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WO2001088135A2 - Proteines et acides nucleiques codant pour de telles proteines - Google Patents

Proteines et acides nucleiques codant pour de telles proteines Download PDF

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Publication number
WO2001088135A2
WO2001088135A2 PCT/US2001/015526 US0115526W WO0188135A2 WO 2001088135 A2 WO2001088135 A2 WO 2001088135A2 US 0115526 W US0115526 W US 0115526W WO 0188135 A2 WO0188135 A2 WO 0188135A2
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Prior art keywords
nucleic acid
polypeptide
amino acid
seq
protein
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PCT/US2001/015526
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English (en)
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WO2001088135A3 (fr
Inventor
Muralidhara Padigaru
William M. Grosse
Edward D. Szerkeres
Stacie J. Casman
John P. Alsobrook, Iii
Catherine E. Burgess
Sarah Taylor
Corine A. M. Vernet
Jason C. Baumgartner
Velizar T. Tchernev
Kimberly A. Spytek
Li Li
Suresh Shenoy
Ramesha Kekuda
Esha A. Gangolli
David J. Stone
Glennda Smithson
John R. Macdougall
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Curagen Corporation
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Priority to AU2001268060A priority Critical patent/AU2001268060A1/en
Publication of WO2001088135A2 publication Critical patent/WO2001088135A2/fr
Publication of WO2001088135A3 publication Critical patent/WO2001088135A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention generally relates to nucleic acids and polypeptides. More particularly, the invention relates to nucleic acids encoding novel G-protein coupled receptor (GPCR) polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.
  • GPCR G-protein coupled receptor
  • the invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides.
  • novel nucleic acids and polypeptides are referred to herein as GPCRX, or GPCR1, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6, GPCR7 and GPCR8 nucleic acids and polypeptides.
  • GPCRX nucleic acid or polypeptide sequences.
  • the invention provides an isolated GPCRX nucleic acid molecule encoding a GPCRX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43.
  • the GPCRX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a GPCRX nucleic acid sequence.
  • the invention also includes an isolated nucleic acid that encodes a GPCRX polypeptide, or a fragment, homolog, analog or derivative thereof.
  • the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44.
  • the nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43.
  • an oligonucleotide e.g., an oligonucleotid which includes at least 6 contiguous nucleotides of a GPCRX nucleic acid (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43) or a complement of said oligonucleotide.
  • a GPCRX nucleic acid e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43
  • GPCRX polypeptides SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44.
  • the GPCRX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human GPCRX polypeptide.
  • the invention also features antibodies that immunoselectively bind to GPCRX polypeptides, or fragments, homologs, analogs or derivatives thereof.
  • the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier.
  • the therapeutic can be, e.g., a GPCRX nucleic acid, a GPCRX polypeptide, or an antibody specific for a GPCRX polypeptide.
  • the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
  • the invention includes a method of producing a polypeptide by culturing a cell that includes a GPCRX nucleic acid, under conditions allowing for expression of the GPCRX polypeptide encoded by the DNA. If desired, the GPCRX polypeptide can then be recovered.
  • the invention includes a method of detecting the presence of a GPCRX polypeptide in a sample.
  • a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound.
  • the complex is detected, if present, thereby identifying the GPCRX polypeptide within the sample.
  • the invention also includes methods to identify specific cell or tissue types based on their expression of a GPCRX.
  • Also included in the invention is a method of detecting the presence of a GPCRX nucleic acid molecule in a sample by contacting the sample with a GPCRX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a GPCRX nucleic acid molecule in the sample.
  • the invention provides a method for modulating the activity of a
  • GPCRX polypeptide by contacting a cell sample that includes the GPCRX polypeptide with a compound that binds to the GPCRX polypeptide in an amount sufficient to modulate the activity of said polypeptide.
  • the compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
  • a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, or other disorders related to cell signal processing and metabolic pathway modulation.
  • the therapeutic can be, e.g., a GPCRX nucleic acid, a GPCRX polypeptide, or a GPCRX-specific antibody, or biologically-active derivatives or fragments thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: developmental diseases, MHCII and III diseases (immune diseases), taste and scent detectability Disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, Retinal diseases including those involving photoreception, Cell growth rate disorders; cell shape disorders, feeding disorders; control of feeding; potential obesity due to over-eating; potential disorders due to starvation (lack of appetite), noninsulin- dependent diabetes mellitus (NTDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HTV-2), pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; Albright Hereditary Ostoeodys
  • polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding GPCRX may be useful in gene therapy, and GPCRX may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uter
  • the invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders or other disorders related to cell signal processing and metabolic pathway modulation.
  • the method includes contacting a test compound with a GPCRX polypeptide and determining if the test compound binds to said GPCRX polypeptide. Binding of the test compound to the GPCRX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
  • Also within the scope of the invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders or other disorders related to cell signal processing and metabolic pathway modulation by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes.
  • the test animal expresses a recombinant polypeptide encoded by a GPCRX nucleic acid.
  • the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a GPCRX polypeptide, a GPCRX nucleic acid, or both, in a subject (e.g., a human subject).
  • the method includes measuring the amount of the GPCRX polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the GPCRX polypeptide present in a control sample.
  • An alteration in the level of the GPCRX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject.
  • the predisposition includes, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders.
  • the expression levels of the new polypeptides of the invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
  • the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a GPCRX polypeptide, a GPCRX nucleic acid, or a GPCRX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition.
  • the disorder includes, e.g., diabetes, metabolic disturbances associated with obesity, the metabolic syndrome X, anorexia, wasting disorders associated with chronic diseases, metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders.
  • the invention can be used in a method to identity the cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
  • the invention is based, in part, upon the discovery of novel nucleic acid sequences that encode novel polypeptides.
  • novel nucleic acids and their encoded polypeptides are referred to individually as GPCR1, GPCR2, GPCR3, GPCR4, GPCR5, GPCR6, GPCR7 and GPCR8.
  • GPCRX The nucleic acids, and their encoded polypeptides, are collectively designated herein as "GPCRX”.
  • novel GPCRX nucleic acids of the invention include the nucleic acids whose sequences are provided in Tables 1 A, ID, 2A, 2D, 2G, 3A, 3D, 4A, 5A, 5E, 5H, 5L, 6A, 6D, 6G, 6K, 7A, 8A, 8D, 8H, 8L and 8P, inclusive ("Tables 1A- 8P"), or a fragment, derivative, analog or homolog thereof.
  • novel GPCRX proteins of the invention include the protein fragments whose sequences are provided in Tables IB, IE, 2B, 2E, 2H, 3B, 3E, 4B, 5B, 5F, 51, 5M, 6B, 6E, 6H, 6L, 7B, 8B, 8E, 81, 8M and 8Q, inclusive ("Tables IB - 8Q").
  • the individual GPCRX nucleic acids and proteins are described below. Within the scope of this invention is a method of using these nucleic acids and peptides in the treatment or prevention of a disorder related to cell signaling or metabolic pathway modulation.
  • GPCRs G-Protein Coupled Receptor proteins
  • G protein-coupled receptors in a number of species. These receptors share a seven transmembrane domain structure with many neurotransmitter and hormone receptors, and are likely to underlie the recognition and G-protein-mediated transduction of various signals.
  • Human GPCR generally do not contain introns and belong to four different gene subfamilies, displaying great sequence variability. These genes are dominantly expressed in olfactory epithelium. See, e.g., Ben-Arie et al., Hum. Mol. Genet. 1994 3:229-235; and, Online Mendelian Inheritance in Man (OMIM) entry # 164342 (http://www.ncbi.nlm.nih.gov/entrez/ dispomim.cgi?).
  • the olfactory receptor (OR) gene family constitutes one of the largest GPCR multigene families and is distributed among many chromosomal sites in the human genome. See
  • Olfactory receptors constitute the largest family among G protein-coupled receptors, with up to 1000 members expected. See Vanderhaeghen et al, Genomics 39(3):239-46 (1997); Xie et al., Mamm. Genome l l(12):1070-78 (2000); Issel-Tarver et al., Proc. Natl. Acad. Sci. USA 93(20):10897-902 (1996).
  • the recognition of odorants by olfactory receptors is the first stage in odor discrimination.
  • chemoreceptors Other examples of seven membrane spanning proteins that are related to GPCRs are chemoreceptors. See Thomas et al., Gene 178(1-2): 1-5 (1996). Chemoreceptors have been identified in taste, olfactory, and male reproductive tissues. See id.; Walensky et al., J. Biol.
  • GPCR1 includes two novel G-protein coupled receptor (“GPCR”) proteins disclosed below.
  • GPCRla G-protein coupled receptor
  • GPCRlb GPCR1 protein coupled receptor
  • a disclosed GPCRla nucleic acid of 964 nucleotides (also referred to as AP001884_A) is shown in Table 1A.
  • the disclosed GPCRla open reading frame (“ORF") begins at the ATG initiation codon at nucleotides 39-41, shown in bold in Table 1 A.
  • the disclosed GPCRla ORF terminates at a TGA codon at nucleotides 933-935.
  • putative untranslated regions 5' to the start codon and 3' to the stop codon are underlined, and the start and stop codons are in bold letters.
  • Table 1A GPCRla nucleotide sequence (SEQ ID NO:l).
  • the Psort profile for GPCRla predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the most likely cleavage site for a GPCRla peptide is between amino acids 46 and 47, at: LTA-IL, based on the SignalP result.
  • GPCRla has a molecular weight of 33106.8.
  • Table IB Encoded GPCRla protein sequence (SEQ ID NO:2).
  • the amino acid sequence of GPCRla had high homology to other proteins as shown in Table IC.
