T1R2 binding assays
The invention relates to binding assays using human T1R2 polypeptides containing the extracellular and/or transmembrane regions of endogenous human T1R2 polypeptide. These assays may be used to identify T1R2 modulators, especially taste modulatory compounds.
1. A method for identifying a compound that putatively modulates or elicits taste in a human subject comprising:(i) screening one or more compounds in a binding assay comprising contacting said one or more compounds with a human T1R2 polypeptide, wherein said T1R2 polypeptide is selected from the group consisting of: (a) a polypeptide which consists of or contains a polypeptide which is at least 90% identical to the entire extracellular or transmembrane regions of the human T1R2 polypeptide in SEQ ID NO: 21 and which polypeptide specifically binds to a ligand that specifically binds to the human T1R2 polypeptide in SEQ ID NO:21; (b) a human T1R2 polypeptide encoded by a nucleic acid sequence that specifically hybridizes to the complement of the hT1R2 nucleic acid sequence of SEQ ID NO: 23 under stringent hybridizations which are: conducting hybridization at 42 degrees C. in a solution comprising 50% formamide, 5XSSC, and 1% SDS, and washing at 65 degrees C. in a solution containing 0.2XSSC and 0.1% SDS, and which human T1R2 polypeptide specifically binds to a ligand that specifically binds to the human T1R2 polypeptide in SEQ ID NO:21; and (c) a human T1R2 polypeptide which has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 21; and (ii) whereby a compound that putatively modulates or elicits taste is identified by its specific binding to a human T1R2 polypeptide according to (a), (b), (c) or its inhibition or enhancement of the specific binding of another compound to a T1R2 polypeptide according to (a), (b), or (c).
2. The method of claim 1 , wherein said T1R2 polypeptide comprises the extracellular or transmembrane regions of the human T1R2 polypeptide in SEQ ID NO:21.
3. The method of claim 1 , wherein said T1R2 polypeptide contains a polypeptide that possesses at least 90% sequence identity to the extracellular regions of the human T1R2 polypeptide of SEQ. ID. NO:21.
4. The method of claim 1 , wherein said T1R2 polypeptide contains a polypeptide that possesses at least 90% sequence identity to the transmembrane regions of the human T1R2 polypeptide of SEQ. ID. NO:21.
5. The method of claim 3 , wherein said T1R2 polypeptide comprises the extracellular regions of a different GPCR.
6. The method of claim 4 , wherein said T1R2 polypeptide comprises the transmembrane regions of a different GPCR.
7. The method of claim 1 , wherein said T1R2 polypeptide contains a polypeptide that possesses at least 95% sequence identity to the extracellular regions of the human T1R2 polypeptide of SEQ. ID. NO:21.
8. The method of claim 1 , wherein said T1R2 polypeptide contains a polypeptide that possesses at least 95% sequence identity to the transmembrane regions of the human T1R2 polypeptide of SEQ. ID. NO:21.
9. The method of claim 1 wherein said T1R2 polypeptide is attached to a solid phase.
10. The method of claim 1 , wherein said T1R2 polypeptide is in solution.
11. The method of claim 1 , wherein T1R2 polypeptide is in a lipid bilayer or vesicle.
12. The method of claim 1 , wherein the T1R2 polypeptide is expressed in an isolated cell.
13. The method of claim 1 , wherein the T1R2 polypeptide is in the cell membrane of an isolated cell.
14. The method of claim 1 , wherein the binding assay comprises detecting binding by detecting a change in T1R2 polypeptide conformation.
15. The method of claim 12 , wherein the cell is a eukaryotic cell.
16. The method of claim 12 , wherein said cell is a yeast, insect, amphibian or mammalian cell.
17. The method of claim 12 , wherein the cell is a CHO cell, HEK-293 cell, COS cell, or Xenopus oocyte.
18. The method of claim 1 , wherein the binding assay detects a change in T1R2 polypeptide conformation upon binding of the compound.
19. The method of claim 18 , wherein said change is detected by NMR spectroscopy.
20. The method of claim 18 , wherein said change is detected by fluorescence spectroscopy.
21. The method of claim 1 , wherein the assay includes the use of a label, wherein said label allows for the identification of a compound that specifically binds to said T1R2 polypeptide or that modulates the specific binding of another compound to said T1R2 polypeptide.
22. The method of claim 21 , wherein said label is an enzyme, radionuclide, chemiluminescent compound or fluorescent compound.
23. The method of claim 1 , wherein the binding assay detects binding of a labeled ligand to said T1R2 polypeptide.
24. The method of claim 1 , wherein said binding assay is a fluorescent polarization or FRET assay.
25. The method of claim 1 , wherein binding of a compound to T1R2 polypeptide is detected by a competitive binding assay.
26. The method of claim 1 , wherein the binding assay detects release of a labeled ligand from said T1R2 polypeptide.
27. The method of claim 1 which is a high throughput binding assay.
28. The method of claim 1 , which further includes step (iii) whereby said putative taste modulating compound is assayed in a human taste test.