Sweet taste receptors

Abstract

This invention provides novel genes and polypeptides of the sweet receptor family, methods for production of the polypeptides, methods for screening compounds that specifically bind to and/or modulate the activity of these polypeptides; and antibodies specific for the polypeptides.

1 . An isolated or recombinant polypeptide that comprises one or more of the following:
 (a.) an amino acid sequence or subsequence that is at least 75% identical to SEQ ID SEQ ID NO.7 as determined by BLASTP using default parameters;   (b.) an amino acid sequence or subsequence that comprises one or more domains of an hT1R3 polypeptide, wherein the hT1R3 polypeptide comprises an amino acid sequence of SEQ ID NO.7;   (c.) an amino acid sequence or subsequence that is at least 75% identical to a domain within SEQ ID NO.7 as determined by BLASTP using default parameters, wherein the domain is selected from the group consisting of: an amino-terminal extracellular domain; an extracellular domain located between TM2 and TM3, between TM4 and TM5, or between TM6 and TM7; a transmembrane (TM) domain; an intracellular domain located between TM1 and TM2, between TM3 and TM4, or between TM5 and TM6; and a carboxyl-terminal intracellular domain;   (d.) an amino acid sequence or subsequence that is specifically bound by an antibody that specifically binds to an amino acid sequence of SEQ ID NO.7, wherein the antibody is not specifically bound by an amino acid selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, and SEQ ID NO. 9;   (e.) an amino acid sequence or subsequence that is encoded by a nucleotide sequence of SEQ ID NO 12, or a complementary sequence thereof;   (f.) an amino acid sequence or subsequence that is encoded by a first nucleic acid that specifically hybridizes to a second nucleic acid, wherein the second nucleic acid comprises a sequence of SEQ ID NO 12, or a complement thereof, under stringent conditions, wherein the first nucleic acid hybridizes to the second nucleic acid under said stringent conditions with at least 5× an affinity that the first nucleic acid hybridizes to a third nucleic acid that is an mT1R3 nucleic acid or an rT1R3 nucleic acid; or,   (g.) an amino acid sequence or subsequence corresponding to a conservative variation an amino acid sequence or subsequence of any one of (a.)-(f.).   
     
     
         2 . The isolated or recombinant polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid sequence of SEQ ID NO.7, or a conservative variation thereof. 
     
     
         3 . The isolated or recombinant polypeptide of  claim 1 , wherein the polypeptide comprises a mature hT1R3 protein. 
     
     
         4 . The isolated polypeptide of  claim 1 , wherein the domain of (b.) is selected from the group consisting of:
 an amino-terminal extracellular domain;   an extracellular domain located between TM2 and TM3, between TM4 and TM5, or between TM6 and TM7;   a transmembrane (TM) domain;   an intracellular domain located between TM1 and TM2, between TM3 and TM4, or between TM5 and TM6; and   a carboxyl-terminal intracellular domain.   
     
     
         5 . The polypeptide of  claim 1 , wherein the polypeptide is a heteromer. 
     
     
         6 . The polypeptide of  claim 1 , wherein the polypeptide is a homomultimer. 
     
     
         7 . The polypeptide of  claim 1 , wherein the polypeptide is a heteromer that comprises more than one polypeptide having an amino acid sequence of SEQ ID NO.7, or a conservative variation thereof. 
     
     
         8 . An isolated or recombinant nucleic acid that encodes the isolated or recombinant polypeptide of  claim 1 , wherein the polypeptide is a substantially full-length a polypeptide, or wherein the nucleic acid is capable of rescuing function of a mutant or recombinant cell that is defective with respect to hT1R3. 
     
     
         9 . The isolated or recombinant nucleic acid of  claim 8 , wherein the nucleic acid is a DNA. 
     
     
         10 . The isolated or recombinant nucleic acid of  claim 8 , wherein the nucleic acid is a cDNA. 
     
     
         11 . The isolated or recombinant nucleic acid of  claim 8 , wherein the nucleic acid is an RNA. 
     
     
         12 . The isolated or recombinant nucleic acid of  claim 8 , wherein the nucleic acid is a coding nucleic acid that encodes an amino acid sequence of SEQ ID NO. 7, or a conservative variation thereof; or wherein the isolated or recombinant nucleic acid is a complementary nucleic acid that is complementary to the coding nucleic acid. 
     
     
         13 . The isolated or recombinant nucleic acid of  claim 8 , wherein the nucleic acid comprises a nucleotide sequence of SEQ ID NO. 12, and a complementary sequence thereof. 
     
     
         14 . The isolated or recombinant nucleic acid of  claim 8 , wherein the nucleic acid comprises or is coded within an expression vector. 
     
     
         15 . A cDNA or mRNA that encodes the isolated or recombinant polypeptide of  claim 1 . 
     
     
         16 . An expression vector encoding the cDNA or mRNA of  claim 15 . 
     
     
         17 . An antibody or fragment thereof which specifically binds the isolated or recombinant polypeptide of  claim 1 . 
     
     
         18 . An antibody fragment according to  claim 17 , wherein the antibody fragment is an Fab or F(ab′)2 fragment. 
     
     
         19 . An antibody according to  claim 17  wherein the antibody is a polyclonal antibody. 
     
     
         20 . An antibody according to  claim 17  which is a monoclonal antibody. 
     