  • the target sequence identified previously, Accession Number AP001884_A was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • a disclosed GPCRlb (also referred to as AP001884_A_dal) nucleic acid of 906 nucleotides is shown in Table ID.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 5-7 and ending with a TGA codon at nucleotides 899-901.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table ID, and the start and stop codons are in bold letters.
  • the disclosed encoded GPCRlb protein has 298 amino acid residues, referred to as the
  • GPCRlb protein is presented in Table IE using the one-letter amino acid code.
  • the Psort profile for GPCRlb predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the most likely cleavage site for a GPCRlb peptide is between amino acids 46 and 47, at: LTA-IL, based on the SignalP result.
  • GPCRlb has a molecular weight of 33068.7.
  • Table IE Encoded GPCRlb protein sequence (SEQ ID NO:4).
  • GPCRla and lb are related to each other as shown in the alignment listed in Table
  • GPCR 1A Protein Sequence (AP00 IHLTAILCAYGPVIVIYLQPNPSAL GSIIQILNNLVTPMLNPLIYSLRNKDVKSDQ)
  • GPCR IB Protein Sequence (AP0O IHLTAI CAYGPVIVIYLQPNPSA LGSIIQILNNLVTPMLNPLIYS RNKDVKSDQ!
  • GPCRla had homology to the amino acid sequences shown in the BLASTP data listed in Table II.
  • GPCR 1A gilll692587
  • CDD Conserved Domain Database
  • Table IK lists the domain description from DOMAIN analysis results against GPCRla. This indicates that the GPCRla sequence has properties similar to those of other proteins known to contain this domain as well as to the 377 amino acid 7tm domain itself.
  • the 7 transmembrane receptor family includes a number of different proteins, including, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • serotonin receptors include, for example, serotonin receptors, dopamine receptors, histamine receptors, andrenergic receptors, cannabinoid receptors, angiotensin II receptors, chemokine receptors, opioid receptors, G-protein coupled receptor (GPCR) proteins, olfactory receptors (OR), and the like.
  • GPCR G-protein coupled receptor
  • OR olfactory receptors
  • Some proteins and the Protein Data Base Ids/gene indexes include, for example: rhodopsin (129209); 5-hydroxytryptamine receptors; (112821, 8488960, 112805, 231454, 1168221, 398971, 112806); G protein-coupled receptors (119130, 543823, 1730143, 132206, 137159, 6136153, 416926, 1169881, 136882, 134079); gustatory receptors (544463, 462208); c-x-c chemokine receptors (416718, 128999, 416802, 548703, 1352335); opsins (129193, 129197, 129203); and olfactory receptor-like proteins (129091, 1171893, 400672, 548417).
  • the nucleic acids and proteins of GPCR1 are useful in potential therapeutic applications implicated in various GPCR- or olfactory receptor (OR)-related pathologies and/or disorders.
  • a cDNA encoding the G-protein coupled receptor-like protein may be useful in gene therapy, and the G-protein coupled receptor-like protein may be useful when administered to a subject in need thereof.
  • the novel nucleic acid encoding GPCR1 protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: developmental diseases, MHC ⁇ and III diseases (immune diseases), Taste and scent detectability Disorders, Burkitt's lymphoma, Corticoneurogenic disease, Signal Transduction pathway disorders, Retinal diseases including those involving photoreception, Cell Growth rate disorders; Cell Shape disorders, Feeding disorders;control of feeding; potential obesity due to over-eating; potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus (NIDDMl), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HTV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypo
  • compositions of the present invention will have efficacy for treatment of patients suffering from neoplasm, adenocarcinoma, lymphoma, prostate cancer, uterus cancer, immune response, AIDS, asthma, Crohn's disease, multiple sclerosis, and
  • the novel nucleic acid encoding the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below.
  • the disclosed GPCRla protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated GPCRla epitope is from about amino acids 160 to 170. In another embodiment, a GPCRla epitope is from about amino acids 220 to 240. In an additional embodiment, GPCRla epitopes are from about amino acids 280 to 290.
  • GPCR2 includes three novel G-protein coupled receptor-like proteins disclosed below. The disclosed proteins have been named GPCR2a, GPCR2b and GPCR2c.
  • GPCR2a A novel nucleic acid of 1051 nucleotides (AP001884_B) encoding a novel G-protein coupled receptor-like protein, referred to herein as GPCR2a, is shown in Table 2A.
  • GPCR2a polypeptide (SEQ ID NO:6) encoded by SEQ ID NO:5 is 311 amino acid residues and is presented using the one-letter code in Table 2B.
  • the Psort profile for GPCR2a predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400.
  • the most likely cleavage site for a GPCR2a peptide is between amino acids 51 and 52, at: VDS-HL., based on the SignalP result.
  • GPCR2a has a molecular weiglit of 34476.4.
  • the amino acid sequence of GPCR2a had high homology to other proteins as shown in Table 2C.
  • a novel nucleic acid of 1101 nucleotides (AP001884_C) encoding a novel G-protein coupled receptor-like protein is shown in Table 2D.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 76-78 and ending with a TAA codon at nucleotides 1009-1011.
  • Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 2D, and the start and stop codons are in bold letters.
  • the disclosed GPCR2b polypeptide (SEQ ID NO:8) encoded by SEQ ID NO:7 is 311 amino acid residues, and is presented using the one-letter code in Table 2E.
  • the Psort profile for GPCR2b predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of0.6400.
  • the most likely cleavage site for a GPCR2b peptide is between amino acids 51 and 52, at: VDS-HL., based on the SignalP result.
  • GPCR2b has a molecular weight of34568.7.
  • the amino acid sequence of GPCR2b had high homology to other proteins as shown in
  • GPCR2c the target sequence identified previously, Accession Number AP001884_C, was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • GPCR2c also referred to as CG54395-02
  • nucleic acid of 1401 nucleotides is shown in Table 2G.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 175-177 and ending with a TAA codon at nucleotides 1108-1110.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 2G, and the start and stop codons are in bold letters.
  • the disclosed nucleic acid sequence has 595 of 949 bases (62%) identical to a Homo sapiens T-cell receptor alpha mRNA (gb:GENBANK-ID:HUAE000658
  • the GPCR2c protein endcoded by SEQ ID NO:9 has 311 amino acid residues, referred to as the GPCR2c protein, is presented in Table 2H using the one-letter amino acid code.
  • the Psort profile for GPCR2c predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400.
  • the most likely cleavage site for a GPCR2c peptide is between amino acids 51 and 52, at: VDS-HL, based on the SignalP result.
  • GPCR2a, 2b and 2c are related to each other as shown in the alignment listed in Table 2J.
  • GPCR 2A Protein Sequence (AP00 iTNLSFIDM FSTV VPKML TLVSPSGRAISFHSCVAQLYFFHFLGSTECF YTVMSY GPCR 2B Protein Sequence (AP00 iTNLSFIDM FSTVTVPKMLMTLVSPSGRAISFHSCVAQLYFFHFLGSTECFLYTVMSYI GPCR 2C Protein Sequence (CG54
  • GPCR 2A Protein Sequence (APOO [53CTS ⁇ «kM ⁇ Ji]F& ⁇ BlB iff ⁇ f3- ⁇ GPCR 2B Protein Sequence (APOO 'PI KLACADTSANV VIFVDIGIVASGCFV IVLSYVSIVCSILRIRTSDGRRRAFQTI GPCR 2C Protein Sequence (CG54 »PILK ACADTSANVMVIFVDEGIVASGCFVLIV SYVSIVCSILRIRTSDGRRRAFQTI
  • GPCR 2A Protein Sequence (APOO fflff ⁇ tt. ff?i- x ffl F ⁇ ti R H w R
  • GPCR 2B Protein Sequence (APOO WIY RPGSMDAMDGVVAIFYTVLTPLLNPVVYTLRNKEVKKAVL
  • GPCR 2C Protein Sequence (CG54 SHCIVVLCFFVPCVVIYLRPGSMDAMDGVVAIFYTVLTPLLNPVVYTLRNKEVKKAV
  • GPCR2a had homology to the amino acid sequences shown in the BLASTP data listed in Table 2K.
  • GPCR 2A g ⁇ LKI ⁇ RD ⁇ 3vAHSQGE gi
  • the GPCR2c gene maps to chromosome 11 and is expressed in at least the following tissues: Apical microvilli of the retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate
  • GPCR2a-c include the nucleic acids whose sequences are provided in Table 2A, 2D and 2G, or a fragment thereof.
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 2A, 2D and 2G while still encoding a protein that maintains its G-Protein Coupled Receptor-like activities and physiological functions, or a fragment of such a nucleic acid.
  • GPCR2a-c further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described.
  • GPCR2a-c additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
  • modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject, hi the mutant or variant nucleic acids, and their complements, up to about 38 percent of the bases may be so changed.
  • nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • a protein therapeutic such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), (v) an agent promoting tissue regeneration in vitro and in vivo, and (vi) a biological defense weapon.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: developmental diseases, MHCII and III diseases (immune diseases), Taste and scent detectability Disorders, Burkitt's lymphoma, Corticoneurogenic disease, Signal Transduction pathway disorders, Retinal diseases including those involving photoreception, Cell Growth rate disorders; Cell Shape disorders, Feeding disorders;control of feeding; potential obesity due to over-eating; potential disorders due to starvation (lack of apetite), noninsulin-dependent diabetes mellitus (NTDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HTV-l or HTV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the OR -like protein may be useful in gene therapy, and the OR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HTV-l or H ⁇ V-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma
  • novel nucleic acid encoding the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the
  • GPCR2a-c proteins have multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated GPCR2a-c epitope is from about amino acids 55 to 65.
  • a GPCR2a-c epitope is from about amino acids 120 to 130.
  • GPCR2a-c epitopes are from about amino acids 165 to 185, from about amino acids 220 to 240 and from about amino acids 285 to 310.
