Received for publication October 6, 2006.
Revised December 15, 2006.
Accepted for publication January 2, 2007.

Uncovering the pharmacology of the G protein-coupled receptor GPR40: high apparent constitutive activity in [³⁵S]GTPS binding studies reflects binding of an endogenous agonist

Abstract

In cells lacking expression of Ca²⁺-mobilizing G proteins co-expression of human GPR40 and G_q allowed medium- and long-chain fatty acids to elevate intracellular [Ca²⁺]. This was also observed when HEK293 cells were transfected with a GPR40-G_q fusion protein. The kinetic of elevation of intracellular [Ca²⁺]slowed with increasing fatty acid chain length, suggesting different ligand on-rates, whilst addition of fatty acid-free bovine serum albumin reduced signals, presumably by binding the fatty acids. To allow effective ligand equilibration GPR40-G_q was employed in [³⁵S]GTPS binding assays. Following expression of GPR40-G_q in HEK293 cells and membrane preparation basal binding of [³⁵S]GTPS in G_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 [³⁵S]GTPS binding in a concentration-dependent manner and now allowed the responsiveness and pharmacology at GPR40 of each of 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-G_q was also reversed by a small molecule GPR40 antagonist and basal [³⁵S]GTPS binding was prevented by the selective G_q/G₁₁inhibitor YM-254890. The current studies provide novel insights into the pharmacology of GPR40 and indicate that G protein-coupled receptors that respond to fatty acids, and potentially to other lipid ligands, can be occupied by endogenous agonists prior to assay, that this may mask the pharmacology of the receptor and may be mistaken for high levels of constitutive activity.

Key words: Orphan, Gq/11 family, Ca imaging, Endocrine cells

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