Use of T1R3 venus flytrap region polypeptide to screen for taste modulators
The invention relates to compounds that specifically bind a T1R1/T1R3 or T1R2/T1R3 receptor or fragments or sub-units thereof. The present invention also relates to the use of hetero-oligomeric and chimeric 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.
1. A method for identifying a putative taste modulator compound comprising conducting a binding or functional assay using one or more compounds and identifying a putative taste modulatory compound based on its specifically interacting with extracellular loop 2 and extracellular loop 3 of the human transmembrane domain of a human T1R3 subunit that is at least 90% identical to the human T1R3 polypeptide of SEQ ID NO: 7.
2. The method of claim 1 , wherein said T1R3 polypeptide is comprised in a heteromeric taste receptor that comprises said T1R3 polypeptide and further comprises a T1R1 polypeptide that is at least 90% identical to the hT1R1 polypeptide of SEQ ID NO:5 or a hT1R2 polypeptide that is at least 90% identical to the hT1R2 polypeptide of SEQ ID NO:6.
3. The method of claim 2 , wherein the assay uses cells that co-express said T1R3 polypeptide and said T1R1 or T1R2 polypeptide that respectively is at least 90% identical to the hT1R1 polypeptide of SEQ ID NO:5 or at least 90% identical to the hT1R2 polypeptide of SEQ ID NO:6.
4. The method of claim 1 , wherein said T1R3 polypeptide is comprised on a membrane or is linked to a solid phase.
5. The method of claim 1 , wherein said T1R3 polypeptide is expressed by a cell.
6. The method of claim 5 , wherein said cell is selected from the group consisting of yeast, insect, mammalian, amphibian and worm cells.
7. The method of claim 1 , wherein the assay detects changes in the activity of said T1R3 polypeptide that is measured by detecting changes in intracellular calcium ion levels, by detecting changes in fluorescence polarization, by detecting changes in second messenger levels, or by detecting changes in intracellular cyclic nucleotides.
8. The method of claim 1 , which further includes a step whereby said putative taste modulating compound is assayed in a human taste test.
9. The method of claim 2 , which is a binding assay that detects the specific binding of said compound to the Venus flytrap domain of said T1R3 polypeptide.
10. The method of claim 1 , which is a binding assay that detects the specific binding of said compound to extracellular loop 2 and extracellular loop 3 of said T1R3 polypeptide.
11. The method of claim 2 , which is a binding assay that detects the specific binding of said compound to extracellular loop 2 and extracellular loop 3 of said T1R3 polypeptide.