Cell lines that stably or transiently express a functional sweet (T1R2/T1R3) taste receptor
View Patent ↗Cells are engineered to stably or transiently express T1R2 and T1R3 polypeptides, desirably in association with a G protein. The resultant heteromeric taste receptor is a sweet taste receptor and responds to sweet taste stimuli such as artificial and natural sweeteners, e.g., saccharin. The invention provides a preferred mammalian cell lines, e.g., HEK-293 cells that stably express T1R2/T1R3 and G α15 under inducible conditions. These cells are useful in cell-based assays for identifying compounds that elicit or modulate sweet taste.
1. An isolated recombinant cell that expresses a heteromeric taste receptor, wherein said receptor is comprised of at least one T1R2 polypeptide and at least one T1R3 polypeptide, wherein said T1R2 polypeptide is (i) encoded by a nucleic acid sequence comprising SEQ. ID. NO: 10, (ii) encoded by a nucleic acid sequence comprising a nucleic acid that hybridizes to SEQ. ID. NO: 10 under stringent hybridization conditions which are conducting the hybridization reaction at 42° C. in a solution comprising 50% formamide, 5×SSC, and 1% SDS and washing at 65° C. in a solution comprising 0.2×SSC and 0.1% SDS, or (iii) a T1R2 polypeptide possessing at least 90% sequence identity to the T1R2 polypeptide of SEQ. ID. NO: 6;
wherein said T1R3 polypeptide is (i) encoded by a nucleic acid sequence comprising SEQ. ID. NO: 9; (ii) encoded by a nucleic acid sequence that hybridizes to SEQ. ID. NO: 9 under stringent hybridization conditions which are conducting the hybridization reaction at 42° C. in a solution comprising 50% formaniide, 5×SSC, 10% SDS; and washing at 65° C. in a solution comprising 0.2×SCC and 0.1% SDS, or (iii) a T1R3 polypeptide possessing at least 90% sequence identity to the T1R3 polypeptide of SEQ. ID. NO: 7;
and wherein said isolated cell expresses a heteromeric taste receptor that specifically binds to a ligand that specifically binds to an endogenous (wild-type) human heteromeric T1R2/T1R3 receptor comprised of at least one endogenous T1R2 polypeptide and at least one endogenous T1R3 polypeptide.
2. The cell of claim 1 , which is selected from the group consisting bacterial, yeast, mammalian, amphibian and insect cells.
3. The cell of claim 1 , wherein said cell is a prokaryotic cell.
4. The cell of claim 1 , wherein said cell is a eukaryotic cell.
5. The cell of claim 4 , wherein said eukaryotic cell is a CHO, HEK-293, COS or Xenopus oocyte.
6. The cell of claim 1 , wherein said T1R2 and T1R3 are of the same species.
7. The cell of claim 1 , wherein T1R2 and T1R3 nucleic acid sequences are stably integrated into said cell.
8. The cell of claim 1 , wherein T1R2 and T1R3 nucleic acid sequences comprise an extrachromosomal element.
9. The cell of claim 1 , which comprises T1R2 and T1R3 nucleic acid sequences that are operably linked to a constitutive promoter.
10. The cell of claim 1 , which comprises T1R2 and T1R3 nucleic acid sequences that are operably linked to an inducible promoter.
11. The cell of claim 1 , which further expresses a G protein.
12. The cell of claim 11 , wherein said G protein is G a15 , G a16 or gustducin.
13. The cell of claim 1 , wherein said T1R2 polypeptide has the amino acid sequence of SEQ. ID. NO: 6.
14. The cell of claim 1 , wherein said T1R2 polypeptide has an amino acid sequence that possesses at least 90% sequence identity to the amino acid sequence of SEQ. ID. NO: 6.
15. The cell of claim 1 , wherein said T1R2 polypeptide has at least 95% sequence identity to the polypeptide of SEQ. ID. NO: 6.
16. The cell of claim 1 , wherein said T1R2 polypeptide has at least 96% sequence identity to the polypeptide of SEQ. ID. NO: 6.
17. The cell of claim 1 , wherein said T1R2 polypeptide has at least 97% sequence identity to the polypeptide of SEQ. ID. NO: 6.
18. The cell of claim 1 , wherein said T1R2 polypeptide has at least 98% sequence identity to the polypeptide of SEQ. ID. NO: 6.
