Method for identifying compounds that modulate a T2R taste receptor
The invention provides nucleic acid and amino acid sequences for a novel family of taste transduction G-protein coupled receptors, antibodies to such receptors, methods of detecting such nucleic acids and receptors, and methods of screening for modulators of taste transduction G-protein coupled receptors.
1. A method for identifying a compound that modulates taste signaling in taste cells, the method comprising the steps of:
(i) contacting a taste transduction G-protein coupled receptor polypeptide with the compound, the polypeptide comprising at least 90% amino acid identity to SEQ ID NO:26 and comprising a sequence selected from the group consisting of SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168; SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:171, wherein said receptor detects bitter tastants; and
determining the functional effect on G-protein coupled receptor activity upon the polypeptide by contacting the polypeptide with the compound and measuring the functional effect on the G-protein coupled receptor activity, wherein a modulation in the functional effect identifies the compound as a compound that modulates taste signaling.
2. The method of claim 1 , wherein the polypeptide has G-protein coupled receptor activity.
3. The method of claim 1 , wherein the functional effect is determined by measuring binding of the compound to an extracellular domain of the polypeptide.
4. The method of claim 1 , wherein the functional effect is determined by measuring binding of radiolabeled GTP to the polypeptide.
5. The method of claim 1 , wherein the polypeptide is recombinant.
6. The method of claim 1 , wherein the polypeptide is from a rat, a mouse, or a human.
7. The method of claim 1 , wherein the polypeptide is expressed in a cell or cell membrane.
8. The method of claim 7 , wherein the functional effect is measured by determining changes in the electrical activity of a cell expressing the polypeptide.
9. The method of claim 7 , wherein the functional effect is determined by measuring changes in intracellular cAMP, cGMP, IP3, or Ca 2+ .
10. The method of claim 9 , wherein a change in intracellular Ca 2+ is detected by detecting a change in FURA-2-dependent fluorescence in the cell.
11. The method of claim 7 , wherein the cell is a eukaryotic cell.
12. The method of claim 11 , wherein the cell is an HEK-293 cell.
13. The method of claim 7 , wherein the polypeptide is a fusion protein comprising at least about 20 consecutive N-terminal amino acids of a rhodopsin protein.
14. The method of claim 13 , wherein the rhodopsin protein is a bovine rhodopsin.
15. The method of claim 7 , wherein the cell comprises Gα15.
16. The method of claim 7 , wherein the polypeptide is contacted with the compound in the presence of a bitter tastant, and wherein a difference in the functional effect of the bitter tastant on the cell in the presence of the compound and the functional effect of the bitter tastant on the cell in the absence of the compound indicates that the compound is capable of modulating taste signaling in taste cells.