Binding and functional assays that use the T1R3 receptor to screen for taste modulatory compounds
View Patent ↗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.
1. An in vitro method for identifying a compound that specifically binds to a human T1R3 extracellular domain polypeptide, the method comprising:
(1) contacting a compound with a human T1R3 extracellular domain polypeptide, wherein said T1R3 extracellular domain polypeptide possesses at least 95% sequence identity to amino acid residues 1 to 563 of SEQ ID NO: 4; and
(2) detecting whether said compound specifically binds to said T1R 3 extracellular domain polypeptide, thereby determining whether said compound binds to said T1R3 extracellular domain.
2. The method of claim 1 , wherein said human T1R3 extracellular domain polypeptide has an amino acid sequence that possesses at least 96% sequence identity to amino acid residues 1 to 563 of SEQ ID NO: 4.
3. The method of claim 1 , wherein said human T1R3 extracellular domain polypeptide has an amino acid sequence that possesses at least 97% sequence identity to amino acid residues 1 to 563 of SEQ ID NO: 4.
4. The method of claim 1 , wherein said human T1R3 extracellular domain polypeptide has an amino acid sequence that possesses at least 98% sequence identity to amino acid residues 1 to 563 of SEQ ID NO: 4.
5. The method of claim 1 , wherein the human T1R3 extracellular domain polypeptide possesses at least 99% sequence identity to amino acid residues 1 to 563 of SEQ ID NO: 4.
6. The method of claim 1 , wherein said assay uses a cell that expresses said human T1R3 extracellular domain polypeptide.
7. The method of claim 6 , wherein said cell is intact or permeabilized.
8. The method of claim 1 , wherein said human T1R3 extracellular domain polypeptide is contained in a membrane extract.
9. The method of claim 6 , wherein said human T1R3 extracellular domain polypeptide is expressed on the surface of said cell.
10. The method of claim 6 , wherein the cell is a eukaryotic cell.
11. The method of claim 6 , wherein said cell is a yeast, insect, amphibian or mammalian cell.
12. The method of claim 6 , wherein the cell is a CHO, human kidney two hundred and ninety-three, COS or Xenopus oocyte.
13. The method of claims 6 , wherein said cell further expresses a G protein.
14. The method of claim 13 , wherein said G protein is G α15 , G α16 or gustducin.
15. The method of claim 1 , wherein said human T1R3 extracellular domain polypeptide is stably or transiently expressed by a cell.
16. The method of claim 1 , wherein the binding assay is a high throughput assay.
17. The method of 1 , wherein said binding assay screens a library of compounds.
18. The method of claim 17 , wherein said library is a combinatorial chemical library.
19. The method of claim 17 , wherein said library comprises at least 1000 compounds.
20. The method of claim 1 , further comprising assaying in vivo said compound that specifically binds to said T1R3 extracellular domain polypeptide for its effect on taste.
21. The method of claim 20 which assays the effect of a compound on the taste of a particular compound.
22. The method of claim 21 , wherein said assay detects the effect of a compound on sweet or umami taste.
23. The method of claim 1 , wherein said human T1R3 extracellular domain polypeptide comprises the amino acid sequence of amino acid residues 1 to 563 of SEQ ID NO: 4.