAC CAATCATAGC GACCAACCGC CACAAAACTT CTCAGCAACA 120 CCAAATGTTA CTACCTGTCC CATGGATGAA AAATTGCTAT CTACTGTGTT AACCACATCC 180 TACTCTGTTA TTTTCATCGT GGGACTGGTT GGGAACATAA TCGCCCCCTA TGTATTTCTG 240 GGTATTCACC GTAAAAGAAA TTCCATTCAA ATTTATCTAC TTAACGTAGC CATTGCAGAC 300 CTCCTACTCA TCTTCTGCCT CCCTTTCCGA ATAATGTATC ATATTAACCA AAACAAGTGG 360 ACACTAGGTG TGATTCTGTG CAAGGTTGTG GGAACACTGC TTTATATGAA CATGTACATT 420 AGCATTATTT TGCTTGGATT CATCAGTTTG GATCGCTATA TAAAAATTAA TCGGTCTATA 480 CAGCAACGGA AGGCAATAAC AACCAAACAA AGTATTTATG TCTGTTGTAT AGTATGGATG 540 CTTGCTCTTG GTGGATTCCT AACTATGATT ATTTTAACAC TTAAGAAAGG AGGGCATAAT 600 TCCACAATGT GTTTCCATTA CAGAGATAAG CATAACGCAA AAGGAGAAGC CATTTTTAAC 660 TTCATTCTTG TGGTAATGTT CTGGCTAATT TTCTTACTAA TAATCCTTTC ATATATTAAG 720 ATTGGGAAGA ATCTATTGAG GATTTCTAAA AGGAGGTCAA AATTTCCTAA TTCTGGTAAA 7Í0 TATGCCACTA CAGCTCGTAA CTCCTTTATT GTACTTATCA mTTACTAT ATGTGTGGGT 8 0 CCCTATCATG CCTTTCGATT CATCTACATT TCTTCACAGC TAAATGTATC ATCTTGCTAC 900 TGGAAAGAAA TTGTTCACAA AACCAATGAG ATCATGCTGG TTCTCTCATC TTTCAATAGT 960 TGGTTAGATC CAGTCATGTA TTTCCTGATG TCCAGTAACA TTCGCAAAAT AATGTGCCAA 1020 CTTCTTTTTA GACGATTTCA AGGTGAACCA AGTAGGAGTG AAAGCACTTC AGAATTTAAA 1080 CCAGGATACT CCCTGCATGA TACATCTGTG GCAGGGAAAA TACAGTCTAG TTCTGAAAGT 1140 ACT 1143 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 381 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (vii) IMMEDIATE SOURCE : (A) COLLECTION: Liver (B) CLON: 86700 (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Mee Arg Ser His Thr He Thr Met Thr Thr Thr Ser Val Ser Ser Trp 1 5 10 15 Pro Tyr Ser Ser His Arg Met Arg Phe He Thr Asn His Ser Asp Gln 20 25 30 Pro Pro Gln Asn Phe Ser Wing Thr Pro Asn Val Thr Thr Cys Pro Met 35 40 45 Asp Glu Lys Leu Leu Ser Thr Val Leu Thr Thr Ser Tyr Ser Val He 50 55 60 Phe He Val Gly Leu Val Gly Asn He He Ala Pro Tyr Val Phe Leu 65 70 75 80 Gly He His Arg Lys Arg Asn Ser He Gln He Tyr Leu Leu Asn Val 85 90 95 Wing He Wing Asp Leu Leu Leu He Phe Cys Leu Pro Phe Arg He Met 100 105 lyr Tyr His He Asn Gln Asn Lys Trp Thr Leu Gly Val He Leu Cys Lys 115 120 125 Val Val Gly Thr Leu Leu Tyr Met Asn Met Tyr He Ser He He Leu 130 135 140 Leu Gly Phe He Ser Leu Aßp A rg Tyr He Lys He Asn Arg Ser He 145 150 1S5 160 Gln Gln Arg Lys Wing He Thr Thr Lys Gln Ser He Tyr Val Cys Cys 165 170 175 He Val Trp Met Leu Ala Leu Gly Gly Phe Leu Thr Met He He Leu 180 185 190 Thr Leu Lys Lys Gly Gly H s Asn Ser Thr Met Cys Phe His Tyr Arg 195 200 205 Asp Lys His Asn? The Lys Gly Glu Wing He Phe Asn Phe He Leu Val 210 215 220 Val Met Phe Trp Leu He Phe Leu Leu He He Leu Ser Tyr He Lys 225 230 235 240 He Gly Lys Asn Leu Leu Arg He Ser Lys Arg Arg Ser Lys Phe Pro 245 250 255 Asn Ser Gly Lys Tyr Wing Thr Thr Wing Arg Asn Being Phe He Val Leu 260 265 270 He He Phe Thr He Cys Val Gly Pro