  • GPCR3 includes two nucleic acids disclosed below.
  • the disclosed nucleic acids encode a GPCR-like protein.
  • the disclosed GPCR3a (also referred to herein as 645i8A) is encoded by a nucleic acid, 1011 nucleotides long. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 20-22 and ending with a TAA codon at nucleotides 977-979. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 3 A, and the start and stop codons are in bold letters.
  • GPCR3a polypeptide (SEQ ID NO:12) encoded by SEQ ID NO:l l has 319 amino acid residues, referred to as GPCR3a protein, is presented using the one-letter amino acid code in Table 3B.
  • the Psort profile for GPCR3a predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the most likely cleavage site for a GPCR3a peptide is between amino acids 46 and 47, at: ALG-AN., based on the SignalP result.
  • GPCR3a has a molecular weight of 35453.0.
  • the amino acid sequence of GPCR3a had high homology to other proteins as shown in Table 3C.
  • the disclosed GPCR3b (also referred to herein as CG52784-02) is encoded by a nucleic acid, 973 nucleotides long. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 5-7 and ending with a TAA codon at nucleotides 962-964. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 3D, and the start and stop codons are in bold letters.
  • the disclosed nucleic acid sequence has 504 of 808 bases (62%) identical to a Mus musculus odorant receptor S18 mRNA (gb:GENBANK-ID:AF121975
  • the GPCR3b polypeptide (SEQ ID NO:14) encoded by SEQ ID NO:13 is presented using the one-letter amino acid code in Table 3E.
  • the SignalP, Psort and/or Hydropathy profile for the disclosed GPCR3b GPCR-like protein predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the most likely cleavage site for a GPCR3b peptide is between amino acids 46 and 47, at: ALG-AN., based on the SignalP result.
  • GPCR3a also has 184 of 290 residues (63%) identical to and 219 of 290 residues (75%) positive with olfactory receptor S83 from mouse [Mus musculus] (GenBank Ace. No.:AAK00590.1).
  • Table 3H lists the domain description from DOMAIN analysis results against GPCR3a. This indicates that the GPCR3a sequence has properties similar to those of other proteins known to contain this domain as well as to the 377 amino acid 7tm domain itself.
  • the GPCR3b is expressed in at least the following tissues: Apical microvilli of the retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogemc cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and ITI nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum
  • tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
  • the nucleic acids and proteins of GPCR3a and GPCR3b are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR- related pathological disorders, described further below.
  • a cDNA encoding the olfactory receptor-like protein may be useful in gene therapy, and the olfactory receptor-like protein may be useful when administered to a subject in need thereof.
  • the protein similarity information, expression pattern, and map location for the Olfactory Receptor-like protein and nucleic acid disclosed herein suggest that this Olfactory Receptor may have important structural and/or physiological functions characteristic of the Olfactory Receptor family. Therefore, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: developmental diseases, MHCII and III diseases (immune diseases), taste and scent detectability disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, retinal diseases including those involving photoreception, cell growth rate disorders, cell shape disorders, feeding disorders, potential obesity due to over-eating, potential disorders due to starvation (lack of appetite), noninsulin-dependent diabetes mellitus (NTDDMl), bacterial, fungal, protozoal and viral infections (particularly infections caused by HTV-l or HTV-2), pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension,
  • polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the OR -like protein may be useful in gene therapy, and the OR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome.
  • cancer including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer
  • GPCR-3 diseases and disorders are contemplated.
  • the novel nucleic acid encoding the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below.
  • the disclosed GPCR3a and 3b proteins have multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated GPCR3a and 3b epitope is from about amino acids 165 to 185.
  • a GPCR3a and 3b epitope is from about amino acids 235 to 245.
  • GPCR3a and 3b epitopes are from amino acids 310 to 320.
  • the disclosed novel GPCR4 nucleic acid of 1103 nucleotides (also referred to as 645i8_B) is shown in Table 4A.
  • An ORF begins with an ATG initiation codon at nucleotides 42-44 and ends with a TGA codon at nucleotides 1014-1016.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 4A, and the start and stop codons are in bold letters.
  • the GPCR4 protein encoded by SEQ ID NO: 15 has 324 amino acid residues and is presented using the one-letter code in Table 4B.
  • the Psort profile for GPCR4 predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • GPCR4 has a molecular weight of 36125.5.
  • GPCR4 also has homology to the proteins shown in the BLASTP data in Table 4D.
  • This BLASTP data is displayed graphically in the Clustal W in Table 4E.
  • Table 4F lists the domain description from DOMAIN analysis results against GPCR4. This indicates that the GPCR4 sequence has properties similar to those of other proteins known to contain this domain as well as to the 377 amino acid 7tm domain itself.
  • GPCR4 The nucleic acids and proteins of GPCR4 are useful in potential therapeutic applications implicated in various GPCR-related pathological disorders and/or OR-related pathological disorders, described further below.
  • a cDNA encoding the GPCR (or olfactory- receptor) like protein may be useful in gene therapy, and the receptor -like protein may be useful when administered to a subject in need thereof.
  • the nucleic acids and proteins of the invention are also useful in potential therapeutic applications used in the treatment of developmental diseases, MHC ⁇ and III diseases (immune diseases), taste and scent detectability disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, retinal diseases including those involving photoreception, cell growth rate disorders, cell shape disorders, feeding disorders, potential obesity due to over-eating, potential disorders due to starvation (lack of appetite), noninsulin-dependent diabetes mellirus (NiDDMl), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, allergies, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease, multiple sclerosis, Albright hereditary ostoeodystrophy,
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the GPCR-like protein may be useful in gene therapy, and the GPCR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from developmental diseases, MHCII and III diseases (immune diseases), taste and scent detectability disorders, Burkitt's lymphoma, corticoneurogenic disease, signal transduction pathway disorders, retinal diseases including those involving photoreception, cell growth rate disorders, cell shape disorders, feeding disorders, potential obesity due to over-eating, potential disorders due to starvation (lack of appetite), noninsulin-dependent diabetes mellitus (NTDDM1), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, allergies, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease, multiple sclerosis, Albright hereditary ostoeodyst
  • novel nucleic acid encoding GPCR-like protein, and the GPCR- like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods..
  • novel nucleic acid encoding the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below.
  • the disclosed GPCR4 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated GPCR4 epitope is from about amino acids 10 to 20.
  • a contemplated GPCR4 epitope is from about amino acids 10 to 20.
  • a contemplated GPCR4 epitope is from about amino acids 10 to 20.
  • a contemplated GPCR4 epitope is from about amino acids 10 to 20.
  • a contemplated GPCR4 epitope is from about amino acids 10
  • GPCR4 epitope is from about amino acids 175 to 190. In additional embodiments, GPCR4 epitopes are from about amino acids 230 to 255, from about amino acids 270 to 280 and from about amino acids 300 to 320. GPCR5
  • the disclosed novel GPCR5a nucleic acid of 1241 nucleotides (also referred to as 645i8_C) is shown in Table 5A.
  • An open reading frame begins with an ATG initiation codon at nucleotides 129-131 and ends with a TAA codon at nucleotides 1089-1091.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 5A, and the start and stop codons are in bold letters.
  • the GPCR5a protein encoded by SEQ ID NO: 17 has 320 amino acid residues, and is presented using the one-letter code in Table 5B (SEQ ID NO: 18).
  • the Psort profile for GPCR5a predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the most likely cleavage site for a GPCR5a peptide is between amino acids 51 and 52, at: ALP-AV., based on the SignalP result.
  • GPCR5a has a molecular weight of 34413.5.
  • the target sequence identified previously, Accession Number 645i8_C was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a wide range of cDNA libraries.
  • the resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the sequence reported below, winch is designated Accession Number 645i8C_dal .
  • the disclosed novel GPCR5b nucleic acid of 974 nucleotides (also referred to as 645i8C_dal) is shown in Table 5E.
  • An open reading frame begins with an ATG initiation codon at nucleotides 6-8 and ends with a TAA codon at nucleotides 966-968.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 5E, and the start and stop codons are in bold letters.
  • Table 5E GPCR5b Nucleotide Sequence (SEQ ID NO:19)
  • the GPCR5b protein encoded by SEQ ID NO: 19 has 320 amino acid residues, and is presented using the one-letter code in Table 5F (SEQ ID NO:20).
  • the Psort profile for GPCR5b predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the most likely cleavage site for a peptide is between amino acids 51 and 52, at: ALP-AV, based on the SignalP result.
  • the molecular weight of GPCR5b is 34483.6 Daltons.
  • the target sequence identified previously, Accession Number 645i8_C was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a wide range of cDNA libraries. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the sequence reported below, which is designated Accession Number 645i8Cl.
  • the disclosed novel GPCR5c nucleic acid of 1241 nucleotides (also referred to as 645i8Cl) is shown in Table 5H.
  • An open reading frame begins with an ATG initiation codon at nucleotides 129-131 and ends with a TAA codon at nucleotides 1089-1091.
  • Aputative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 5H, and the start and stop codons are in bold letters.
  • the GPCR5c protein encoded by SEQ ID NO:21 has 320 amino acid residues, and is presented using the one-letter code in Table 51 (SEQ ID NO:22).
  • the Psort profile for GPCR5c predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the most likely cleavage site for a peptide is between amino acids 51 and 52, at: ALP-AV, based on the SignalP result.
  • the molecular weight of GPCR5c is 34383.5 Daltons.
  • the target sequence identified previously, Accession Number 645i8_C was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a wide range of cDNA libraries.
  • the resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the sequence reported below, which is designated Accession Number AL078595_da2.
  • the disclosed novel GPCR5d nucleic acid of 1240 nucleotides (also referred to as AL078595_da2) is shown in Table 5L.