19. The cell of claim 1 , wherein said T1R2 polypeptide has at least 99% sequence identity to the polypeptide of SEQ. ID. NO: 6.
20. The cell of claim 1 , wherein said T1R2 polypeptide is encoded by the nucleic acid sequence of SEQ. ID. NO: 10.
21. The cell of claim 1 , wherein said T1R2 polypeptide is encoded by a nucleic acid sequence that hybridizes to SEQ. ID. NO: 10 under stringent hybridization conditions which are conducting the hybridization reaction at 42° C. in a solution comprising 50% formamide, 5×SSC, and 1% SDS and washing at 65° C. in a solution comprising 0.2×SSC and 0.1% SDS.
22. The cell of claim 1 , wherein said T1R3 polypeptide has the amino acid sequence of SEQ. ID. NO: 7.
23. The cell of claim 1 , wherein said T1R3 polypeptide has an amino acid sequence that possesses at least 90% sequence identity to the amino acid sequence of SEQ. ID. NO: 7.
24. The cell of claim 1 , wherein said T1R3 polypeptide exhibits at least 95% sequence identity to the polypeptide of SEQ. ID. NO:7.
25. The cell of claim 1 , wherein said T1R3 polypeptide exhibits at least 96% sequence identity to the polypeptide of SEQ. ID. NO:7.
26. The cell of claim 1 , wherein said T1R3 polypeptide exhibits at least 97% sequence identity to the polypeptide of SEQ. ID. NO:7.
27. The cell of claim 1 , wherein said T1R3 polypeptide exhibits at least 98% sequence identity to the polypeptide of SEQ. ID. NO:7.
28. The cell of claim 1 , wherein said T1R3 polypeptide exhibits at least 99% sequence identity to the polypeptide of SEQ. ID. NO:7.
29. The cell of claim 1 , wherein said T1R3 polypeptide is encoded by the nucleic acid sequence of SEQ. ID. NO: 9.
30. The cell of claim 1 , wherein said T1R3 polypeptide is encoded by a nucleic acid sequence that hybridizes to SEQ. ID. NO: 9 under stringent hybridization conditions which are conducting the hybridization reaction at 42° C. in a solution comprising 50% formamide, 5×SSC, and 1% SDS and washing at 65° C. in a solution comprising 0.2×SSC and 0.1% SDS.
31. The cell of claim 1 , which expresses a T1R2 polypeptide comprising SEQ. ID. NO: 6 and a T1R3 polypeptide comprising SEQ. ID. NO: 7.
32. The cell of claim 31 , which is a eukaryotic cell.
33. The cell of claim 32 , which is a mammalian, yeast, amphibian or insect cell.
34. The cell of claim 32 , which is a CHO, COS, HEK-293 or Xenopus oocyte.
35. The cell of claim 34 , which is an HEK-293 cell.
36. The cell of claim 34 , which stably expresses said T1R2 and T1R3 polypeptides.
37. The cell of claim 34 , which transiently expresses said T1R2 and T1R3 polypeptides.
38. The cell of claim 31 , which further expresses a G protein.
39. The cell of claim 38 , wherein said G protein is G a15 and G a15 or gustducin.
40. An isolated eukaryotic recombinant cell that expresses a heteromeric taste receptor, wherein said receptor is comprised of at least one T1R2 polypeptide and at least one T1R3 polypeptide, wherein said T1R2 polypeptide is (i) encoded by a nucleic acid sequence comprising SEQ. ID. NO: 10, (ii) encoded by a nucleic acid sequence comprising a nucleic acid that hybridizes to SEQ. ID. NO: 10 under stringent hybridization conditions which are conducting the hybridization reaction at 42° C. in a solution comprising 50% formamide, 5×SSC, and 1% SDS and washing at 65° C. in a solution comprising 0.2×SSC and 0.1% SDS, or (iii) a T1R2 polypeptide possessing at least 90% sequence identity to the T1R2 polypeptide of SEQ. ID. NO: 6;
wherein said T1R3 polypeptide is (i) encoded by a nucleic acid sequence comprising SEQ. ID. NO: 9; (ii) encoded by a nucleic acid sequence that hybridizes to SEQ. ID. NO: 9 under stringent hybridization conditions which are conducting the hybridization reaction at 42° C. in a solution comprising 50% formamide, 5×SSC, 10% SDS; and washing at 65° C. in a solution comprising 0.2×SCC and 0.1% SDS, or (iii) a T1R3 polypeptide possessing at least 90% sequence identity to the T1R3 polypeptide of SEQ. ID. NO: 7;
and wherein said isolated cell expresses a heteromeric taste receptor that specifically binds to a ligand that specifically binds to an endogenous (wild-type) human heteromeric T1R2/T1R3 receptor comprised of at least one endogenous T1R2 polypeptide and at least one endogenous T1R3 polypeptide.
