PT  - JOURNAL ARTICLE
AU  - Leigh A. Stoddart
AU  - Andrew J. Brown
AU  - Graeme Milligan
TI  - Uncovering the Pharmacology of the G Protein-Coupled Receptor GPR40: High Apparent Constitutive Activity in Guanosine 5′-<em>O</em>-(3-[<sup>35</sup>S]thio)triphosphate Binding Studies Reflects Binding of an Endogenous Agonist
AID  - 10.1124/mol.106.031534
DP  - 2007 Apr 01
TA  - Molecular Pharmacology
PG  - 994--1005
VI  - 71
IP  - 4
4099  - http://molpharm.aspetjournals.org/content/71/4/994.short
4100  - http://molpharm.aspetjournals.org/content/71/4/994.full
SO  - Mol Pharmacol2007 Apr 01; 71
AB  - In cells lacking expression of Ca2+-mobilizing G proteins, coexpression of human GPR40 and Gαq 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-Gαq 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 effective ligand equilibration, GPR40-Gαq was used in guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assays. After expression of GPR40-Gαq in HEK293 cells and membrane preparation basal binding of [35S]GTPγSinGαq immunoprecipitates was high and not elevated substantially by fatty acids. However, treatment of membranes with fatty acid-free bovine serum albumin reduced the basal [35S]GTPγS 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-Gαq was also reversed by a small-molecule GPR40 antagonist, and basal [35S]GTPγS binding was prevented by the selective Gαq/Gα11 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. The American Society for Pharmacology and Experimental Therapeutics