  • An open reading frame begins with an ATG initiation codon at nucleotides 129-131 and ends with a TAA codon at nucleotides 1089-1091.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 5L, and the start and stop codons are in bold letters.
  • the disclosed nucleic acid sequence for GPCR5d has 582 of 943 bases (61%) identical to a Mus musculus odorant receptor S19 mRNA (gb:GENBANK- ID:AF121976
  • the GPCR5d protein encoded by SEQ ID NO:23 has 320 amino acid residues, and is presented using the one-letter code in Table 5M (SEQ ID NO:24).
  • the Psort and Hydropathy profile for GPCR5d predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the most likely cleavage site for a peptide is between amino acids 51 and 52, at: ALP-AV, based on the SignalP result.
  • GPCR5a also has homology to the proteins shown in the BLASTP data in Table 5P.
  • Table 5R lists the domain description from DOMAIN analysis results against GPCR5a. This indicates that the GPCR5a sequence has properties similar to those of other proteins known to contain this domain as well as to the 377 amino acid 7tm domain itself.
  • GPCR 5A 7 transmembrane receptor (rhod _ The GPCR5 family members have strong homology for each other as shown in Table
  • GPCR 5A Protein Sequence (645i lAETLQLNSTFLHPNFFILTGFPGLGSAQTWLTLVFGPIYLLALLGNGALPAVVWIDST]
  • GPCR 5B Protein Sequence (645i ⁇ AET QLNSTFLHPNFFILTGFPGLGSAQTWLTLVFGPIY LALLGNGALPAVV IDST1
  • GPCR 5C Protein Sequence (6451 IAETLQNSTFLHPNFFI TGFPG GSAQTWLTLVFGPIY AL GNGALPAVVWIDSTJ
  • GPCR 5D Protein Sequence (AL07 1AETLQLNSTFLHPNFFILTGFPGLGSAQTWLTLVFGPIYLLALLGNGALPAVVWIDST]
  • GPCR 5A Protein Sequence (645i FPLLVA FEHFQAKTIG GPCR 5B Protein Sequence (645i JAMACDRAAAIGRP HYPVLVTKACVGYAALALALKAVAIVVPFPL VAKFEHFQAKTIG GPCR 5C Protein Sequence (6451 .AMACDRAAAIGRPLHYPV VTKACVGYAALALALKAVAIVVPFPLLVAKFEHFQAKTIG GPCR 5D Protein Sequence (AL07 AMACDRAAAIGRPLHYPVLVTKACVGYAALALALKAVAIVVPFPLLVAKFEHFQAKTIG
  • GPCR 5A Protein Sequence (645i GPCR 5B Protein Sequence (645i CAFGTCSSHICVI@AFYIPG FSYLMHRFGHHTVPKPVHILLSNIY LLPPALNPLIYG2
  • GPCR 5C Protein Sequence 6451 CAFGTCSSHICVILAFYIPGLFSYL0HRFGHHTVPKPVHILLSNIYLLLPPALNPLIYGA
  • GPCR5d is expressed in at least the following tissues: Apical microvilli of the retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leulcocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal
  • GPCR5a-d may have important structural and/or physiological functions characteristic of the Olfactory Receptor family and the GPCR family. Therefore, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: developmental diseases, MHCII and III diseases (immune diseases), Taste and scent detectability Disorders, Burkitt's lymphoma, Corticoneurogenic disease, Signal Transduction pathway disorders, Retinal diseases including those involving photoreception, Cell Growth rate disorders; Cell Shape disorders, Feeding disorders; control of feeding; potential obesity due to over-eating; potential disorders due to starvation (lack of appetite), noninsulin-dependent diabetes mellitus (NTDDMl), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeody
  • DPLA Dentatorubro-pallidoluysian atrophy
  • Acrocallosal syndrome and dyskinesias such as Huntington's disease or Gilles de la
  • polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the OR -like protein may be useful in gene therapy, and the OR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-l or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer
  • novel nucleic acid encoding OR-like protein, and the OR-like protein of the invention, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti- GPCRX Antibodies" section below.
  • GPCR5a and 5d proteins have multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated GPCR5a and 5d epitope is from about amino acids 170 to 180.
  • a GPCR5a and 5d epitope is from about amino acids 230 to 245.
  • GPCR5a and 5d epitopes are from about amino acids 260 to 280 and about amino acids 290-320.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
  • a disclosed novel GPCR6a nucleic acid of 1201 nucleotides (also referred to as 645i_8D) is shown in Table 6A.
  • An open reading frame begins with an ATG initiation codon at nucleotides 111-113 and ends with a TGA codon at nucleotides 1104-1106.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 6 A, and the start and stop codons are in bold letters.
  • the GPCR6a protein encoded by SEQ ID NO:25 has 331 amino acid residues, and is presented using the one-letter code in Table 6B (SEQ ID NO:26).
  • the Psort profile for GPCR6a predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400.
  • the most likely cleavage site for a GPCR6a peptide is between amino acids 51 and 52, at: VLG-NS., based on the SignalP result.
  • GPCR6a has a molecular weight of 37159.3.
  • GPCR6a protein sequence (SEQ ID NO:26).
  • GPCR6a The amino acid sequence of GPCR6a had high homology to other proteins as shown in Table 6C. Table 6C. BLASTX results for GPCR6a
  • the target sequence identified previously, Accession Number 645i8_D was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in PCR amplifications based on a library containing a wide range of cDNA species.
  • GPCR6b Accession Numbers 6458iD_dal
  • GPCR6c CG50231-01
  • GPR6d CG50171-01
  • a disclosed novel GPCR6b nucleic acid of 1004 nucleotides (also referred to as 6458iD_dal) is shown in Table 6D.
  • An open reading frame begins with an ATG initiation codon at nucleotides 5-7 and ends with a TGA codon at nucleotides 998-1000.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 6D, and the start and stop codons are in bold letters.
  • Table 6D GPCR6b Nucleotide Sequence (SEQ ID NO-.27)
  • the GPCR6b protein encoded by SEQ ID NO:27 has 331 amino acid residues, and is presented using the one-letter code in Table 6E (SEQ ID NO:28).
  • the SignalP, Psort and/or Hydropathy profile for GPCR6b predict that GPCR ⁇ b has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400.
  • SignalP results predict that GPCR ⁇ b is cleaved between position 51 and 52 of SEQ ID NO:28, i.e., in the amino acid sequence VLG-NS. This is typical of this type of membrane protein.
  • the molecular weight of GPCR ⁇ b is 37088.2 Daltons.
  • the disclosed, novel GPCR ⁇ c nucleic acid of 1124 nucleotides also referred to as
  • CG50231-01 is shown in Table 6G.
  • An open reading frame begins with an ATG initiation codon at nucleotides 90-92 and ends with a TGA codon at nucleotides 1059-1061.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 6G, and the start and stop codons are in bold letters.
  • the disclosed nucleic acid sequence for GPCR ⁇ c has 688 of 996 bases (69%) identical to a Mus musculus odorant receptor S18 mRNA (gb:GENBANK- ID:AF121975
  • the GPCR ⁇ c protein encoded by SEQ ID NO:29 has 323 amino acid residues, and is presented using the one-letter code in Table 6H (SEQ ID NO:30).
  • the SignalP, Psort and/or Hydropathy profile for GPCR ⁇ c predict that GPCR ⁇ c has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400.
  • the SignalP shows a signal sequence is coded for in the first 43 amino acids with a cleavage site between amino acids 43 and 44 of SEQ ID NO:30, i.e., in the amino acid sequence VLG-NS. This is typical of this type of membrane protein.
  • the full amino acid sequence of the protein of the invention was found to have 220 of 315 amino acid residues (69%) identical to, and 254 of 315 amino acid residues (80%) similar to, the 321 amino acid residue ODORANT RECEPTOR SI 8 from Mus musculus (SPTREMBL-ACC:Q9 U89) BLASTP results include those listed in Table 61.
  • the disclosed novel GPCR ⁇ d nucleic acid of 1006 nucleotides (also referred to as CG50171-01) is shown in Table 6K.
  • An open reading frame begins with an ATG initiation codon at nucleotides 6-8 and ends with a TGA codon at nucleotides 999-1001.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 6K, and the start and stop codons are in bold letters.
  • the disclosed nucleic acid sequence for GPCR ⁇ d has 680 of 984 bases (69%) identical to a Mus musculus odorant receptor S18 mRNA (gb:GENBANK- ID:AF121975
  • the GPCR ⁇ d protein encoded by SEQ ID NO:31 has 331 amino acid residues, and is presented using the one-letter code in Table 6L (SEQ ID NO:32).
  • the SignalP, Psort and/or Hydropathy profile for GPCR ⁇ d predict that GPCR ⁇ d has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6400.
  • the most likely cleavage site for a peptide is between amino acids 51 and 52, at: VLG-NS, based on the SignalP result. This is typical of this type of membrane protein.
  • the full amino acid sequence of the protein of the invention was found to have 221 of 318 amino acid residues (69%>) identical to, and 254 of 318 amino acid residues (79%) similar to, the 321 amino acid residue ODORANT RECEPTOR S 18 from Mus musculus (SPTREMBL-ACC:Q9WU89)
  • GPCR ⁇ a also has homology to the proteins shown in the BLASTP results in Table 6O.
  • Table 6Q lists the domain description from DOMAIN analysis results against GPCR ⁇ a. Tins indicates that the GPCR ⁇ a sequence has properties similar to those of other proteins known to contain this domain as well as to the 377 amino acid 7tm domain itself.
