Methods of screening T1R1/T1R3 heteromeric receptors for compounds that modulate umami taste signaling
View Patent ↗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.
1. A method of screening for a compound that putatively blocks or activates umami taste signaling, the method comprising the steps of:
(a) contacting cells comprising isolated or cultured mammalian cells with one or more compounds, wherein said cells express a heteromeric T1R1/T1R3 taste receptor; and
(b) detecting whether said one or more compounds specifically bind to said heteromeric T1R1/T1R3 taste receptor and/or specifically activate said heteromeric T1R1/T1R3 taste receptor and, based thereon, identifying said one or more compounds as compounds that putatively block or activate umami taste signaling, wherein
(1) said heteromeric T1R1/T1R3 taste receptor expressed by said cells specifically binds to a ligand that specifically binds to an endogenous (wild-type) human heteromeric T1R1/T1R3 receptor comprised of at least one endogenous T1R1 polypeptide and at least one endogenous T1R3 polypeptide; and
(2) said heteromeric T1R1/T1R3 taste receptor expressed by said cells comprises at least one T1R1 polypeptide that (i) is encoded by a nucleic acid sequence comprising SEQ ID NO: 8, or (ii) possesses at least 90% sequence identity to the T1R1 polypeptide of SEQ ID NO: 5; and
(3) said heteromeric T1R1/T1R3 taste receptor expressed by said cells comprises at least one T1R3 polypeptide that (i) is encoded by a nucleic acid sequence comprising SEQ ID NO: 9; or (ii) possesses at least 90% sequence identity to the T1R3 polypeptide of SEQ ID NO: 7; and
(4) at least one of said expressed T1R1 polypeptide or said expressed T1R3 polypeptide is expressed from a gene that is endogenous to said cells.
2. A method of screening for a compound that putatively modulates umami taste signaling, the method comprising the steps of:
(a) contacting cells comprising isolated or cultured mammalian cells with one or more compounds, wherein said cells express a heteromeric T1R1/T1R3 taste receptor; and
(b) detecting whether said one or more compounds affect the binding of another compound to said heteromeric T1R1/T1R3 taste receptor and/or modulate the activation of said heteromeric T1R1/T1R3 taste receptor by another compound and, based thereon, identifying said one or more compounds as compounds that putatively modulate umami taste signaling, wherein
(1) said heteromeric T1R1/T1R3 taste receptor expressed by said cells specifically binds to a ligand that specifically binds to an endogenous (wild-type) human heteromeric T1R1/T1R3 receptor comprised of at least one endogenous T1R1 polypeptide and at least one endogenous T1R3 polypeptide; and
(2) said heteromeric T1R1/T1R3 taste receptor expressed by said cells comprises at least one T1R1 polypeptide that (i) is encoded by a nucleic acid sequence comprising SEQ ID NO: 8, or (ii) possesses at least 90% sequence identity to the T1R1 polypeptide of SEQ ID NO: 5; and
(3) said heteromeric T1R1/T1R3 taste receptor expressed by said cells comprises at least one T1R3 polypeptide that (i) is encoded by a nucleic acid sequence comprising SEQ ID NO: 9; or (ii) possesses at least 90% sequence identity to the T1R3 polypeptide of SEQ ID NO: 7; and
(4) at least one of said expressed T1R1 polypeptide or said expressed T1R3 polypeptide is expressed from a gene that is endogenous to said cells.
3. The method of claim 1 that further includes a taste test to determine the effect of the identified compound on umami taste and, based on the result of the taste test, identifying the compound as a compound that blocks or activates umami taste signaling.
4. The method of claim 2 that further includes a taste test to determine the effect of the identified compound on umami taste and, based on the result of the taste test, identifying the compound as a compound that modulates umami taste signaling.
5. The method of claim 1 , wherein said expressed T1R1 polypeptide is expressed from a gene that is endogenous to said cells and said expressed T1R3 polypeptide is expressed from a gene that is endogenous to said cells.
