In vitro methods of detecting heteroligomeric T1R2/T2R3-expressing cells that are potentially sensitive to sweet tastants
The present invention relates to the discovery that the T1R receptors assemble to form functional taste receptors. Particularly, it has been discovered that co-expression of T1R1 and T1R3 results in a taste receptor that responds to umami taste stimuli, including monosodium glutamate. Also, it has been discovered that co-expression of the T1R2 and T1R3 receptors results in a taste receptor that responds to sweet taste stimuli including naturally occurring and artificial sweeteners. Also the present invention relates to the use of hetero-oligomeric 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. Finally, the invention relates to the discovery that some compounds, e.g., lactisole, inhibit both the activities of human T1R2/T1R3 and T1R1/T1R3 receptors, and accordingly the sweet and umami taste, suggesting that these receptors may be the only sweet and umami receptors.
1. An in vitro method of detecting a human or monkey cell that is potentially sensitive to sweet tastants, the method comprising: (a) detecting the expression of a T1R2 polypeptide at least 90% identical to the polypeptide of SEQ ID NO: 6; and (b) detecting expression of a T1R3 polypeptide at least 90% identical to the polypeptide of SEQ ID NO: 7; and (c) based on the results of the detecting of steps (a) and (b), identifying a cell that expresses said T1R2 and T1R3 polypeptides which therefore is potentially sensitive to sweet tastants.
2. The in vitro method of claim 1 wherein the detected cell expresses a T1R2 polypeptide at least 95% identical to the polypeptide of SEQ ID NO: 6 and expresses a T1R3 polypeptide at least 95% identical to the polypeptide of SEQ ID NO: 7.
3. The in vitro method of claim 1 wherein the detected cell expresses a T1R2 polypeptide at least 98% identical to the polypeptide of SEQ ID NO: 6 and expresses a T1R3 polypeptide at least 98% identical to the polypeptide of SEQ ID NO: 7.
4. The in vitro method of claim 1 wherein the detected cell expresses a T1R2 polypeptide at least 99% identical to the polypeptide of SEQ ID NO: 6 and expresses a T1R3 polypeptide at least 99% identical to the polypeptide of SEQ ID NO: 7.
5. The in vitro method of claim 1 wherein the detected cell expresses a T1R2 polypeptide at identical to the polypeptide of SEQ ID NO: 6 and expresses a T1R3 polypeptide identical to the polypeptide of SEQ ID NO: 7.
6. The method of claim 1 , further comprising confirming that the identified cell is sensitive to sweet tastants, and using said identified cell in a high-throughput screening assay that detects compounds that specifically bind, activate or modulate the activation of a receptor comprising said T1R2 and T1R3 polypeptides or which modulate the binding, or activation of a receptor comprising said T1R2 and T1R3 polypeptides by another compound.
7. The method of claim 1 , wherein the identified cell is isolated from a human tissue or human cell culture.
8. The method of claim 7 , wherein said human tissue comprises tongue, oral cavity tissue, gastrointestinal epithelium, or epiglottis cells.
9. The method of claim 1 , wherein the identified cell is transfected with a heterologous nucleic acid encoding a detectable label that provides for the identification of compounds that specifically bind or modulate the activity of a receptor comprising said T1R2 polypeptide and said T1R3 polypeptide.
10. The method of claim 1 , wherein the detected cell endogenously expresses a G protein or is transfected with a heterologous nucleic acid encoding a G protein.
11. The method of claim 10 , wherein said G protein comprises gustducin, a promiscuous G protein, Gα15, Gα16, or a chimeric G protein.
12. The method of claim 1 , wherein said T1R2 polypeptide or a nucleic acid encoding said T1R2 polypeptide, is expressed from a gene that is endogenous to said detected cell, and said T1R3 polypeptide or a nucleic acid encoding said T1R3 polypeptide is expressed from a gene that is endogenous to said identified cell.
13. The method of claim 1 , wherein at least one probe is used in step (a) to detect expression of T1R2 which comprises a nucleic acid that specifically detects a nucleic acid encoding said T1R2 polypeptide or comprises an antibody that specifically detects said T1R2 polypeptide and wherein said at least one probe is used in step (b) to detect expression of T1R3 which comprises a nucleic acid that specifically detects a nucleic acid encoding said T1R3 polypeptide or comprises an antibody that specifically detects said T1R3 polypeptide.
14. The method of claim 1 , further comprising using said identified cell in a high-throughput screening assay that detects compounds that specifically activate or modulate sweet taste signaling or detects activators, inhibitors, blockers, stimulators, enhancers, agonists, or antagonists of sweet taste signaling.
15. The method of claim 1 , further comprising using said identified cell in a high-throughput screening assay that detects compounds that specifically bind, activate or modulate the activation of a receptor comprising said T1R2 and T1R3 polypeptides or which modulate the binding, or activation of a receptor comprising said T1R2 and T1R3 polypeptides by another compound.
16. The method of claim 15 , wherein the assay screens for sweet enhancers.