  • GPCR 6A Protein Sequence (645i ⁇ ERTIKCIMSHTNVTIFHPAVFVLPGIPG EAYHIW SIPLC IYITAVLGNSI IVVIV GPCR 6B Protein Sequence (6458 IERTIKCIMSHTNVTIFHPAVFVLPGIPGS5@AYHIWLSIP CLIYITAVLGNSILIVVIV GPCR 6C Protein Sequence (CG50 4SHTNVTIFHPAVFVLPGIPG EAYHIWLSIP CLIYITAV GNSI IVVI 1 GPCR 6D Protein Sequence (CG50 IERTIKCIMSHTNVTIFHPAVFV PGIPG EAYHIWLSIP CLIYITAVLGNSILIVVI ⁇ I
  • proteins that are homologous to any one member of the family are also largely homologous to the other members except where the sequences are different as shown above in Table 6R.
  • GPCR ⁇ is expressed in at least the following tissues: Apical microvilli of the retinal pigment epithelium, arterial (aortic), basal forebrain, brain, Burkitt lymphoma cell lines, corpus callosum, cardiac (atria and ventricle), caudate nucleus, CNS and peripheral tissue, cerebellum, cerebral cortex, colon, cortical neurogenic cells, endothelial (coronary artery and umbilical vein) cells, palate epithelia, eye, neonatal eye, frontal cortex, fetal hematopoietic cells, heart, hippocampus, hypothalamus, leukocytes, liver, fetal liver, lung, lung lymphoma cell lines, fetal lymphoid tissue, adult lymphoid tissue, Those that express MHC II and III nervous, medulla, subthalamic nucleus, ovary, pancreas, pituitary, placenta, pons, prostate, putamen, serum, skeletal muscle,
  • GPCR6a-d may have important structural and/or physiological functions characteristic of the Olfactory Receptor family and the GPCR family. Therefore, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: developmental diseases, MHCII and III diseases (immune diseases), Taste and scent detectability Disorders, Burkitt's lymphoma, Corticoneurogenic disease, Signal Transduction pathway disorders, Retinal diseases including those involving photoreception, Cell Growth rate disorders; Cell Shape disorders, Feeding disorders; control of feeding; potential obesity due to over-eating; potential disorders due to starvation (lack of appetite), noninsulin-dependent diabetes mellitus (NIDDMl), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-l or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension,
  • DPLA Dentatorubro-pallidoluysian atrophy
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the OR -like protein may be useful in gene therapy, and the OR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-l or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below.
  • the disclosed GPCR6a-d proteins have multiple hydrophilic regions, each of which can be used as an immunogen.
  • contemplated GPCR6a-d epitopes are from about amino acids 225 to 245.
  • GPCR6a-d epitopes are from about amino acids 260 to 270. In additional embodiments, GPCR6a-d epitopes are from amino acids 275 to 280 and 290 to 320. These novel proteins also have value in the development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
  • a disclosed novel GPCR7 nucleic acid of 1102 nucleotides (also referred to as 645i8_E) is shown in Table 7 A.
  • An open reading frame begins with an ATG initiation codon at nucleotides 69-71 and ends with a TAG codon at nucleotides 954-956.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 7A, and the start and stop codons are in bold letters.
  • the GPCR7 protein encoded by SEQ ID NO:33 has 295 amino acid residues, and is presented using the one-letter code in Table 7B (SEQ ID NO:34).
  • the Psort profile for GPCR7 predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • GPCR7 has a molecular weight of 32704.3.
  • the amino acid sequence of GPCR7 had high homology to other proteins as shown in Table 7C.
  • GPCR7 also has strong homology to the following proteins shown in the BLASTP data in Table 7D.
  • the homology is displayed graphically in the Clustal W shown in Table 7E.
  • Table 7F lists the domain description from DOMAIN analysis results against GPCR7.
  • GPCR 7 [vPVLAAQgHYCSRlSEI-DQCLJ8sN GVTSLACDDTT ⁇ NRF YQlTflfflvWVVfflGSD Gffl
  • GPCR7 may have important structural and/or physiological functions characteristic of the Olfactory Receptor family and the GPCR family. Therefore, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • the novel nucleic acid encoding GPCR7, and the GPCR7 protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • GPCR7 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in bacterial, fungal, protozoal and viral infections
  • Neoplasm adenocarcinoma; lymphoma; prostate cancer; uterus cancer
  • anorexia bulimia
  • asthma Parkinson's disease
  • acute heart failure hypotension
  • hypertension hypertension
  • urinary retention osteoporosis
  • Crohn's disease Crohn's disease
  • multiple sclerosis multiple sclerosis
  • Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-l or HTV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias,
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • the novel nucleic acid encoding the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below.
  • the disclosed GPCR7 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated GPCR7 epitope is from about amino acids 160 to 180.
  • a GPCR7 epitope is from about amino acids 185 to 195.
  • GPCR7 epitopes are from about amino acids 230 to 240 and from about amino acids 260 to 270.
  • a disclosed novel GPCR8a nucleic acid of 1201 nucleotides (also referred to as 17e20) is shown in Table 8A.
  • An open reading frame begins with an ATG initiation codon at nucleotides 105-107 and ends with a TGA codon at nucleotides 1053-1055.
  • Aputative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 8A, and the start and stop codons are in bold letters.
  • the GPCR8a protein encoded by SEQ ID NO:35 has 345 amino acid residues, and is presented using the one-letter code in Table 8B (SEQ ID NO:36).
  • the Psort profile for GPCR8a predicts that this sequence has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.8000.
  • the most likely cleavage site for a GPCR8a peptide is between amino acids 21 and 22, at: STG-RN., based on the SignalP result.
  • GPCR ⁇ a has a molecular weight of 38472.5.
  • GPCR8a The amino acid sequence of GPCR8a had high homology to other proteins as shown in Table 8C. Table 8C. BLASTX results for GPCR8a
  • the target sequence identified previously, Accession Number 17e20 was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • 17e20_A_dal is shown in Table 8D.
  • An open reading frame begins with an ATG initiation codon at nucleotides 5-7 and ends with a TGA codon at nucleotides 953-955.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 8D, and the start and stop codons are in bold letters.
  • the GPCR8b protein encoded by SEQ ID NO:37 has 316 amino acid residues, and is presented using the one-letter code in Table 8E (SEQ ID NO:38).
  • the SignalP, Psort and/or Hydropathy profile for GPCR8b predict that GPCR8b has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the SignalP predicts a cleavage site at the sequence LLG-NS between amino acids 41 and 42.
  • the Molecular weight of GPCR8b is 35028.5 Daltons.
  • GPCR8c In the present invention, the target sequence identified previously, Accession Number
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • An open reading frame begins with an ATG initiation codon at nucleotides 6-8 and ends with a TGA codon at nucleotides 954-956.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 8H, and the start and stop codons are in bold letters.
  • the GPCR8c protein encoded by SEQ ID NO:39 has 316 amino acid residues, and is presented using the one-letter code in Table 81 (SEQ ID NO:40).
  • the SignalP, Psort and/or Hydropathy profile for GPCR8c predict that GPCR8c has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the SignalP predicts a cleavage site at the sequence LLG-NS between amino acids 41 and 42.
  • the Molecular weight of GPCR8c is 35044.6 Daltons.
  • the target sequence identified previously, Accession Number 17e20 was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • the disclosed novel GPCR8d nucleic acid of 963 nucleotides (also referred to as CG50343-01) is shown in Table 8L.
  • An open reading frame begins with an ATG initiation codon at nucleotides 6-8 and ends with a TGA codon at nucleotides 954-956.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 8L, and the start and stop codons are in bold letters.
  • the disclosed nucleic acid sequence for GPCR8d has 544 of 784 bases (69%) identical to a Mus musculus or37a gene mRNA (gb:GENBANK-ID:MMU133424
  • the GPCR8d protein encoded by SEQ ID NO:41 has 316 amino acid residues, and is presented using the one-letter code in Table 8M (SEQ ID NO:42).
  • the SignalP, Psort and/or Hydropathy profile for GPCR8d predict that GPCR8d has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the SignalP predicts a cleavage site at the sequence LLG-NS between amino acids 41 and 42.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • the disclosed novel GPCR8e nucleic acid of 1115 nucleotides (also referred to as 17e20_dal) is shown in Table 8P.
  • An open reading frame begins with an ATG initiation codon at nucleotides 50-52 and ends with a TGA codon at nucleotides 998-1000.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon are underlined in Table 8P, and the start and stop codons are in bold letters.
  • the disclosed nucleic acid sequence for GPCR8e has 551 of 796 bases (69%) identical to a Mus niusculus or37a gene mRNA (gb:GENBANK-ID:MMU133424
  • the GPCR8e gene maps to chromosome 9.
  • the GPCR ⁇ e protein encoded by SEQ ID NO:43 has 316 amino acid residues, and is presented using the one-letter code in Table 8Q (SEQ ID NO:44).
  • the SignalP, Psort and/or Hydropathy profile for GPCR8e predict that GPCR8e has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • the SignalP predicts a cleavage site at the sequence LLG-NS between amino acids 41 and 42.
