T1R3 receptor binding assays for identification of taste modulatory compounds
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Abstract
Newly identified mammalian taste-cell-specific G protein-coupled receptors, and the genes and cDNA encoding said receptors are described. Specifically. T1R G protein-coupled receptors active in taste signaling, and the genes and cDNA encoding the same, are described, along with methods for isolating such genes and for isolating and expressing such receptors. Methods for representing taste perception of a particular tastant in a mammal are also described, as are methods for generating novel molecules or combinations of molecules that elicit a predetermined taste perception in a mammal, and methods for simulating one or more tastes. Further, methods for stimulating or blocking taste perception in a mammal are also disclosed.
73 Citations
299 Claims
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1-271. -271. (canceled)
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272. A method for identifying a compound that putatively modulates the T1R3 receptor polypeptide in a human subject comprising:
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(1) screening one or more compounds in a binding assay which identifies compounds that specifically bind to a human T1R3 polypeptide or which modulate (inhibit or enhance) the specific binding of another compound that specifically binds to said human T1R3 polypeptide, wherein said T1R3 polypeptide is selected from the group consisting of; (a) a human T1R3 polypeptide having the amino acid sequence encoded by SEQ. ID. NO;
4;(b) a human T1R3 polypeptide encoded by a nucleic acid sequence that specifically hybridizes to the hT1R3 nucleic acid sequence in SEQ. ID. NOS;
2, 3 or 20 under stringent hybridization conditions which are 50% formamide, 5×
SSC and 1% SDS, incubating at 42 degrees C. with wash in 0.2×
SSC and 0.1% SDS at 65 degrees C. and which T1R3 polypeptide binds to a ligand bound by native human T1R3 polypeptide; and(c) a human T1R3 polypeptide which has possesses at least 90% sequence identity to the amino acid sequence in SEQ. ID. NO;
4 and which T1R3 polypeptide specifically binds to a ligand bound by native human T1R3 polypeptide; and(2) identifying compounds that putatively modulate a human T1R3 polypeptide according to (a), (b), or (c), or its modulation (inhibition or enhancement) of the specific binding of another compound to a T1R3 polypeptide according to (a), (b), or (c). - View Dependent Claims (273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 293, 294, 295, 296)
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297. An isolated nucleic acid selected from the group consisting of:
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(i) a genomic DNA sequence consisting essentially of a nucleic acid sequence coding for a T1R mammalian G protein-coupled receptor polypeptide active in taste signaling, wherein said nucleic acid sequence consists essentially of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 20; (ii) a genomic DNA sequence consisting essentially of a nucleic acid sequence coding for a T1R mammalian G protein-coupled receptor polypeptide have an amino acid sequence selected from the group consisting of SEQ ID NOs 4, 14, and 17; (iii) a genomic DNA sequence having at least about 50% identity to a nucleic acid sequence coding for a T1R mammalian G protein-coupled receptor polypeptide, wherein said nucleic acid sequence consists essentially of a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 20;
a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 20(iv) a genomic DNA sequence consisting essentially of a nucleic acid sequence coding for a T1R mammalian G protein-coupled receptor polypeptide having an amino acid sequence that is at least about 40% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs 4, 14, and 17; (v) a genomic DNA sequence consisting essentially of a sequence coding for a T1R mammalian G protein-coupled receptor polypeptide comprising a consensus sequence selected from the group consisting of SEQ ID NOs 18 and 19, and sequences having at least about 75% identity to SEQ ID NOs 18 or 19; (vi) a cDNA sequence having the same nucleic acid sequence as the T1R mammalian G protein-coupled receptor polypeptide coding region in the genomic DNA sequence selected from the group consisting of SEQ ID NOs 15 and 20; (vii) a cDNA sequence coding for a T1R mammalian G protein-coupled receptor polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 4, 14, and 17; (viii) a cDNA sequence coding for a T1R mammalian G protein-coupled receptor polypeptide comprising a consensus sequence selected from the group consisting of SEQ ID NOs 18 and 19, and sequences having at least about 75% identity to SEQ ID NOs 18 or 19; (ix) a cDNA sequence selected from the group consisting of SEQ ID NOs 3, 13 and 16; (x) a cDNA sequence having at least about 50% sequence identity to the T1R G protein-coupled receptor polypeptide coding region in the genomic DNA sequence selected from the group consisting of SEQ ID NOs 15 and 20; (xi) a cDNA sequence having at least about 50% sequence identity to a sequence encoding a T1R mammalian G protein-coupled receptor polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 4, 14, and 17; (xii) a cDNA sequence having at least about 50% identity to a sequence selected from the group consisting of SEQ ID NOs 3, 13, and 16; (xiii) a variant of a nucleotide sequence selected from the group consisting of SEQ ID NOs 3, 13, 15, 16, and 20, containing at least one conservative substitution in a region coding for a T1R G protein-coupled receptor polypeptide active in taste signaling; (xiv) a variant of a nucleotide sequence encoding a T1R G protein-coupled receptor polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 4, 14, and 17, containing at least one conservative substitution in a T1R mammalian G protein-coupled receptor polypeptide coding region; and (xv) a variant of a cDNA sequence selected from the group consisting of SEQ ID NOs 3, 13, and 16, containing at least one conservative substitution.
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- 298. An isolated genomic DNA molecule consisting essentially of a nucleic acid sequence coding for a mammalian G protein-coupled receptor polypeptide, wherein said nucleic acid sequence consists essential of SEQ ID NO 15 or 20.
Specification