T1R hetero-oligomeric taste receptors, cell lines that express said receptors, and taste compounds
The invention relates to compounds that specifically bind a T1R1/T1R3 or T1R2/T1R3 receptor or fragments or sub-units thereof. The present invention also relates to the use of hetero-oligomeric and chimeric taste receptors comprising T1R1/T1R3 and T1R2/T1R3 in assays to identify compounds that respectively respond to umami taste stimuli and sweet taste stimuli. Further, the invention relates to the constitutive of cell lines that stably or transiently co-express a combination of T1R1 and T1R3; or T1R2 and T1R3; under constitutive or inducible conditions. The use of these cells lines in cell-based assays to identify umami and sweet taste modulatory compounds is also provided, particularly high throughput screening assays that detect receptor activity by use of fluorometric imaging.
1. A functional heteromeric T1R2/T1R3 receptor comprising a T1R2 subunit and a T1R3 subunit,
wherein the T1R2 subunit comprises a Venus Flytrap Domain that has at least 90% sequence identity to amino acid residues 1 to 560 of the human T1R2 polypeptide in SEQ ID NO: 6 and a transmembrane domain and C-terminal domain each having at least 90% sequence identity to the transmembrane and C-terminal domains of a rat T1R1, T1R2 , T1R3 , or mGluR1 polypeptide, and
wherein the T1R3 subunit comprises at least 90% sequence identity to the human T1R3 polypeptide in SEQ ID NO: 7.
2. The functional heteromeric T1R2/T1R3 receptor of claim 1 , wherein the T1R2 subunit comprises the transmembrane domain and C-terminal domain from the rat T1R1 polypeptide.
3. The functional heteromeric T1R2/T1R3 receptor of claim 1 , wherein the T1R2 subunit comprises the transmembrane domain and C-terminal domain from the rat T1R2 polypeptide.
4. The functional heteromeric T1R2/T1R3 receptor of claim 1 , wherein the T1R2 subunit comprises the transmembrane domain and C-terminal domain from the rat T1R3 polypeptide.
5. The functional heteromeric T1R2/T1R3 receptor of claim 1 , wherein the T1R2 subunit comprises the transmembrane domain and C-terminal domain from the rat mG1uR1 polypeptide.
6. A method for identifying a putative taste modulatory compound comprising
(i) contacting the functional heteromeric T1R2/T1R3 receptor of claim 1 with the putative taste modulatory compound, and
(ii) determining whether said compound specifically binds or modulates the activity of the heteromeric T1R2/T1R3 receptor.
7. The method of claim 6 , wherein the heteromeric T1R2/T1R3 receptor is attached to a solid phase.
8. The method of claim 6 , wherein the heteromeric T1R2/T1R3 receptor is expressed in a cell.
9. The method of claim 8 , wherein said cell is selected from the group consisting of yeast, insect, mammalian, amphibian and worm cells.
10. The method of claim 9 , wherein said cell is a mammalian cell selected from the group consisting of CHO, HeLa and HEK-293 cells.
11. The method of claim 8 , wherein said cell further expresses a G protein that couples to the heteromeric T1R2/T1R3 receptor.
12. The method of claim 8 , wherein changes in the activity of the heteromeric T1R2/T1R3 receptor are measured by detecting changes in intracellular calcium ion levels.
13. The method of claim 12 , wherein calcium ion levels are detected using an ion sensitive dye or a membrane voltage fluorescent indicator.
14. The method of claim 12 , wherein calcium ion levels are detected by fluorescence imaging.
15. The method claim 8 , wherein changes in the activity of the heteromeric T1R2/T1R3 receptor are measured by detecting changes in second messenger levels.
16. The method of claim 15 , wherein said second messenger is IP3.
17. The method of claim 8 , wherein changes in the activity of the heteromeric T1R2/T1R3 receptor are measured by detecting changes in intracellular cyclic nucleotides.
18. The method of claim 17 , wherein said cyclic nucleotides are cAMP or cGMP.
19. The method of claim 6 , wherein changes in the activity of the heteromeric T1R2/T1R3 receptor are detected by monitoring changes in fluorescence polarization.
20. The method of claim 6 , wherein changes in the activity of the heteromeric T1R2/T1R3 receptor are detected by measuring changes in G protein binding of GTP gamma S.
21. The method of claim 6 , wherein said putative taste modulating compound is further evaluated in a human taste test.