Assays for detecting T2R76 taste modulatory compounds
Isolated nucleic acids encoding T2R76 polypeptides, recombinantly expressed T2R76 polypeptides, heterologous expression systems for recombinant expression of T2R76 polypeptides, assay methods employing the same, and methods for altering taste perception via administration of a T2R76 modulator. These T22R76 polypeptides can be expressed alone or co-expressed with another T2R polypeptide, preferably a different human T2R polypeptide.
1. An in vitro method for identifying a compound that putatively elicits or modulates bitter taste in a human subject based on its specific binding or modulatory effect on the activation of a T2R polypeptide comprising:
(1) effecting a binding or functional assay that detects for at least one compound which specifically binds or which modulates the activation of a taste receptor comprising a human T2R polypeptide, which polypeptide possesses at least 95% sequence identity to the human T2R polypeptide of SEQ ID NO:2 wherein said human T2R polypeptide specifically binds to a bitter ligand that specifically binds the human T2R polypeptide of SEQ ID NO:2; and
(2) identifying at least one compound in said assay which specifically binds or modulates the activation of said human T2R polypeptide, which at least one compound putatively elicits or modulates bitter taste in humans.
2. The method of claim 1 , wherein said T2R polypeptide has the amino acid sequence of SEQ ID NO:2.
3. The method of claim 1 , wherein said T2R polypeptide has an amino acid sequence that possesses 95-99% sequence identity to the amino acid sequence of SEQ ID NO:2.
4. The method of claim 1 , wherein said T2R polypeptide has an amino acid sequence that possesses at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2.
5. The method of claim 1 , wherein said assay uses a cell that expresses said T2R polypeptide.
6. The method of claim 1 , wherein said T2R polypeptide is comprised in a membrane extract.
7. The method of claim 5 , wherein said T2R polypeptide is expressed on the surface of said cell.
8. The method of claim 7 , wherein said cell is a eukaryotic cell.
9. The method of claim 7 , wherein said cell is a yeast, insect, amphibian or mammalian cell.
10. The method of claim 7 , wherein said cell is a HEK-293, COS or Xenopus oocyte.
11. The method of claim 7 , wherein said cell further expresses a G protein.
12. The method of claim 11 , wherein said G protein is Gα15 or Gα16 or gustducin.
13. The method of claim 1 , wherein said functional assay detects the effect of said at least one compound on phosphorylation of said T2R polypeptide.
14. The method of claim 1 , wherein said functional assay detects the effect of said at least one compound on the dissociation of said T2R polypeptide and a G protein.
15. The method of claim 1 , wherein said functional assay detects the effect of said at least one compound on second messengers.
16. The method of claim 1 , wherein said functional assay detects the effect of said at least one compound on signal transduction.
17. The method of claim 1 , wherein said functional assay detects GTPγS.
18. The method of claim 1 , wherein said functional assay detects changes in cAMP, cGMP, or IP3.
19. The method of claim 1 , wherein said functional assay detects whether said at least one compound results in a detectable change in intracellular calcium ion concentration.
20. The method of claim 19 , which uses a calcium-sensitive dye.
21. The method of claim 1 , wherein said functional assay detects the effect of said at least one compound on G protein activation of said T2R polypeptide.
22. The method of claim 21 , wherein said G protein is Gα15, Gα16 or gustducin.
23. The method of claim 1 , wherein said T2R polypeptide is stably or transiently expressed by a cell.
24. The method of claim 1 , wherein said functional assay detects the activation of the T2R polypeptide fluorescently.
25. The method of claim 1 , wherein said functional assay detects a change in ligand dependent coupling of said T2R polypeptide with a G protein.
26. The method of claim 1 , wherein said functional assay detects the effect of said at least one compound on the transcription of a gene of interest.
27. The method of claim 1 , wherein said assay is a high throughput assay.
28. The method of claim 1 , wherein at least one compound identified as putatively modulating bitter taste is further assayed in a taste test.