Chimeric T1R taste receptor polypeptides and nucleic acid sequences encoding and cell lines that express said chimeric T1R polypeptides
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 putative sweet taste enhancer compounds, comprising conducting a binding or functional assay using one or more compounds and identifying putative sweet enhancer compounds based on their specifically interacting with the Venus Flytrap Region of the human TIR3 receptor polypeptide of SEQ ID NO:7 or the corresponding region in a polypeptide that is at least 90% identical to the human T1R3 polypeptide in SEQ ID NO:7, wherein said Venus Flytrap Region consists of residues 1-563 of said T1R3 polypeptide of SEQ ID NO: 7.
2. The assay of claim 1 , wherein the assay uses cells that co-express said T1R3 polypeptide and a T1R2 taste receptor polypeptide that is at least 90% identical to the hT1R2 polypeptide in SEQ ID NO:6.
3. The method of claim 1 , wherein said T1R3 polypeptide is comprised on a membrane or is linked to a solid phase.
4. The method of claim 1 , wherein said T1R3 polypeptide is expressed by a cell.
5. The method of claim 4 , wherein said cell is selected from the group consisting of yeast, insect, mammalian, amphibian and worm cells.
6. The method of claim 1 , wherein the assay detects changes in the activity of said T1R3 polypeptide is measured by detecting changes in intracellular calcium ion levels, by fluorescence polarization, by detecting second messenger levels or by detecting changes in intracellular cyclic nucleotides.
7. The method of claim 1 , which further includes a step whereby said putative taste modulating compound is assayed in a human taste test.
8. The method of claim 1 , which is a binding assay that detects the specific binding of said compound to the Venus flytrap domain of said T1R3 polypeptde.