  • GPCR8a also had high homology to the proteins described in the BLASTP data in
  • GPCR 8A Coding Sequence ⁇ 17e20 AGTATTTTCCTAACTAAATGAATATCACACTTTATTCATTTCTTTATGTCCTCAGATTAA GPCR 8B Coding Sequence (17e20 GPCR 8C Coding Sequence (ba386 GPCR 8D Coding Sequence (CG503 GPCR 8E Coding Sequence (17e20 ATTAA
  • GPCR 8A Coding Sequence (I7e20 ATGAAGGGAATAGTACTGGAAGAAATATAGATGAAAGAAAI GAAAATGCAAGGAGAAAACT,
  • GPCR 8A Coding Sequence (17e20 llUft UftlrTl ⁇ iAfciU ⁇ T11 riTT TTbbAUbUA.1.TXTCUCAi rAUUUAb ⁇ TTAbAAbTUb
  • GPCR 8B Coding Sequence (17e20 TCACCATTTGGAGCATTTTTTTCTTGGAGGGATTTTCCCAGTACCCAGGBTTAGAAGTGG
  • GPCR 8C Coding Sequence (ba386 TCACCATTTGGAGCATTTTTTTCTTGGAGGGATTTTCCCAGTACCCAGGGTTAGAAGTGG
  • GPCR 8D Coding Sequence (CG503 CACCATTTGGAGCATTTTTTTCTTGGAGGGATTTTCCCAGTACCCAGGGTTAGAAGTGG
  • GPCR 8A Coding Sequence (17e20 TTCTCTTCGTCTTCAGCCTTGTAATGTATCTGACAACGCTCTTGGGCAACAGCACTCTTA
  • GPCR 8A Coding Sequence (17e20 TTTTGATCACTATCCTAGATTCACGCCTTAAAACCCCCATGTACTTATTCCTTGGAAATC
  • GPCR 8B Coding Sequence (17e20 TTTTGATCACTATCCTAGATTCACGCCTTAAAACCCCCATGTACTTATTCCTTGGAAATC
  • GPCR 8C Coding Sequence (ba386 TTTTGATCACTATCCTAGATTCACGCCTTAAAACCCCCATGTACTTATTCCTTGGAAATC
  • GPCR 8D Coding Sequence (CG503 TTTTGATCACTATCCTAGATTCACGCCTTAAAACCCCCATGTACTTATTCCTTGGAAATC
  • GPCR 8E Coding Sequence (17e20 TTTTGATCACTATCCTAGATTCACGCCTTAAAACCCCCATGTACTTATTCCTTGGAAATC
  • GPCR 8A Coding Sequence (17e20 TTTGTAACCCGCTGAGATACTCCATCATCATGAACAGGTGCGTCTGTGCACGGATGGCCA
  • GPCR 8B Coding Sequence (17e20 TTTGTAACCCGCTGAGATACTCCATCATCATGAACAGGTGCGTCTGTGCACGGATGGCJ3A
  • GPCR 8C Coding Sequence (ba386 TTTGTAACCCGCTGAGATACTCCATCATCATGAACAGGTGCGTCTGTGCACGGATGGCCA
  • GPCR 8D Coding Sequence (CG503 TTTGTAACCCGCTGAGATACTCCATCATCATGAACAGGTGCGTCTGTGCACGGATGGCCA
  • GPCR 8E Coding Sequence (17e20 TTTGTAACCCGCTGAGATACTCCATCATCATGAACAGGTGCGTCTGTGCACGGATGGCCA
  • GPCR 8A Coding Sequence (17e20 CGGTCTCCTGGGTGACGGGTTGCCTGACCGCTCTGCTGGAAACCAGTTTTGCCCTGCAG?
  • GPCR 8B Coding Sequence (17e20 CGGTCTCCTGGGTGACGGGTTGCCTGACCGCTCTGCTGGAAACCAGTTTTGCCCTGCAGA
  • GPCR 8C Coding Sequence (ba386 CGGTCTCCTGGGTGACGGGTTGCCTGACCGCTCTGCTGGAAACCAGTTTTGCCCTGCAGA
  • GPCR 8E Coding Sequence (17e20 CGGTCTCCTGGGTGACGGGTTGCCTGACCGCTCTGCTGGAAACCAGTTTTGCCCTGCAGA
  • GPCR 8B Coding Sequence (17e20 TGCCAATTCCAATGCTCTTAGTTTGCATCTCTTACATCTTCATCCTTTCCACTATTCTGA
  • GPCR 8C Coding Sequence (ba386 TGCCAATTCCAATGCTCTTAGTTTGCATCTCTTACATCTTCATCCTTTCCACTATTCTGA
  • GPCR 8D Coding Sequence (CG503 TGCCAATTCCAATGCTCTTAGTTTGCATCTCTTACATCTTCATCCTTTCCACTATTCTGA
  • GPCR 8A Coding Sequence (17e20 CACAAAAAATAGACAAAATCATCTCGTTGCTTTACGGAGTGCTTACCCCTATGTTGAACC
  • GPCR 8C Coding Sequence (ba386 CACAAAAAATAGACAAAATCATCTCGTTGCTTTACGGAGTGCTTACCCCTATGTTGAACC
  • GPCR 8D Coding Sequence (CG503 CACAAAAAATAGACAAAATCATCTCGTTGCTTTACGGAGTGCTTACCCCTATGTTGAACC
  • GPCR 8A Coding Sequence (17e20 CCATAATTTACAGTTTAAGAAACAAGGAAGTCAAAGATGCTATGAAGAAATTGCTGGGCA
  • GPCR 8B Coding Sequence (17e20 CCATAATTTACAGTTTAAGAAACAAGGAAGTCAAAGATGCTATGAAGAAATTGCTGGGCA
  • GPCR 8C Coding Sequence (ba386 CCATAATTTACAGTTTAAGAAACAAGGAAGTCAAAGATGCTATGAAGAAATTGCTGGGCA
  • GPCR 8D Coding Sequence (CG503 CCATAATTTACAGTTTAAGAAACAAGGAAGTCAAAGATGCTATGAAGAAATTGCTGGGCA
  • GPCR 8E Coding Sequence (17e20 CCATAATTTACAGTTTAAGAAACAAGGAAGTCAAAGATGCTATGAAGAAATTGCTGGGCA
  • GPCR 8A Coding Sequence 17e20 k A ⁇ AMATJA ⁇ AiWCAMTTiGtiC ⁇ AMTCMAAMAC ⁇ A ⁇ C ⁇ AMCG ⁇ AMAC ⁇ AATiC ⁇ TMCTMGAMTTiG ⁇ GGATic TRTGG ⁇ T ⁇ T ⁇ C ⁇ CiAG
  • GPCR 8A Coding Sequence 17e20 ATGTGCCCCTGGCAGAGCTCATCAGAGAAATTCGAGACAACATACAACCTCTTAGAACTC
  • GPCR 8B Coding Sequence 17e20 GPCR 8C Coding Sequence (ba386 GPCR 8D Coding Sequence (CG503 GPCR 8E Coding Sequence ( 17e20 ATGTGCCCCTGGCAGAGCTCATCAGAGAAATTCGAGACAACATACAACCTCTTAGAACTC 1150 1160 1170 1180 1190 1200 ....
  • GPCR 8A Coding Sequence 17e20 TGATCGGATCTTATCTCTATATAATTTCACAGTTATGAGCTGCATACACAGAGTGATTGC
  • Table 8W lists the domain description from DOMAIN analysis results against GPCR8a. This indicates that the GPCR8a sequence has properties similar to those of other proteins known to contain this domain as well as to the 377 amino acid 7tm domain itself.
  • GPCR8a-e may have important structural and/or physiological functions characteristic of the Olfactory Receptor family and the GPCR family. Therefore, the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications and as a research tool.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo (vi) biological defense weapon.
  • novel nucleic acid encoding GPCR8a-e, and the GPCR8a-e protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • the nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies.
  • compositions of the present invention will have efficacy for treatment of patients suffering from: developmental diseases, MHCII and III diseases (immune diseases), Taste and scent detectability Disorders, Burkitt's lymphoma, Corticoneurogenic disease, Signal Transduction pathway disorders, Retinal diseases including those involving photoreception, Cell Growth rate disorders; Cell Shape disorders, Feeding disorders; control of feeding; potential obesity due to over-eating; potential disorders due to starvation (lack of appetite), noninsulin-dependent diabetes mellitus (NTDDMl), bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-l or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoe
  • DPLA Dentatorubro-pallidoluysian atrophy
  • Acrocallosal syndrome and dyskinesias such as Huntington's disease or Gilles de la
  • polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding the OR -like protein may be useful in gene therapy, and the OR-like protein may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-l or HIV-2), pain, cancer (including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer), anorexia, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Crohn's disease; multiple sclerosis; and Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, asthma, allergies, benign prostatic hypertrophy, and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles de la Tourette syndrome and/or other pathologies and disorders.
  • cancer including but not limited to Neoplasm; adenocarcinoma; lymphoma; prostate cancer
  • novel nucleic acid encoding GPCR-like protein, and the GPCR-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-GPCRX Antibodies" section below.
  • the disclosed GPCR ⁇ a, d and GPCR8e proteins have multiple hydrophilic regions, each of which can be used as an immunogen.
  • GPCR8a epitopes are from about amino acids 10 to 40.
  • contemplated GPCR8d and GPCR8e epitopes are from about amino acids 125 to 130. In another embodiment, GPCR8a epitopes are from about amino acids 250 to 260. In another embodiment, GPCR8d and GPCR8e epitopes are from about amino acids 225 to 240. In additional embodiments, GPCR8a epitopes are from amino acids 275 to 290 and 320 to 340. hi additional embodiments, GPCR8d and GPCR8e epitopes are from amino acids 255 to 270 and 285 to 310. These novel proteins also have value in the development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
  • nucleic acid molecules that encode GPCRX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify GPCRX-encoding nucleic acids (e.g., GPCRX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of GPCRX nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double-stranded DNA.
  • An GPCRX nucleic acid can encode a mature GPCRX polypeptide.
  • a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein.
  • the naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein.
  • the product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises.
  • processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence.
  • a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining.
  • a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, byway of non-limiting example, glycosylation, myristoylation or phosphorylation.
  • a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
  • probes refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter- length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
  • isolated nucleic acid molecule is one, which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid
  • the isolated GPCRX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.).
  • an "isolated" nucleic acid molecule such as a cDNA molecule
  • a nucleic acid molecule of the invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • GPCRX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et ⁇ l, (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et ⁇ l., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to GPCRX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
  • an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43, or a complement thereof. Oligonucleotides maybe chemically synthesized and may also be used as probes.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43, or aportio of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an GPCRX polypeptide).