     
         21 . An antibody according to  claim 17 , wherein the antibody does not bind to any protein selected from the group consisting of: mT1R1, mT1R2, mT1R3, rT1R1, rT1R2 and rT1R3. 
     
     
         22 . An expression vector that encodes a polypeptide of  claim 1 . 
     
     
         23 . A cell comprising the expression vector of  claim 22 . 
     
     
         24 . A biosensor comprising the polypeptide of  claim 1 . 
     
     
         25 . A method for producing a recombinant or isolated polypeptide, comprising:
 (a.) culturing a cell comprising an expression vector encoding the recombinant or isolated polypeptide of  claim 1 , under conditions suitable for expression of the isolated or recombinant polypeptide; and,   (b.) purifying the polypeptide such that the polypeptide is enriched at least 5× as compared to the polypeptide present in step (a).   
     
     
         26 . The method of  claim 25 , wherein (b.) comprises purifying the polypeptide such that the polypeptide is enriched at least 100× as compared to the polypeptide present in step (a.). 
     
     
         27 . An isolated or recombinant polypeptide made by the method of  claim 25 . 
     
     
         28 . A method of identifying compounds which bind to and/or modulate an activity of the isolated or recombinant polypeptide of  claim 1 , the method comprising:
 (a.) contacting a biological sample comprising the isolated or recombinant polypeptide with a test compound; and,   (b.) detecting binding and/or modulation of the activity of the polypeptide by the compound, thereby identifying a compound which binds to and/or modulates the activity of the polypeptide.   
     
     
         29 . The method of  claim 28 , wherein step (b.) includes detecting binding of an antibody to the isolated or recombinant polypeptide. 
     
     
         30 . The method of  claim 28 , wherein step (b.) includes detecting a signal produced by the isolated or recombinant polypeptide. 
     
     
         31 . The method of  claim 30 , wherein the signal is a conformation-dependent signal, wherein a conformation of the isolated or recombinant polypeptide is modified by binding of the test compound to the isolated or recombinant polypeptide. 
     
     
         32 . The method of  claim 28 , wherein the biological sample comprises a cell which expresses the recombinant polypeptide. 
     
     
         33 . The method of  claim 28 , wherein detecting binding comprises one or more of: a Ca 2+  flux assay, a cAMP assay, a GTPgammaS binding assay, a melanophore assay, a phospholipase C assay, a beta-arrestin FRET assay, and a transcriptional reporter assay. 
     
     
         34 . The method of  claim 28 , wherein the transcriptional reporter assay comprises detecting an activity of one or response element selected from the group consisting of: a CRE, a SRE, an MRE, a TRE, an NFAT, and an NFkB-response element, wherein the response element directs expression of an operably coupled reporter gene. 
     
     
         35 . The method of  claim 28 , wherein the biological sample comprises a biosensor. 
     
     
         36 . The method of  claim 35 , wherein the biosensor comprises a Chem-FET. 
     
     
         37 . A method of identifying compounds which bind to and/or modulate an activity of a polypeptide comprising hT1R3, the method comprising:
 (a.) contacting a biological sample comprising hT1R3 with a test compound; and,   (b.) detecting binding and/or modulation of the activity of the polypeptide by the compound, thereby identifying a compound which binds to and/or modulates the activity of the polypeptide.   
     
     
         38 . The method of  claim 37 , wherein step (b.) includes detecting binding of an antibody to the isolated or recombinant polypeptide. 
     
     
         39 . The method of  claim 37 , wherein step (b.) includes detecting a signal produced by the isolated or recombinant polypeptide. 
     
     
         40 . The method of  claim 37 , wherein the signal is a conformation-dependent signal, wherein a conformation of the isolated or recombinant polypeptide is modified by binding of the test compound to the isolated or recombinant polypeptide. 
     
     
         41 . The method of  claim 37 , wherein the biological sample comprises a cell which expresses the recombinant polypeptide. 
     
     
         42 . The method of  claim 37 , wherein detecting binding comprises one or more of: a Ca 2+  flux assay, a cAMP assay, a GTPgammaS binding assay, a melanophore assay, a phospholipase C assay, a beta-arrestin FRET assay, and a transcriptional reporter assay. 
     
     
         43 . The method of  claim 42 , wherein the transcriptional reporter assay comprises detecting an activity of one or response element selected from the group consisting of: a CRE, a SRE, an MRE, a TRE, an NFAT, and an NFkB-response element, wherein the response element directs expression of an operably coupled reporter gene. 
     
     
         44 . The method of  claim 37 , wherein the biological sample comprises a biosensor. 
     
     
         45 . The method of  claim 44 , wherein the biosensor comprises a Chem-FET. 
     
     
         46 . A method of rescuing a cell that has altered or missing T1R3 function, comprising introducing a nucleic acid into the cell, wherein the nucleic acid encodes the recombinant polypeptide of  claim 1 , and expressing recombinant polypeptide, thereby providing hT1R3 function to the cell. 
     
     
         47 . The method of  claim 46 , wherein the cell is in cell culture. 
     
     
         48 . The method of  claim 46 , wherein the cell is in a tissue. 
     
     
         49 . The method of  claim 46 , wherein the cell is in a taste bud. 
     
     
         50 . The method of  claim 46 , wherein the cell is in taste bud in a mammal.