Methods of screening compounds using a T1R3 hetero-oligomeric taste receptor
View Patent ↗Newly identified mammalian taste-cell-specific G protein-coupled receptors which function as hetero-oligomeric complexes in the sweet taste transduction pathway, and the genes and cDNA encoding said receptors are described. Specifically, T1R G protein-coupled receptors active in sweet taste signaling as hetero-oligomeric complexes, and the genes and cDNA encoding the same, are described, along with methods for isolating such genes and for isolating and expressing such receptors. Methods for identifying putative taste modulating compounds using such hetero-oligomeric complexes also described, as is a novel surface expression facilitating peptide useful for targeting integral plasma membrane proteins to the surface of a cell.
1. A method for identifying a compound that modulates sweet or umami taste elicited by a receptor comprising a T1R3 polypeptide sequence comprising: (1) screening one or more compounds in a binding assay which identifies compounds that specifically bind to a receptor comprising a T1R3 polypeptide that is at least 90% identical to the polypeptide contained in SEQ ID NO:6 or which specifically modulate (enhances or inhibits) the specific binding of another compound to said receptor comprising said T1R3 polypeptide; and (2) identifying whether said compound potentially modulates umami or sweet taste elicited by a taste receptor comprising a polypeptide having a sequence which is at least 90% identical to the T1R3 polypeptide contained in SEQ ID NO:6 based on its (a) specific binding to a T1R3 receptor or (b) modulation of the specific binding of another compound to a T1R3 receptor, wherein said T1R3 polypeptide possesses at least 90% sequence identity to the T1R3 polypeptide of SEQ. ID. NO: 6 and wherein said T1R3 receptor specifically binds to a ligand that specifically binds to an endogenous (wild-type) human T1R3 receptor and (3) effecting a taste test to confirm whether said identified compound modulates sweet or umami taste.
2. The method of claim 1 wherein said T1R3 is a human T1R3 that exhibits at least 95% sequence identity to the polypeptide of SEQ. ID. NO: 6.
3. The method of claim 1 wherein said T1R3 is a human T1R3 that exhibits at least 96% sequence identity to the polypeptide of SEQ. ID. NO: 6.
4. The method of claim 1 wherein said T1R3 is a human T1R3 that exhibits at least 97% sequence identity to the polypeptide of SEQ. ID. NO: 6.
5. The method of claim 1 wherein said T1R3 is a human T1R3 that exhibits at least 98% sequence identity to the polypeptide of SEQ. ID. NO: 6.
6. The method of claim 1 wherein said T1R3 is a human T1R3 that exhibits at least 99% sequence identity to the polypeptide of SEQ. ID. NO: 6.
7. The method of claim 1 wherein said T1R3is encoded by the nucleic acid sequence of SEQ. ID. NO: 5.
8. The method of claim 1 wherein said T1R3 receptor is expressed by a cell.
9. The method of claim 1 wherein a membrane extract comprises said T1R3 receptor.
10. The method of claim 1 wherein said T1R3 receptor is attached to a solid phase.
11. The method of claim 1 wherein said T1R3 receptor is in solution.
12. The method of claim 1 wherein a liquid bilayer or vesicle comprises said T1R3 receptor.
13. The method of claim 8 wherein said cell is an intact or permeabilized cell.
14. The method of claim 8 wherein said cell further expresses a G protein.
15. The method of claim 8 wherein said cell is a prokaryotic cell.
16. The method of claim 8 wherein said cell is a eukaryotic cell.
17. The method of claim 16 wherein said cell is an insect, yeast, amphibian or mammalian cell.
18. The method of claim 16 wherein said cell is a CHO cell, HEK-293 cell, COS cell or Xenopus oocyte.
19. The method of claim 1 wherein the binding assay detects changes in the conformation of the T1R3 receptor.
20. The method of claim 19 wherein said change is detected by NMR spectroscopy.
21. The method of claim 19 wherein said change is detected by fluorescence spectroscopy.
22. The method of claim 1 wherein said T1R3 receptor further comprises a G protein.
23. The method of claim 22 wherein said G protein is Galpha15, Galpha16 or gustducin.
24. The method of claim 1 wherein said binding assay includes the use of a detectable label.
25. The method of claim 24 wherein said label is an enzyme, radionuclide, chemiluminescent compound or fluorescent compound.
26. The method of claim 1 wherein the binding assay detects displacement of a labeled ligand at said T1R3 receptor.
27. The method of claim 1 wherein said binding assay is a fluorescence polarization or FRET assay.
28. The method of claim 1 wherein the binding assay detects conformational changes in the T1R1/T1R3 taste receptor based on altered susceptibility to proteolysis.
29. The method of claim 1 wherein the binding assay is a competitive binding assay.
30. The method of claim 1 wherein the binding assay is a non-competitive binding assay.
31. The method of claim 1 wherein the binding assay detects the effect of said compound on the specific binding of another compound to said receptor.
32. The method of claim 1 wherein said binding assay detects the effect of said compound on the binding of L-glutamate or L-aspartate to said receptor.
33. The method of claim 1 wherein said binding assay uses a eukaryotic cell that stably expresses the T1R3 receptor on its surface.
34. The method of claim 1 which said binding assay uses a cell that transiently expresses said T1R3 receptor on its surface.
35. The method of claim 1 wherein the binding assay uses an HEK-293 cell that stably expresses T1R3 and further expresses Galpha15.
36. The method of claim 35 wherein said binding assay detects the effect of said compound on the binding of a radioactively or fluorescently labeled ligand to said receptor.
37. The method of claim 1 wherein said binding assay detects binding based on a detectable change in fluorescence absorbance or refractive index.
38. The method of claim 1 wherein the binding assay is a high-throughput screening assay.
39. The method of claim 36 wherein the assay screens a combinatorial chemical library.
40. The method of claim 36 wherein the assay screens a randomized small compound library.
41. The method of claim 1 wherein step (3) assays the effect of said compound in a human taste test.
42. The method of claim 8 , wherein the cell is an endogenous taste cell.
43. The method of claim 42 , wherein the cell is a taste cell present in foliate, circumvallate or fungiform papillae.
44. The method of claim 42 , wherein the cell is a taste cell present in geschmackstreifen, oral cavity, gastrointestinal epithelium or epiglottis.
45. The method of claim 44 , wherein the cell is a taste cell present in gastrointestinal epithelium.
46. The method of claim 8 wherein said T1R3 receptor sequence is expressed on the surface of a mammalian cell.
47. The method of claim 46 wherein the cell is a taste cell.
48. The method of claim 47 , wherein the cell is a taste cell present in foliate, circumvallate or fungiform papillae.
49. The method of claim 47 , wherein the cell is a taste cell present in geschmackstreifen, oral cavity, gastrointestinal epithelium or epiglottis.
50. The method of claim 49 , wherein the cell is a taste cell present in gastrointestinal epithelium.