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43, thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
  • Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
  • Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions.
  • a "homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of GPCRX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • homologous nucleotide sequences include nucleotide sequences encoding for an GPCRX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
  • homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human GPCRX protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43, as well as a polypeptide possessing GPCRX biological activity. Various biological activities of the GPCRX proteins are described below.
  • An GPCRX polypeptide is encoded by the open reading frame ("ORF") of an GPCRX nucleic acid.
  • An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide.
  • a stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon.
  • An ORF that represents the coding sequence for a full protein begins with an ATG
  • an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate
  • a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
  • the nucleotide sequences determined from the cloning of the human GPCRX genes allows for the generation of probes and primers designed for use in identifying and/or cloning GPCRX homologues in other cell types, e.g. from other tissues, as well as GPCRX homologues from other vertebrates.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43; or an anti-sense strand nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43; or of a naturally occurring mutant of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43.
  • Probes based on the human GPCRX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis- express an GPCRX protein, such as by measuring a level of an GPCRX-encoding nucleic acid in a sample of cells from a subject e.g., detecting GPCRX mRNA levels or determining whether a genomic GPCRX gene has been mutated or deleted.
  • a polypeptide having a biologically-active portion of an GPCRX polypeptide refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
  • a nucleic acid fragment encoding a "biologically- active portion of GPCRX” can be prepared by isolating a portion SEQ JD NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 that encodes a polypeptide having an GPCRX biological activity (the biological activities of the GPCRX proteins are described below), expressing the encoded portion of GPCRX protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of GPCRX.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, ⁇ 8 31, 33, 35, 37, 39, 41 and 43due to degeneracy of the genetic code and thus encode the same GPCRX proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44.
  • SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44 is a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44.
  • Such genetic polymorphism in the GPCRX genes may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an GPCRX protein, preferably a vertebrate GPCRX protein.
  • ORF open reading frame
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the GPCRX genes. Any and all such nucleotide variations and resulting amino acid polymo ⁇ hisms in the GPCRX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the GPCRX polypeptides, are intended to be within the scope of the invention.
  • nucleic acid molecules encoding GPCRX proteins from other species and thus that have a nucleotide sequence that differs from the human sequence SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 are intended to be within the scope of the invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the GPCRX cDNAs of the invention can be isolated based on their homology to the human GPCRX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
  • nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length.
  • an isolated nucleic acid molecule of the invention hybridizes to the coding region.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • Homologs i.e., nucleic acids encoding GPCRX proteins derived from species other than human
  • other related sequences e.g., paralogs
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • Tm thermal melting point
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • a non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequences of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or fragments, analogs or derivatives thereof, under conditions of moderate stringency.
  • moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C.
  • Other conditions of moderate stringency that may be used are well-known within the art.
  • nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided.
  • low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1 % SDS at 50°C.
  • Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).
  • non-essential amino acid residues can be made in the sequence of SEQ ED NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the GPCRX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity.
  • amino acid residues that are conserved among the GPCRX proteins of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
  • nucleic acid molecules encoding GPCRX proteins that contain changes in amino acid residues that are not essential for activity.
  • GPCRX proteins differ in amino acid sequence from SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44 yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44.
  • the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44; more preferably at least about 70% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44; still more preferably at least about 80% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44; even more preferably at least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44; and most preferably at least about 95% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44.
  • An isolated nucleic acid molecule encoding an GPCRX protein homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,' 39, 41 and 43 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
  • conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted non-essential amino acid residue in the GPCRX protein is replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an GPCRX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for GPCRX biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
  • amino acid families may also be determined based on side chain interactions.
  • Substituted amino acids may be fully conserved "strong” residues or fully conserved “weak” residues.
  • the "strong” group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other.
  • a mutant GPCRX protein can be assayed for (i) the ability to form protei protein interactions with other GPCRX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant GPCRX protein and an GPCRX ligand; or (Hi) the ability of a mutant GPCRX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
  • a mutant GPCRX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
  • Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43, or fragments, analogs or derivatives thereof.
  • An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an RNA sequence).
  • antisense nucleic acid molecules comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire GPCRX coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an GPCRX protein.
  • coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the GPCRX protein.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of GPCRX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of GPCRX mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GPCRX mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxyhnethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-me
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an GPCRX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation).
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens).
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol IT or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl. Acids Res. 15: 6625-6641.
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (see, e.g., frioue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (see, e.g., frioue, et al, 1987. FEES Lett. 215: 327-330.
  • Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
  • an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591
  • a ribozyme having specificity for an GPCRX-encoding nucleic acid can be designed based upon the nucleotide sequence of an GPCRX cDNA disclosed herein (i.e., SEQ TD NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an GPCRX-encoding mRNA.
  • GPCRX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al, (1993) Science 261:1411-1418.
  • GPCRX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the GPCRX nucleic acid (e.g., the GPCRX promoter and/or enhancers) to form triple helical structures that prevent transcription of the GPCRX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6: 569-84;
  • nucleotide sequences complementary to the regulatory region of the GPCRX nucleic acid e.g., the GPCRX promoter and/or enhancers
  • the GPCRX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al, 1996. BioorgMed Chem 4: 5-23.
  • peptide nucleic acids refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al, 1996. supra; Perry-O'Keefe, et al, 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.
  • PNAs of GPCRX can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs of GPCRX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S] nucleases (see, Hyrup, et al, 1996. supra); or as probes or primers for DNA sequence and hybridization (see, Hyrup, et al, 1996, supra; Perry-O'Keefe, et al, 1996. supra).
  • PNAs of GPCRX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of GPCRX can be generated that may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996. supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al, 1996. supra and Finn, et al, 1996. Nucl Acids Res 24: 3357-3363.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al, 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al, 1996. supra.
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al, 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al, 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
  • other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemai
  • oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al, 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549).
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
  • a polypeptide according to the invention includes a polypeptide including the amino acid sequence of GPCRX polypeptides whose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44 while still encoding a protein that maintains its GPCRX activities and physiological functions, or a functional fragment thereof.
  • an GPCRX variant that preserves GPCRX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence.
  • Any amino acid substitution, insertion, or deletion is encompassed by the invention, hi favorable circumstances, the substitution is a conservative substitution as defined above.
  • GPCRX proteins and biologically- active portions thereof, or derivatives, fragments, analogs or homologs thereof.
  • polypeptide fragments suitable for use as immunogens to raise anti-GPCRX antibodies are provided.
  • native GPCRX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • GPCRX proteins are produced by recombinant DNA techniques.
  • an GPCRX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the GPCRX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of GPCRX proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced.
  • the language "substantially free of cellular material” includes preparations of GPCRX proteins having less than about 30% (by dry weight) of non-GPCRX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-GPCRX proteins, still more preferably less than about 10% of non-GPCRX proteins, and most preferably less than about 5% of non-GPCRX proteins.
  • non-GPCRX proteins also referred to herein as a "contaminating protein”
  • the GPCRX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the GPCRX protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of GPCRX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein, h one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of GPCRX proteins having less than about 30% (by dry weight) of chemical precursors or non-GPCRX chemicals, more preferably less than about 20% chemical precursors or non-GPCRX chemicals, still more preferably less than about 10% chemical precursors or non-GPCRX chemicals, and most preferably less than about 5% chemical precursors or non-GPCRX chemicals.
  • Biologically-active portions of GPCRX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the GPCRX proteins (e.g., the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44) that include fewer amino acids than the full- length GPCRX proteins, and exhibit at least one activity of an GPCRX protein.
  • biologically-active portions comprise a domain or motif with at least one activity of the GPCRX protein.
  • a biologically-active portion of an GPCRX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
  • other biologically-active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native GPCRX protein.
  • the GPCRX protein has an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44.
  • the GPCRX protein is substantially homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44, and retains the functional activity of the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below.
  • the GPCRX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44, and retains the functional activity of the GPCRX proteins of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the nucleic acid sequence homology may be determined as the degree of identity between two sequences.
  • the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMol Biol 48: 443-453.
  • GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3
  • the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15,
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I, in the case of nucleic acids
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • an GPCRX "chimeric protein” or “fusion protein” comprises an GPCRX polypeptide operatively- linked to a non-GPCRX polypeptide.
  • GPCRX polypeptide refers to a polypeptide having an amino acid sequence corresponding to an GPCRX protein (SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44), whereas a "non-GPCRX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the GPCRX protein, e.g., a protein that is different from the GPCRX protein and that is derived from the same or a different organism.
  • an GPCRX fusion protein can correspond to all or a portion of an GPCRX protein, hi one embodiment, an GPCRX fusion protein comprises at least one biologically-active portion of an GPCRX protein. In another embodiment, an GPCRX fusion protein comprises at least two biologically-active portions of an GPCRX protein, hi yet another embodiment, an GPCRX fusion protein comprises at least three biologically-active portions of an GPCRX protein.
  • the term "operatively-linked" is intended to indicate that the GPCRX polypeptide and the non-GPCRX polypeptide are fused in-frame with one another. The non-GPCRX polypeptide can be fused to the N-terminus or C-terminus of the
  • the fusion protein is a GST-GPCRX fusion protein in which the
  • GPCRX sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant GPCRX polypeptides.
  • the fusion protein is an GPCRX protein containing a heterologous signal sequence at its N-terminus.
  • GPCRX protein containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of GPCRX can be increased through use of a heterologous signal sequence.
  • the fusion protein is an GPCRX-immunoglobulin fusion protein in which the GPCRX sequences are fused to sequences derived from a member of the immunoglobulin protein family.
  • the GPCRX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an GPCRX ligand and an GPCRX protein on the surface of a cell, to thereby suppress GPCRX-mediated signal transduction in vivo.
  • the GPCRX-immunoglobulin fusion proteins can be used to affect the bioavailability of an GPCRX cognate ligand.
