Identification and modulation of a G-protein coupled receptor (GPCR), RAI-3, associated with chronic obstructive pulmonary disease (COPD) and NF-kappaB and E-selectin regulation

Abstract

The present invention describes a G-protein coupled receptor (GPCR) family member newly identified as being modified, e.g., phosphorylated, and associated with tyrosine phosphorylated activation complexes, following exposure of cells to smoke from tobacco burning substances, namely, cigarette smoke. This GPCR protein is RAI-3, which was first found to be phosphorylated in cells treated with cigarette smoke and to be associated with other proteins activated in cigarette smoke treated cells by virtue of the present invention. Because cigarette smoke is considered to be a major causative factor of chronic obstructive pulmonary disease (COPD) and disorders and conditions related thereto, the RAI-3 protein is newly provided as a cellular drug target for screening, discovering, and identifying modulators for the treatment and/or prevention of COPD and its related disorders and conditions, such as emphysema and chronic bronchitis. In accordance with the present invention RAI-3 modulators, e.g., agonists and antagonists, can be used as therapeutics in the treatment of COPD and numerous other diseases and disorders that are associated with regulation of NF-κB and/or its associated or interacting signaling molecules. This invention further provides SNPs of RAI-3, e.g., for determining COPD association in individuals.

What is claimed is:  
     
         1 . An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: 
 (a) a polynucleotide fragment of SEQ ID NO:2 or a polynucleotide fragment of the cDNA sequence included in ATCC Deposit No: ______, which is hybridizable to SEQ ID NO:2;    (b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:3 or a polypeptide fragment encoded by the cDNA sequence included in ATCC Deposit No: ______, which is hybridizable to SEQ ID NO:2;    (c) a polynucleotide encoding a polypeptide domain of SEQ ID NO:3 or a polypeptide domain encoded by the cDNA sequence included in ATCC Deposit No: ______, which is hybridizable to SEQ ID NO:2;    (d) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:3 or a polypeptide epitope encoded by the cDNA sequence included in ATCC Deposit No: ______, which is hybridizable to SEQ ID NO:2;    (e) a polynucleotide encoding a polypeptide of SEQ ID NO:3 or the cDNA sequence included in ATCC Deposit No: ______, which is hybridizable to SEQ ID NO:2, having biological activity;    (f) a polynucleotide which is a variant of SEQ ID NO:2;    (g) a polynucleotide which is an allelic variant of SEQ ID NO:2;    (h) an isolated polynucleotide comprising nucleotides 251 to 1324 of SEQ ID NO:2, wherein said nucleotides encode a polypeptide corresponding to amino acids 2 to 357 of SEQ ID NO:3 minus the start methionine;    (i) an isolated polynucleotide comprising nucleotides 254 to 1324 of SEQ ID NO:2, wherein said nucleotides encode a polypeptide corresponding to amino acids 2 to 357 of SEQ ID NO:3 including the start codon;    (j) an isolated polynucleotide comprising nucleotides 521 to 565 of SEQ ID NO:2, wherein said nucleotides encode a polypeptide corresponding to amino acids 90 to 104 of SEQ ID NO:3;    (k) an isolated polynucleotide comprising nucleotides 1055 to 1105 of SEQ ID NO:2, wherein said nucleotides encode a polypeptide corresponding to amino acids 269 to 284 of SEQ ID NO:3;    (l) an isolated polynucleotide comprising nucleotides 1271 to 1312 of SEQ ID NO:2, wherein said nucleotides encode a polypeptide corresponding to amino acids 340 to 353 of SEQ ID NO:3;    (m) an isolated polynucleotide comprising nucleotides 716 to 787 of SEQ ID NO:2, wherein said nucleotides encode a polypeptide corresponding to amino acids 155 to 178 of SEQ ID NO:3;    (n) an isolated polynucleotide comprising nucleotides 947 to 997 of SEQ ID NO:2, wherein said nucleotides encode a polypeptide corresponding to amino acids 232 to 248 of SEQ ID NO:3;    (o) an isolated polynucleotide comprising nucleotides 1106 to 1165 of SEQ ID NO:2, wherein said nucleotides encode a polypeptide corresponding to amino acids 285 to 304 of SEQ ID NO:3;    (p) a polynucleotide which represents the complimentary sequence (antisense) of SEQ ID NO:2; and    (q) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(p), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.    
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein the polynucleotide fragment consists of a nucleotide sequence encoding a human G-protein coupled receptor.  
     
     
         3 . A recombinant vector comprising the isolated nucleic acid molecule of  claim 1 .  
     
     
         4 . A recombinant host cell comprising the vector sequences of  claim 3 .  
     
