Stoddart Leigh A, Brown Andrew J, Milligan Graeme
Davidson Building University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
Mol Pharmacol. 2007 Apr;71(4):994-1005. doi: 10.1124/mol.106.031534. Epub 2007 Jan 2.
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.
在缺乏钙动员型G蛋白表达的细胞中，共表达人GPR40和Gαq可使中链和长链脂肪酸升高细胞内[Ca2+]。当用人GPR40 - Gαq融合蛋白转染人胚肾（HEK）293细胞时也观察到了这一现象。细胞内[Ca2+]升高的动力学随着脂肪酸链长度的增加而减慢，提示不同的配体结合速率，而添加无脂肪酸的牛血清白蛋白可降低信号，推测是通过结合脂肪酸。为了实现有效的配体平衡，GPR40 - Gαq用于鸟苷5'-O-(3-[(35)S]硫代)三磷酸([(35)S]GTPγS)结合试验。在HEK293细胞中表达GPR40 - Gαq并制备膜后，[(35)S]GTPγS在Gαq免疫沉淀中的基础结合很高，且脂肪酸并未使其显著升高。然而，用无脂肪酸的牛血清白蛋白处理膜以浓度依赖的方式降低了基础[(35)S]GTPγS结合，并揭示了噻唑烷二酮类脂肪酸和一种新型小分子激动剂在GPR40上的反应性和药理学特性。内源性表达GPR40的大鼠INS-1E细胞的膜也得到了类似的观察结果。GPR40 - Gαq的高表观组成活性也被一种小分子GPR40拮抗剂逆转，并且基础[(35)S]GTPγS结合被选择性Gαq/Gα11抑制剂YM - 254890所阻断。目前的研究为GPR40的药理学提供了新的见解，并表明对脂肪酸以及可能对其他脂质配体有反应的G蛋白偶联受体在测定前可能被内源性激动剂占据，这可能掩盖受体的药理学特性，并可能被误认为是高水平的组成活性。
