Patent ID: 6800445
Filing Date: 2004-10-05
Classification: G01N

Abstract:
A method of assessing the effect of a test condition on G-protein-coupled receptor (GPCR) pathway activity, comprising:a) providing a cell that expresses a GPCR as a fusion protein to a first mutant form of a reporter enzyme and an arrestin as a fusion protein to a second mutant form of the reporter enzyme complementary to the first mutant form of the reporter enzyme, wherein the arrestin is modified to enhance binding of said arrestin to the GPCR, wherein said enhanced binding between said arrestin and the GPCR increases sensitivity of detection of said effect of the test condition; b) exposing the cell to a ligand for said GPCR under the test condition; and c) monitoring activation of said GPCR by complementation of the first and second mutant forms of the reporter enzyme; wherein increased reporter enzyme activity in the cell compared to that which occurs in the absence of the test condition indicates increased GPCR interaction with the modified arrestin compared to that which occurs in the absence of the test condition, and decreased reporter enzyme activity in the cell compared to that which occurs in the absence of the test condition indicates decreased GPCR interaction with the modified arrestin compared to that which occurs in the absence of the test condition; and wherein the GPCR and the first mutant form of reporter enzyme are linked together by a polypeptide linker represented by the formula â€”(GGGGS)nâ€” (SEQ ID NO:10).