6. The method of claim 2 , wherein said expressed T1R1 polypeptide is expressed from a gene that is endogenous to said cells and said expressed T1R3 polypeptide is expressed from a gene that is endogenous to said cells.
7. The method of claim 1 , wherein said cells include human cells.
8. The method of claim 2 , wherein said cells include human cells.
9. The method of claim 1 , wherein said cells are identified by a method comprising detecting the expression of a T1R1 and/or T1R3 polypeptide or T1R1- and/or T1R3-encoding nucleic acid by said cells.
10. The method of claim 2 , wherein said cells are identified by a method comprising detecting the expression of a T1R1 and/or T1R3 polypeptide or T1R1- and/or T1R3-encoding nucleic acid by said cells.
11. The method of claim 1 , wherein said cells are identified using a probe that specifically detects the expression of T1R1 and/or T1R3 genes by said cells.
12. The method of claim 2 , wherein said cells are identified using a probe that specifically detects the expression of T1R1 and/or T1R3 genes by said cells.
13. The method of claim 11 , wherein the probe comprises a nucleic acid that specifically detects a T1R1 or T1R3 nucleic acid expressed by said cells or comprises an antibody that specifically detects a T1R1 or T1R3 polypeptide expressed by said cells.
14. The method of claim 12 , wherein the probe comprises a nucleic acid that specifically detects a T1R1 or T1R3 nucleic acid expressed by said cells or comprises an antibody that specifically detect a T1R1 or T1R3 polypeptide expressed by said cells.
15. The method of claim 13 , wherein said T1R1 or T1R3 nucleic acid is an RNA and is detected by a method selected from the group consisting of Northern analysis, dot blot, RNase protection, S1 analysis, a nucleic acid amplification technique, and in situ hybridization.
16. The method of claim 14 , wherein said T1R1 or T1R3 nucleic acid is an RNA and is detected by a method selected from the group consisting of Northern analysis, dot blot, RNase protection, S1 analysis, a nucleic acid amplification technique, and in situ hybridization.
17. The method of claim 11 , wherein said at least one probe comprises a T1R1- and/or T1R3-specific primer.
18. The method of claim 12 , wherein said at least one probe comprises a T1R1- and/or T1R3-specific primer.
19. The method of claim 1 , wherein step (b) comprises detecting an alteration in said cells in one or more of intracellular Ca 2+ , intracellular IP3, an intracellular cyclic nucleotide, intracellular cAMP, intracellular cGMP, membrane voltage, and G protein binding to GTPγS.
20. The method of claim 2 , wherein step (b) comprises detecting an alteration in said cells in one or more of intracellular Ca 2+ , intracellular IP3, an intracellular cyclic nucleotide, intracellular cAMP, intracellular cGMP, membrane voltage, and G protein binding to GTPγS.
21. The method of claim 1 , wherein said cells are obtained from a human tissue or a human cell culture.
22. The method of claim 2 , wherein said cells are obtained from a human tissue or a human cell culture.
23. The method of claim 21 , wherein said human tissue comprises tongue, oral cavity tissue, gastrointestinal epithelium, or epiglottis.
24. The method of claim 22 , wherein said human tissue comprises tongue, oral cavity tissue, gastrointestinal epithelium, or epiglottis.
25. The method of claim 1 , wherein said expressed T1R1 polypeptide or said expressed T1R3 polypeptide is expressed from a nucleic acid heterologous to said cells.
26. The method of claim 2 , wherein said expressed T1R1 polypeptide or said expressed T1R3 polypeptide is expressed from a nucleic acid heterologous to said cells.
27. The method of claim 3 , wherein said compound that blocks or activates umami taste signaling is an activator, inhibitor, blocker, agonist, or antagonist of umami taste signaling.
28. The method of claim 4 , wherein said compound that modulates umami taste signaling is an activator, inhibitor, blocker, stimulator, enhancer, agonist, or antagonist of umami taste signaling.