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 Ca(2+)-mobilizing G proteins, coexpression of human GPR40 and Galpha(q) allowed medium- and long-chain fatty acids to elevate intracellular [Ca(2+)]. This was also observed when human embryonic kidney (HEK) 293 cells were transfected with a GPR40-Galpha(q) fusion protein. The kinetic of elevation of intracellular [Ca(2+)] 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 effective ligand equilibration, GPR40-Galpha(q) was used in guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays. After expression of GPR40-Galpha(q) in HEK293 cells and membrane preparation basal binding of [(35)S]GTPgammaSinGalpha(q) immunoprecipitates was high and not elevated substantially by fatty acids. However, treatment of membranes with fatty acid-free bovine serum albumin reduced the basal [(35)S]GTPgammaS binding in a concentration-dependent manner and allowed the responsiveness and pharmacology at GPR40 of each of the fatty acids thiazolidinediones and a novel small-molecule agonist to be uncovered. Membranes of rat INS-1E cells that express GPR40 endogenously provided similar observations. The high apparent constitutive activity of GPR40-Galpha(q) was also reversed by a small-molecule GPR40 antagonist, and basal [(35)S]GTPgammaS binding was prevented by the selective Galpha(q)/Galpha(11) inhibitor YM-254890. The current studies provide novel insights into the pharmacology of GPR40 and indicate that G protein-coupled receptors which respond to fatty acids, and potentially to other lipid ligands, can be occupied by endogenous agonists before assay and that this may mask the pharmacology of the receptor and may be mistaken for high levels of constitutive activity.