     
         5 . An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: 
 (a) a polypeptide fragment of SEQ ID NO:3 or the encoded sequence included in ATCC Deposit No: ______;    (b) a polypeptide fragment of SEQ ID NO:3 or the encoded sequence included in ATCC Deposit No: ______, having coupling activity;    (c) a polypeptide domain of SEQ ID NO:3 or the encoded sequence included in ATCC Deposit No: ______;    (d) a polypeptide epitope of SEQ ID NO:3 or the encoded sequence included in ATCC Deposit No: ______;    (e) a full length protein of SEQ ID NO:3 or the encoded sequence included in ATCC Deposit No: ______;    (f) a polypeptide comprising amino acids 2 to 357 of SEQ ID NO:3, wherein said amino acids 2 to 357 comprising a polypeptide of SEQ ID NO:3 minus the start methionine;    (g) a polypeptide comprising amino acids 1 to 357 of SEQ ID NO:3;    (h) a polypeptide comprising amino acids 90 to 104 of SEQ ID NO:3;    (i) a polypeptide comprising amino acids 269 to 284 of SEQ ID NO:3;    (j) a polypeptide comprising amino acids 340 to 353 of SEQ ID NO:3;    (k) a polypeptide comprising amino acids 155 to 178 of SEQ ID NO:3;    (l) a polypeptide comprising amino acids 232 to 248 of SEQ ID NO:3; and    (m) a polypeptide comprising amino acids 285 to 304 of SEQ ID NO:3.    
     
     
         6 . The isolated polypeptide of  claim 5 , wherein the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.  
     
     
         7 . An isolated antibody that binds specifically to the isolated polypeptide of  claim 5 .  
     
     
         8 . A recombinant host cell that expresses the isolated polypeptide of  claim 5 .  
     
     
         9 . A method of making an isolated polypeptide comprising: 
 (a) culturing the recombinant host cell of  claim 8  under conditions such that said polypeptide is expressed; and    (b) recovering said polypeptide.    
     
     
         10 . The polypeptide produced by  claim 9 .  
     
     
         11 . A method for preventing, treating, or ameliorating a medical condition, comprising the step of administering to a mammalian subject a therapeutically effective amount of the polypeptide of  claim 5 , or a modulator thereof.  
     
     
         12 . A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising: 
 (a) determining the presence or absence of a mutation in the polynucleotide of  claim 1;  and    (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.    
     
     
         13 . A method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising: 
 (a) determining the presence or amount of expression of the polypeptide of  claim 5  in a biological sample; and    (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.    
     
     
         14 . An antisense compound 8 to 30 nucleotides in length that specifically hybridizes to a nucleic acid molecule encoding the human RAI-3 polypeptide having the sequence set forth in SEQ ID NO:3, wherein said antisense compound inhibits the expression of the human RAI-3 polypeptide.  
     
     
         15 . The antisense compound according to  claim 14  wherein said antisense compound is selected from the group consisting of: SEQ ID NO:52, 53, 54, 55, 56, 57, 58, 59, 60, and 61.  
     
     
         16 . A method of inhibiting the expression of the human RAI-3 polypeptide having the sequence set forth in SEQ ID NO:3 in human cells or tissues comprising contacting said cells or tissues in vitro with an antisense compound of  claim 15  so that expression of the RAI-3 polypeptide is inhibited.  
     
     
         17 . A method for preventing, treating, or ameliorating a medical condition, comprising the step of administering to a mammalian subject a therapeutically effective amount of the antisense compound according to  claim 15 .  
     
     
         18 . The antisense compound according to  claim 14 , wherein said antisense compound is double stranded.  
     
     
         19 . The antisense compound according to  claim 18 , wherein said antisense compound is a DNA/RNA hybrid.  
     
     
         20 . The antisense compound according to  claim 19  wherein said antisense compound is selected from the group consisting of: SEQ ID NO:94, and 95.  
     
     
         21 . An isolated polynucleotide comprising one or more polymorphic alleles selected from the group consisting of: SEQ ID NO:18, 22, 23, 24, 25, 26, 27, 28, 29, 65, 66, 67, 68, 69, and 70.  
     
     
         22 . The isolated polynucleotide comprising one or more polymorphic alleles of  claim 21  comprising a reference allele at one or more polymorphic loci.  
     
     
         23 . The isolated polynucleotide comprising one or more polymorphic alleles of  claim 21  comprising an alternate allele at one or more polymorphic loci.  
     
     
         24 . An isolated polypeptide comprising one or more polymorphic alleles selected from the group consisting of: SEQ ID NO:19, 8, 9, 13, 15, 17, 20, and 21.  
     
     
         25 . The isolated polypeptide comprising one or more polymorphic alleles of  claim 24  comprising a reference allele at one or more polymorphic loci.  
     
     
         26 . The isolated polypeptide comprising one or more polymorphic alleles of  claim 24  comprising an alternate allele at one or more polymorphic loci.  
     
