Methods of detecting T1R hetero-oligomeric taste receptor expression to identify cells that are potentially sensitive to sweet tastants

Patent metadata

Field	Value
Publication number	US-10114009-B2
Application number	US-201715613312-A
Country	US
Kind code	B2
Filing date	Jun 5, 2017
Priority date	Mar 7, 2001
Publication date	Oct 30, 2018
Grant date	Oct 30, 2018

Abstract

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.

First claim

What is claimed:

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) further 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 (a) and (b), identifying a cell that expresses said T1R2 and T1R3 polypeptides (“detected cell”) which, therefore, is potentially sensitive to sweet tastants (“detected cell”).

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/or 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/or 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/or 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/or expresses a T1R3 polypeptide identical to the polypeptide of SEQ ID NO: 7.

6. The method of claim 1 , further comprising confirming that the detected cell is sensitive to sweet tastants, and using said detected 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 detected 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 detected 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 is gustducin, a promiscuous G protein, Gα15, Gα16, or a chimeric G protein.

12. The method of claim 1 , wherein said T1R2 polypeptide and/or said nucleic acid encoding said T1R2 polypeptide is endogenous to said detected cell, and/or said T1R3 polypeptide and/or said nucleic acid encoding said T1R3 polypeptide is endogenous to said detected cell.

13. The method of claim 1 , wherein at least one probe is used in step (a) to detect expression of T1R2 probe which comprises a nucleic acid that specifically detects the 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 probe comprises a nucleic acid that specifically detects the 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 detected 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 detected 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.

17. The method of claim 15 , wherein the isolated detected cell is human and the assay screens for sweet enhancers.

Assignees

• Senomyx Inc

Inventors

• Zoller Mark
• Li Xiaodong
• Staszewski Lena
• O'Connell Shawn
• Zozulya Sergey
• Adler Jon Elliot
• Xu Hong
• Echeverri Fernando

Classifications

• C12Q2600/158

  Expression markers · CPC title

• G01N2333/705

  Assays involving receptors, cell surface antigens or cell surface determinants · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K14/705

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• A01K2217/05

  Animals comprising random inserted nucleic acids (transgenic) · CPC title