Tyr His Wing Phe Arg Phe He 275 280 285 Tyr He Ser Ser Gln Leu Asn Val Ser Ser Cys Tyr Trp Lys Glu He Val His Lys Thr Asn Glu He Met Leu Val Leu Ser Ser Phe Asn Ser 305 310 315 320 Trp Leu Asp Pro Val Met Tyr Phe Leu Met Ser Ser Asn He Arg Lys 325 330 335 He Met Cys Gln Leu Leu Phe Arg Arg Phe Gln Gly Glu Pro Ser Arg 340 345 350 Ser Glu Ser Thr Ser Glu Phe Lys Pro Gly Tyr Ser Leu Has Asp Thr 355 360 365 Ser Val Wing Gly Lys He Gln Ser Ser Ser Glu Ser Thr 370 375 380 (2) INFORMATION FOR SEQ ID NO "3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH 425 amino acids (B) TYPE-amino acid (C) STRING STRUCTURE" individual (D) linear TOPOLOGY (ii) TI PO DE MOLÉCU LA: peptide (vii) IMMEDIATE ENTITY: (A) COLLECTION: GenBank (B) CLON: 339677 (ix) SEQUENCE DESCRITION: SEC ID NO: 3: Met Gly Pro Arg Arg Leu Leu Leu Val Ala? Cys Phe Ser Leu Cys 1 5 10 15 Gly Pro Leu Leu Be Wing Arg Thr Arg Wing Arg Arg Pro Glu Ser Lys 20 25 30 Wing Thr Asn Wing Thr Leu? Sp Pro? Rg Ser Phe Leu Leu Arg Asn Pro 35 40 45 Asn? Sp Lys Tyr Glu Pro Phe Trp Glu Glu Glu Lys? sn Glu Ser 50 55 60 Gly Leu Thr Glu Tyr? rg Leu Val Ser He? sn Lys Ser Ser Pro Leu 65 70 75 80 Gln Lys Gln Leu Pro? The Phe He Ser Glu? Sp? The Ser Gly Tyr Leu 85 90 95 Thr Ser Ser Trp Leu Thr Leu Phe Val Pro Ser Val Tyr Thr Gly Val 100 105 110 Phe Val Val Ser Leu Pro Leu? Sn He Met? La He Val Val Phe He 115 120 125 Leu Lys Met Lys Val Lys Lys Pro Wing Val Val Tyr Met Leu His Leu 130 135 140 Wing Thr Wing Asp Val Leu Phe Val Ser Val Leu Pro Phe Lys He Ser 145 150 155 160 Tyr Tyr Phe Ser Gly Ser Asp Trp Gln Phe Gly Ser Glu Leu Cys Arg 165 170 175 Phe Val Thr Wing Wing Phe Tyr Cys Asn Met Tyr Wing Being He Leu Leu 180 185 190 Met Thr Val He Ser He Asp Arg Phe Leu Wing Val Val Tyr Pro Met 195 200 205 Gln Ser Leu Ser Trp Arg Thr Leu Gly Arg Wing Phe Thr Cys Leu 210 215 220 Wing He Trp Wing Leu Wing He Wing Gly Val Val Pro Leu Val Leu Lys 225 230 235 240 Glu Gln Thr He Gln Val Pro Gly Leu? Sn He Thr Thr Cys His? Sp 245 250 255 Val Leu? Sn Glu Thr Leu Leu Glu Gly Tyr Tyr? Tyr Tyr Phe Ser 260 265 270? Phe Ser? Val Phe Phe Phe Val Pro Leu He He Ser Thr Val 275 280 285 Cys Tyr Val Ser He He? Rg Cys Leu Being Ser? The Val? La? Sn 290 295 300? Rg Ser Lys Lys Ser? Rg? The Leu Phe Leu Ser? La? The Val Phe Cys 305 310 315 320 He Phe He He Cys Phe Gly Pro Thr? Sn Val Leu Leu He? His His 32S 330 335 Tyr Ser Phe Leu Ser His Thr Ser Thr Thr Glu? La? Tyr Phe? 340 345 350 Tyr Leu Leu Cys Val Cys Val Ser Ser He Ser Ser Cys He? Sp Pro 355 360 365 Leu He Tyr Tyr Tyr? The Ser Ser Glu Cys Gln? Rg Tyr Val Tyr Ser 370 375 380 He Leu Cys Cys Lys Glu Ser Ser? Sp Pro Ser Ser Tyr? Sn Ser Ser 385 390 395 400 Gly Gln Leu Met the Ser Lys Met? Sp Thr Cys Ser Ser? Sn Leu? Sn 405 410 415? Sn Ser He Tyr Lys Lys Leu Leu Thr 420 425