     
         27 . The method of diagnosing a pathological condition of  claim 13  wherein the condition is a member of the group consisting of: a disorder related to aberrant G-protein coupled signaling; a disorder related to aberrant cell cycle regulation; pulmonary disorders, inflammatory lung disorders, COPD, the underlying symptoms of COPD, COPD-related disorders and/or conditions, autoimmune disorders, disorders related to hyperimmune activity, inflammatory conditions, disorders related to aberrant acute phase responses, hypercongenital conditions, birth defects, necrotic lesions, wounds, organ transplant rejection, conditions related to organ transplant rejection, renal diseases, ischemia-reperfusion injury, heart disorders, disorders related to aberrant signal transduction, proliferation disorders, cancers, such as lung cancer, stomach cancer, testicular cancer, breast cancer, etc., metastases, HIV infection, or HIV propagation in cells infected with other viruses, asthma, cystic fibrosis and pulmonary fibrosis, ulcerative colitis, cerebral infarct, myocardial infarct, diabetic nephropathy, allergic rhinitis, Crohn's disease, atherosclerosis, rheumatoid arthritis, inflammatory/auto-immune disorders outside of the lung in addition to COPD, glioblastoma, pulmonary small cell undifferentiated carcinoma, carcinoma of the breast, colon, lung, ovary, pancreas, prostate, non-Hodgkin's lymphoma, disorders associated with aberrant cell adhesion, disorders associated with aberrant I-CAM function and/or regulation, disorders associated with aberrant E-selectin function and/or regulation, disorders associated with aberrant NF-κB function and/or regulation.  
     
     
         28 . The method for preventing, treating, or ameliorating a medical condition of  claim 11 , wherein the medical condition is selected from the group consisting of: a disorder related to aberrant G-protein coupled signaling; a disorder related to aberrant cell cycle regulation; pulmonary disorders, inflammatory lung disorders, COPD, the underlying symptoms of COPD, COPD-related disorders and/or conditions, autoimmune disorders, disorders related to hyperimmune activity, inflammatory conditions, disorders related to aberrant acute phase responses, hypercongenital conditions, birth defects, necrotic lesions, wounds, organ transplant rejection, conditions related to organ transplant rejection, renal diseases, ischemia-reperfusion injury, heart disorders, disorders related to aberrant signal transduction, proliferation disorders, cancers, such as lung cancer, stomach cancer, testicular cancer, breast cancer, etc., metastases, HIV infection, or HIV propagation in cells infected with other viruses, asthma, cystic fibrosis and pulmonary fibrosis, ulcerative colitis, cerebral infarct, myocardial infarct, diabetic nephropathy, allergic rhinitis, Crohn's disease, atherosclerosis, rheumatoid arthritis, inflammatory/auto-immune disorders outside of the lung in addition to COPD, glioblastoma, pulmonary small cell undifferentiated carcinoma, carcinoma of the breast, colon, lung, ovary, pancreas, prostate, non-Hodgkin's lymphoma, disorders associated with aberrant cell adhesion, disorders associated with aberrant I-CAM function and/or regulation, disorders associated with aberrant E-selectin function and/or regulation, disorders associated with aberrant NF-κB function and/or regulation.  
     
     
         29 . A method of screening for candidate compounds capable of modulating the activity of a G-protein coupled receptor polypeptide, comprising: 
 (a) contacting a test compound with a cell or tissue comprising an expression vector capable of expressing a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:3, under conditions in which said polypeptide is expressed; and    (b) selecting as candidate modulating compounds those test compounds that modulate activity of the G-protein coupled receptor polypeptide.    
     
     
         30 . The method according to  claim 29  wherein said cells comprise a vector comprising the coding sequence of the beta lactamase gene under the control of NFAT response elements.  
     
     
         31 . The method according to  claim 31  wherein said cells further comprise a vector comprising the coding sequence of G alpha 15 under conditions wherein G alpha 15 is expressed.  
     
     
         32 . The method according to  claim 31  wherein said cells express the G-protein coupled receptor polypeptide at low levels relative to an internal control polypeptide.  
     
     
         33 . The method according to  claim 26  wherein said cells express the G-protein coupled receptor polypeptide at high levels relative to an internal control polypeptide.  
     
     
         34 . A method of predicting the likelihood that an individual will be diagnosed as being at risk of developing COPD, a COPD-like disorder, or one or more of the underlying symptoms of COPD, upon exposure to cigarette smoke, wherein said method comprises the steps of: a.) determining the level of RAI-3 expression relative to a control; and b.) associating said level with the likelihood of being at risk of developing COPD, a COPD-like disorder, or one or more of the underlying symptoms of COPD, upon exposure to cigarette smoke.