Methods of using T1R hetero-oligomeric taste receptors to screen for compounds which modulate sweet or umami taste signaling
Newly identified mammalian taste-cell-specific G protein-coupled receptors which function as hetero-oligomeric complexes in the sweet taste transduction pathway, and the genes and cDNA encoding said receptors are described. Specifically, T1R G protein-coupled receptors active in sweet taste signaling as hetero-oligomeric complexes, and the genes and cDNA encoding the same, are described, along with methods for isolating such genes and for isolating and expressing such receptors. Methods for identifying putative taste modulating compounds using such hetero-oligomeric complexes also described, as is a novel surface expression facilitating peptide useful for targeting integral plasma membrane proteins to the surface of a cell.
1. A method of screening for compounds that modulate sweet or umami taste signaling comprising:
(i) contacting a human cell which co-expresses at least two different T1R receptors or polypeptides on its surface, wherein one of said T1R receptors or polypeptides comprises human T1R3, with a putative taste modulating compound; and
(ii) measuring an activity from the T1R receptors or polypeptides expressed on the cell surface in response to the contacting, wherein the expressed T1R receptors or polypeptides act as a hetero-oligomeric complex, and wherein the activity is indicative of a compound that modulates sweet or umami taste signaling.
2. The method of claim 1 , wherein the T1R receptor or polypeptide activity is measured by assayed changes in intracellular Ca 2+ levels, cAMP, cGMP and IP3, or G protein binding of GTPγS.
3. The method of claim 1 , wherein the cell is transfected with at least one additional nucleic acid construct encoding a gene involved in taste signaling.
4. The method of claim 3 , wherein said at least one additional gene encodes a G protein involved in taste signal transduction.
5. The method of claim 4 , wherein said G protein is a promiscuous G protein.
6. The method of claim 1 , wherein said at least two T1R receptors or polypeptides comprise a T1R1 and T1R3 polypeptide or a T1R2 and T1R3 polypeptide.
7. A method of screening for compounds that modulate taste signaling transduction comprising:
(i) contacting a first human cell co-expressing at least two different human T1R receptors or polypeptides, wherein one of said T1R receptors or polypeptides comprises human T1R3, with a known taste activating compound and a putative taste transduction modulating compound, wherein the expressed T1R receptors or polypeptides act as a hetero-oligomeric complex;
(ii) contacting a second human cell co-expressing at least two different T1R receptors or polypeptides, wherein one of said T1R receptors or polypeptides comprises human T1R3, with a known taste activating compound alone, wherein the expressed T1R receptors or polypeptides act as a hetero-oligomeric complex; and
(iii) comparing a first activity from the T1R receptors or polypeptides expressed on the cell surface of the first human cell with a second activity from the T1R receptors or polypeptides expressed on the cell surface of the second human cell, wherein a difference in the first activity and the second activity is indicative of a compound that modulates taste signaling transduction.
8. The method of claim 7 , wherein the putative taste transduction modulating compound is selected from the group consisting of activators, inhibitors, stimulators, enhancers, agonists, and antagonists.
9. The method of claim 7 , wherein the first activity and the second activity are measured by assayed by changes in intracellular Ca 2+ levels, cAMP, cGMP and IP3, or G protein binding of GTPγS.
10. The method of claim 7 , wherein the first human cell is transfected with at least one additional nucleic acid construct encoding a gene involved in taste signaling.
11. The method of claim 10 , wherein said at least one additional gene encodes a G protein involved in taste signal transduction.
12. The method of claim 11 , wherein said G protein is a promiscuous G protein.