Methods of screening modulators of T2R taste receptors
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 greater than 90% amino acid sequence identity to SEQ ID NO:7, wherein said polypeptide specifically binds to ligands that specifically bind to an endogenous G-protein coupled receptor having the sequence of SEQ ID NO:7; and
(ii) determining the functional effect of the compound upon the polypeptide, thereby identifying a compound that modulates taste signaling in taste cells.
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 a physical effect.
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.
17. The method of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:7.
18. The method of claim 1 , wherein the polypeptide is covalently linked to a heterologous polypeptide, forming a chimeric polypeptide.
19. The method of claim 18 , wherein the polypeptide is linked to a solid phase.
20. The method of claim 19 , wherein the polypeptide is covalently linked to a solid phase.
21. The method of claim 1 , wherein said polypeptide comprises a sequence with at least 95% identity to SEQ ID NO:7.