41. The cell of claim 40 , which is an endogenous taste cell.
42. The cell of claim 41 , wherein the cell is a taste cell present in foliate, circumvallate or fungiform papillae.
43. The cell of claim 41 , wherein the cell is a taste cell present in geschmackstreifen, oral cavity, gastrointestinal epithelium or epiglottis.
44. The cell of claim 43 , wherein the cell is a taste cell present in gastrointestinal epithelium.
45. The cell of claim 40 , which further expresses a G protein.
46. The cell of claim 45 , wherein said G protein is G a15 , G a16 or gustducin.
47. The cell of claim 40 , wherein said T1R2 polypeptide has the amino acid sequence of SEQ. ID. NO: 6.
48. The cell of claim 40 , wherein said T1R2 polypeptide has an amino acid sequence that possesses at least 90% sequence identity to the amino acid sequence of SEQ. ID. NO: 6.
49. The cell of claim 40 , wherein said T1R2 polypeptide has at least 95% sequence identity to the polypeptide of SEQ. ID. NO: 6.
50. The cell of claim 40 , wherein said T1R2 polypeptide has at least 96% sequence identity to the polypeptide of SEQ. ID. NO: 6.
51. The cell of claim 40 , wherein said T1R2 polypeptide has at least 97% sequence identity to the polypeptide of SEQ. ID. NO: 6.
52. The cell of claim 40 , wherein said T1R2 polypeptide has at least 98% sequence identity to the polypeptide of SEQ. ID. NO: 6.
53. The cell of claim 40 , wherein said T1R2 polypeptide has at least 99% sequence identity to the polypeptide of SEQ. ID. NO: 6.
54. The cell of claim 40 , wherein said T1R2 polypeptide is encoded by the nucleic acid sequence of SEQ. ID. NO: 10.
55. The cell of claim 40 , wherein said T1R2 polypeptide is encoded by a nucleic acid sequence that hybridizes to SEQ. ID. NO: 10 under stringent hybridization conditions which are conducting the hybridization reaction at 42° C. in a solution comprising 50% formaxnide, 5×SSC, and 1% SDS and washing at 65° C. in a solution comprising 0.2×SSC and 0.1% SDS.
56. The cell of claim 40 , wherein said T1R3 polypeptide has the amino acid sequence of SEQ. ID. NO: 7.
57. The cell of claim 40 , wherein said T1R3 polypeptide has an amino acid sequence that possesses at least 90% sequence identity to the amino acid sequence of SEQ. ID. NO: 7.
58. The cell of claim 40 , wherein said T1R3 polypeptide exhibits at least 95% sequence identity to the polypeptide of SEQ. ID. NO:7.
59. The cell of claim 40 , wherein said T1R3 polypeptide exhibits at least 96% sequence identity to the polypeptide of SEQ. ID. NO:7.
60. The cell of claim 40 , wherein said T1R3 polypeptide exhibits at least 97% sequence identity to the polypeptide of SEQ. ID. NO:7.
61. The cell of claim 40 , wherein said T1R3 polypeptide exhibits at least 98% sequence identity to the polypeptide of SEQ. ID. NO:7.
62. The cell of claim 40 , wherein said T1R3 polypeptide exhibits at least 99% sequence identity to the polypeptide of SEQ. ID. NO:7.
63. The cell of claim 40 , wherein said T1R3 polypeptide is encoded by the nucleic acid sequence of SEQ. ID. NO: 9.
64. The cell of claim 40 , wherein said T1R3 polypeptide is encoded by a nucleic acid sequence that hybridizes to SEQ. ID. NO: 9 under stringent hybridization conditions which are conducting the hybridization reaction at 42° C. in a solution comprising 50% formamide, 5×SSC, and 1% SDS and washing at 65° C. in a solution comprising 0.2×SSC and 0.1% SDS.
65. The cell of claim 40 , which expresses a T1R2 polypeptide comprising SEQ. ID. NO: 6 and a T1R3 polypeptide comprising SEQ. ID. NO: 7.