Claims (9)

1. - A purified polynucleotide encoding a polypeptide with the amino acid sequence shown in SEQ ID NO:

2. 2. The polynucleotide according to claim 1, wherein the amino acid sequence comprises SEQ ID NO: 1, or its complement.

3. A test for conditions or diseases associated with the expression of the thrombin receptor homolog (calr) in a biological sample comprising the steps of. a) combining the biological sample with the polynucleotide according to claim 1, or a fragment thereof, under conditions suitable for the formation of the hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the expression of the polynucleotide according to claim 1 in the biological sample.

4. An expression vector comprising the polynucleotide according to claim 1. 5 - A host cell transformed with the expression vector according to claim 4. 6 - A method for producing a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2, the method comprises the steps of: a) culturing the host cell according to claim 5, under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the culture of the host cell. 7. An antisense molecule comprising the nucleic acid sequence complementary to at least a portion of the polynucleotide according to claim 1. 8. A pharmaceutical composition comprising the antisense molecule according to claim 7 and a pharmaceutically excipient. acceptable. 9. A method for treating a subject with a condition or disease that involves altered expression of the thrombin receptor homologue, which comprises administering an effective amount of the pharmaceutical composition according to claim 8 to the subject. 10. A purified polypeptide comprising the amino acid sequence of SEQ ID NO: 2. 11. A polypeptide agonist according to claim 10. 12. A pharmaceutical composition comprising the agonist according to claim 11 and a pharmaceutically acceptable excipient. 13. A method for treating a subject with a condition or disease associated with the altered expression of the thrombin receptor homolog comprising administering an effective amount of the composition according to claim 12 to the subject. 14. An inhibitor of the polypeptide according to claim 10. 1

5. A pharmaceutical composition comprising the inhibitor according to claim 14 and a pharmaceutically acceptable excipient. 1

6. A method for treating a subject with a condition or disease associated with the altered expression homolog of the thrombin receptor comprising administering an effective amount of the pharmaceutical composition according to claim 15 to the subject. 1

7. An antibody specific for the purified polypeptide according to claim 10. 1

8. A diagnostic test for a condition or disease associated with the expression of the thrombin receptor homolog in a biological sample comprising the steps of: a) combining the biological sample with the antibody according to claim 17, under conditions suitable for the antibody to bind the polypeptide and form an antibody-polypeptide complex; and b) detecting the complex, wherein the presence of the complex correlates with the expression of the polypeptide in the biological sample. 1

9. A pharmaceutical composition comprising the antibody according to claim 17 and a pharmaceutically acceptable excipient. 20. A method for treating a subject with a condition or disease associated with altered expression of the thrombin receptor homolog comprising administering an effective amount of the pharmaceutical composition according to claim 19 to the subject.