  • GPCRX-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-GPCRX antibodies in a subject, to purify GPCRX ligands, and in screening assays to identify molecules that inhibit the interaction of GPCRX with an GPCRX ligand.
  • An GPCRX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al (eds.) CURRENT PROTOCOLS IN MOLECULAR
  • GPCRX Agonists and Antagonists are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An GPCRX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the GPCRX protein.
  • the invention also pertains to variants of the GPCRX proteins that function as either GPCRX agonists (i.e., mimetics) or as GPCRX antagonists.
  • Variants of the GPCRX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the GPCRX protein).
  • An agonist of the GPCRX protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the GPCRX protein.
  • An antagonist of the GPCRX protein can inhibit one or more of the activities of the naturally occurring form of the GPCRX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the GPCRX protein.
  • GPCRX proteins that function as either GPCRX agonists (i.e., mimetics) or as GPCRX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) of the GPCRX proteins for GPCRX protein agonist or antagonist activity.
  • a variegated library of GPCRX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of GPCRX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential GPCRX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of GPCRX sequences therein.
  • libraries of fragments of the GPCRX protein coding sequences can be used to generate a variegated population of GPCRX fragments for screening and subsequent selection of variants of an GPCRX protein
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an GPCRX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Si nuclease, and ligating the resulting fragment library into an expression vector.
  • expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the GPCRX proteins.
  • Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify GPCRX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al, 1993. Protein Engineering 6:327-331.
  • the invention encompasses antibodies and antibody fragments, such as F a or (F ab ) 2 , that bind immunospecifically to any of the GPCRX polypeptides of said invention.
  • An isolated GPCRX protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind to GPCRX polypeptides using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length GPCRX proteins can be used or, alternatively, the invention provides antigenic peptide fragments of GPCRX proteins for use as immunogens.
  • the antigenic GPCRX peptides comprises at least 4 amino acid residues of the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44 and encompasses an epitope of GPCRX such that an antibody raised against the peptide forms a specific immune complex with GPCRX.
  • the antigemc peptide comprises at least 6, 8, 10, 15, 20, or 30 amino acid residues. Longer antigenic peptides are sometimes preferable over shorter antigenic peptides, depending on use and according to methods well known to someone skilled in the art.
  • At least one epitope encompassed by the antigenic peptide is a region of GPCRX that is located on the surface of the protein (e.g., a hydrophilic region).
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation (see, e.g., Hopp and Woods, 1981. Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle, 1982. J. Mol. Biol. 157: 105-142, each inco ⁇ orated herein by reference in their entirety) .
  • GPCRX protein sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44, or derivatives, fragments, analogs or homologs thereof, may be utilized as immunogens in the generation of antibodies that immunospecifically-bind these protein components.
  • antibody refers to immunoglobulin molecules and immunologically-active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically-binds (immunoreacts with) an antigen, such as GPCRX.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F a and F( a ' ) 2 fragments, and an F a expression library, hi a specific embodiment, antibodies to human GPCRX proteins are disclosed.
  • Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies to an GPCRX protein sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44, or a derivative, fragment, analog or homolog thereof.
  • an appropriate immunogenic preparation can contain, for example, recombinantly-expressed GPCRX protein or a chemically-synthesized GPCRX polypeptide.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • the antibody molecules directed against GPCRX can be isolated from the mammal (e.g. , from the blood) and further purified by well known techniques, such as protein A cliromatography to obtain the IgG fraction.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of GPCRX.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular GPCRX protein with which it immunoreacts.
  • any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized. Such techniques include, but are not limited to, the hybridoma technique (see, e.g., Kohler & Milstein, 1975.
  • techniques can be adapted for the production of single-chain antibodies specific to an GPCRX protein (see, e.g., U.S. Patent No. 4,946,778).
  • methods can be adapted for the construction of F a expression libraries (see, e.g., Huse, et al, 1989. Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F a b fragments with the desired specificity for an GPCRX protein or derivatives, fragments, analogs or homologs thereof.
  • Non-human antibodies can be "humanized" by techniques well known in the art. See, e.g., U.S. Patent No. 5,225,539.
  • Antibody fragments that contain the idiotypes to an GPCRX protein may be produced by techniques known in the art including, but not limited to: (i) an F( a b ' ) fragment produced by pepsin digestion of an antibody molecule; (ii) an F a fragment generated by reducing the disulfide bridges of an F a y )2 fragment; (Hi) an F ab fragment generated by the treatment of the antibody molecule with papain and a reducing agent; and (iv) F v fragments.
  • recombinant anti-GPCRX antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. WO 86/01533; U.S. Patent No. 4,816,567; U.S. Pat. No. 5,225,539; European Patent Application No.
  • methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme- linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art.
  • ELISA enzyme- linked immunosorbent assay
  • selection of antibodies that are specific to a particular domain of an GPCRX protein is facilitated by generation of hybridomas that bind to the fragment of an GPCRX protein possessing such a domain.
  • antibodies that are specific for a desired domain within an GPCRX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
  • Anti-GPCRX antibodies may be used in methods known within the art relating to the localization and/or quantitation of an GPCRX protein (e.g., for use in measuring levels of the GPCRX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like), i a given embodiment, antibodies for GPCRX proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds (hereinafter "Therapeutics").
  • An anti-GPCRX antibody e.g., monoclonal antibody
  • An anti-GPCRX antibody can facilitate the purification of natural GPCRX polypeptide from cells and of recombinantly-produced GPCRX polypeptide expressed in host cells. Moreover, an anti-GPCRX antibody can be used to detect GPCRX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the GPCRX protein. Anti-GPCRX antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • vectors preferably expression vectors, containing a nucleic acid encoding an GPCRX protein, or derivatives, fragments, analogs or homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector is a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Plasmid which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • a viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retro viruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., GPCRX proteins, mutant forms of GPCRX proteins, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of GPCRX proteins in prokaryotic or eukaryotic cells.
  • GPCRX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three pu ⁇ oses: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (Hi) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 61: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N. J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • E. coli expression vectors examples include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET lid (Srudier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
  • nucleic acid sequence of the nucleic acid is altered by e.g., Wada, et al, 1992. Nucl. Acids Res. 20: 2111-2118).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the GPCRX expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al, 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30:
  • GPCRX can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBO J. 6: 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987.
  • lymphoid-specific promoters Calame and Eaton, 1988. Adv. Immunol. 43: 235-275
  • promoters of T cell receptors Winoto and Baltimore, 1989.
  • EMBO J. 8: 729-733 promoters of T cell receptors
  • immunoglobulins Bonerji, et al, 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748
  • neuron-specific promoters e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.
  • pancreas-specific promoters Eslund, et al, 1985. Science 230: 912-916
  • mammary gland-specific promoters e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166
  • Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subj ect cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • GPCRX protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding
  • GPCRX can be introduced on a separate vector.
  • Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) GPCRX protein.
  • the invention further provides methods for producing GPCRX protein using the host cells of the invention, hi one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding GPCRX protein has been introduced) in a suitable medium such that GPCRX protein is produced. In another embodiment, the method further comprises isolating GPCRX protein from the medium or the host cell.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which GPCRX protein-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous GPCRX sequences have been introduced into their genome or homologous recombinant animals in which endogenous GPCRX sequences have been altered.
  • Such animals are useful for studying the function and/or activity of GPCRX protein and for identifying and/or evaluating modulators of GPCRX protein activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous GPCRX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing GPCRX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retro viral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the human GPCRX cDNA sequences of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 can be introduced as a transgene into the genome of a non-human animal.
  • a non-human homologue of the human GPCRX gene such as a mouse GPCRX gene, can be isolated based on hybridization to the human GPCRX cDNA (described further supra) and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably-linked to the GPCRX transgene to direct expression of GPCRX protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of the GPCRX transgene in its genome and/or expression of GPCRX mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene- encoding GPCRX protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of an GPCRX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the GPCRX gene.
  • the GPCRX gene can be a human gene (e.g., the cDNA of SEQ ED NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43), but more preferably, is a non-human homologue of a human GPCRX gene.
  • a mouse homologue of human GPCRX gene of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 can be used to construct a homologous recombination vector suitable for altering an endogenous
  • the vector is designed such that, upon homologous recombination, the endogenous GPCRX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous GPCRX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous GPCRX protein).
  • the altered portion of the GPCRX gene is flanked at its 5'- and 3'-termini by additional nucleic acid of the GPCRX gene to allow for homologous recombination to occur between the exogenous GPCRX gene carried by the vector and an endogenous GPCRX gene in an embryonic stem cell.
  • flanking GPCRX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5'- and 3'-termini
  • the vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced GPCRX gene has homologously-recombined with the endogenous GPCRX gene are selected. See, e.g., Li, et al, 1992. Cell 69: 915.
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras.
  • an animal e.g., a mouse
  • aggregation chimeras See, e.g., Bradley, 1987.
  • STEM CELLS A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152.
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously- recombined DNA by germline transmission of the transgene.
  • Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
  • transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI.
  • cre/loxP recombinase system See, e.g., Lakso, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236.
  • FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al, 1991. Science 251:1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al, 1997. Nature 385: 810-813.

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Abstract

La présente invention concerne des séquences d'acides nucléiques codant pour les polypeptides liés au récepteur de la protéine G. L'invention concerne également les polypeptides codés par ces séquences d'acides nucléiques, et des anticorps, se liant de manière immuno-spécifique au polypeptide, ainsi que les dérivés, variants, mutants, ou fragments dudit polypeptide, ou anticorps. L'invention concerne en outre des procédés thérapeutiques, diagnostiques et de recherche pour le diagnostic, le traitement et la prévention de troubles impliquant l'un de ces acide nucléiques et protéines humains.
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