Human T2R receptors for acetaminophen, ranitidine, strychnine and denatonium and related assays for identifying human bitter taste modulators
The present invention relates to the discovery that specific human taste receptors in the T2R taste receptor family respond to particular bitter ligands, i.e., acetaminophen, ranitidine, strychnine and denatonium. The present invention further relates to the use of these receptors in assays for identifying ligands that modulate the activation of these taste receptors and which may be used as additives in foods, beverages and medicinals for modifying (blocking) T2R-associated bitter taste.
1. An assay for identifying a compound which modulates a human hT2R75 bitter taste receptor polypeptide that possesses at least 90% sequence identity to the polypeptide contained in SEQ ID NO: 15 which comprises:
i. screening a compound for its effect on the specific binding or activation of said hT2R75 polypeptide by a bitter ligand selected from ranitidine, strychnine and denatonium benzoate, and ii. determining whether said compound modulates hT2R75 associated bitter taste based on its effect on the activation of said receptor by said bitter ligand.
2. The assay of claim 1 wherein said human hT27R5 bitter taste receptor polypeptide is expressed on a cell membrane.
3. The assay of claim 1 wherein said human hT2R75 bitter taste receptor polypeptide is expressed on an isolated cell membrane.
4. The assay of claim 1 wherein said human hT2R75 bitter taste receptor polypeptide is expressed on an intact cell.
5. The assay of claim 1 wherein said human hT2R75 bitter taste receptor polypeptide is expressed on a eukaryotic cell.
6. The assay of claim 1 wherein said human hT2R75 bitter taste receptor polypeptide is expressed by an amphibian, mammalian or insect cell.
7. The assay of claim 1 wherein said human hT2R75 bitter taste receptor polypeptide is expressed on a cell selected from an HEK293, BHK, COS, HEK293T, CHO and a Xenopus oocyte.
8. The assay of claim 1 which is a fluorimetric assay.
9. The assay of claim 1 which is a binding assay.
10. The assay of claim 1 which detects the effect on said compound by assaying its effect on an intracellular ion concentration.
11. The assay of claim 1 which detects the effect of said compound on intracellular sodium or calcium.
12. The assay of claim 1 which detects the effect of said compound on cell membrane potential.
13. The assay of claim 1 which detects the effect of said compound on the transcription of said human hT2R75 bitter taste receptor polypeptide.
14. The assay of claim 1 wherein in said compound is selected based on its ability to block interaction of said human hT2R75 bitter taste receptor polypeptide with ranitidine.
15. The assay of claim 1 which detects the effect of said compound on intracellular cAMP, cGMP or IP3.
16. The assay of claim 1 wherein said taste receptor comprises the extracellular domain or transmembrane region of said human hT2R75 bitter taste receptor polypeptide.
17. The assay of claim 1 wherein said assay detects changes in calcium using a calcium specific fluorescent dye.
18. The assay of claim 1 wherein said assay detects changes in intracellular calcium using a dye selected from Fluo-3, Fluo-4 and Fura-2.
19. The assay of claim 1 wherein said human hT2R75 bitter taste receptor polypeptide is in solution.
20. The assay of claim 1 which is a binding assay that detects changes in spectroscopic characteristics, hydrodynamic characteristics or solubility.
21. The assay of claim 1 which detects the effect of said compound on the complexing of said human hT2R75 bitter taste receptor polypeptide with a G protein.
22. The assay of claim 1 which detects the effect of said compound on the complexing of said human hT2R75 bitter taste receptor polypeptide with a G protein selected from transducin, gustducin, Galpha15, and Galpha16.
23. The assay of claim 1 which is a fluorescence polarization assay.
24. The assay of claim 1 wherein said human hT2R75 bitter taste receptor polypeptide is attached to a solid phase substrate.
25. The assay of claim 1 which is a high throughput assay.
26. The assay of claim 1 wherein the human hT2R75 bitter taste receptor polypeptide is expressed by a HEK293 cell.