Uncovering the pharmacology of the G protein-coupled receptor GPR40: high apparent constitutive activity in guanosine 5'-O-(3[35S]thio)triphosphate binding studies reflects binding of an endogenous agonist

In cells lacking expression of Ca2-mobilizing G proteins, co-expression of human GPR40 and Gq allowed medium- and long-chain fatty acids to elevate intracellular [Ca2]. This was also observed when human embryonic kidney (HEK) 293 cells were transfected with a GPR40-Gq fusion protein. The kinetic of elevation of intracellular [Ca2] slowed with increasing fatty acid chain length, suggesting different ligand on-rates, whereas the addition of fatty acid-free bovine serum albumin reduced signals, presumably by binding the fatty acids. To allow effec-tive ligand equilibration, GPR40-Gq was used in guanosine 5-O-(3-[35S]thio)triphosphate ([35S]GTPS) binding assays. Af-ter expression of GPR40-Gq in HEK293 cells and membrane preparation basal binding of [35S]GTPS in Gq immunopre-cipitates was high and not elevated substantially by fatty acids