Assays for sensory modulators using a sensory cell specific G-protein alpha subunit
View Patent ↗The invention identifies nucleic acid and amino acid sequences of a sensory cell specific G-protein alpha subunit that are specifically expressed in sensory cells, e.g., taste cells, antibodies to such G-protein alpha subunits, methods of detecting such nucleic acids and subunits, and methods of screening for modulators of a sensory cell specific G-protein alpha subunit.
1. A method for identifying a compound that modulates signal transduction in a cell that expresses a taste cell specific G protein coupled receptor (GPCR), the method comprising the steps of:
(i) contacting the compound with a cell which expresses:
(a) a taste cell specific G-protein alpha subunit polypeptide, wherein the G-protein alpha subunit polypeptide comprises a sequence with greater than 95% identity to the sequence of SEQ ID NO:2, and the G-protein alpha subunit polypeptide is a subunit of a heterotrimeric G-protein which binds a guanine nucleotide; and
(b) a taste cell specific GPCR comprising a GPCR-B4 polypeptide with greater than 90% identity to the sequence of SEQ ID NO:12, 13, or 14, wherein said GPCR binds said heterotrimeric G-protein; and
(ii) determining a functional effect of the compound upon the cell expressing the G-protein alpha subunit polypeptide and the taste cell specific GPCR, thereby identifying a compound that modulates signal transduction in a cell.
2. The method of claim 1 , wherein the G-protein alpha subunit polypeptide is recombinant.
3. The method of claim 1 , wherein the functional effect is a chemical effect.
4. The method of claim 1 , wherein the G-protein alpha subunit polypeptide is from a mouse, a rat or a human.
5. The method of claim 1 , wherein the G-protein alpha subunit polypeptide comprises the amino acid sequence of SEQ ID NO:2.
6. The method of claim 1 , wherein the functional effect is determined by measuring changes in the level of phosphorylation of G-protein coupled receptor (GPCR)-B4.
7. The method of claim 1 , wherein the cell is attached to a solid substrate.
8. The method of claim 1 , wherein the cell is a eukaryotic cell.
9. The method of claim 8 , wherein the cell is a human cell.
10. The method of claim 1 , wherein the G-protein coupled receptor comprises the GPCR-B4 polypeptide of SEQ ID NO:14.
11. A method for identifying a compound that modulates signal transduction in taste cells, the method comprising the steps of:
(i) expressing a taste cell specific G-protein alpha subunit polypeptide in an HEK 293 host cell, wherein the G-protein alpha subunit polypeptide comprises a sequence with greater than 95% identity to the sequence of SEQ ID NO:2, and the G-protein alpha subunit polypeptide is a subunit of a heterotrimeric G-protein which binds a guanine nucleotide;
(ii) expressing a taste cell specific G-protein coupled receptor (GPCR) comprising a GPCR-B4 polypeptide with greater than 90% identity to the sequence of SEQ ID NO:12, 13, or 14 in the host cell, wherein said GPCR binds said heterotrimeric G-protein;
(iii) contacting the host cell with the compound; and
(iv) determining changes in intracellular calcium levels in the host cell, thereby identifying a compound that modulates signal transduction in taste cells.
12. The method of claim 11 , wherein the G protein alpha subunit polypeptide comprises the sequence of SEQ ID NO:2.
13. The method of claim 11 , wherein the G-protein coupled receptor comprises the GPCR-B4 polypeptide of SEQ ID NO:14.