Flow Cytometry-Based Binding Assay for GPR40 ( FFAR1; Free Fatty Acid Receptor 1)
Flow Cytometry-Based Binding Assay for GPR40 ( FFAR1; Free Fatty Acid Receptor 1)
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DOI：
10.1124/mol.108.052225
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发表时间：
2009-01-01
影响因子：
3.6
通讯作者：
Tsujimoto, Gozoh
中科院分区：
文献类型：
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作者：
Hara, Takafumi;Hirasawa, Akira;Tsujimoto, Gozoh
GPR 40是一种G蛋白偶联受体（GPCR），其内源性配体最近被鉴定为中链和长链游离脂肪酸（FFA），并且被认为在胰岛素释放中起重要作用。尽管最近的研究工作，很多仍然不清楚，在我们的理解其药理学，主要是因为受体-配体相互作用没有被直接分析。为了以更直接的方式研究GPR 40的药理学，我们开发了基于流式细胞术的结合测定。FLAG标记的GPR 40蛋白在Sf 9细胞中表达，溶解，固定在免疫磁珠上，并用荧光探针C1-BODIPY-C12标记。流式细胞术分析显示，C1-BODIPY-C12以可饱和和可逆的方式特异性标记单一类别的结合位点，表观解离常数类似于3 μ M。激活GPR 40的FFA与C1-BODIPY-C12结合竞争;因此，中长链FFA可以竞争，而短链FFA和亚油酸甲酯没有抑制作用。此外，已知激活GPR 40的配体以浓度依赖性方式竞争结合。所有抑制结合的配体都促进了表达GPR 40的人胚肾（HEK）293细胞中细胞外信号调节激酶（ERK）-1/2的磷酸化和天然表达GPR 40的小鼠胰岛素瘤（MIN 6）细胞中的[Ca 2 +]（i）反应;然而，吡格列酮（一种噻唑烷二酮）未能竞争结合，不能激活ERK或[Ca 2 +]（i）反应。这项研究表明，基于流式细胞术的结合试验可以成功地鉴定GPR 40与其配体之间的直接相互作用。这种方法将在研究GPCR的药理学价值。
GPR40 is a G protein-coupled receptor (GPCR) whose endogenous ligands have recently been identified as medium- and long-chain free fatty acids (FFAs), and it is thought to play an important role in insulin release. Despite recent research efforts, much still remains unclear in our understanding of its pharmacology, mainly because the receptor-ligand interaction has not been analyzed directly. To study the pharmacology of GPR40 in a more direct fashion, we developed a flow cytometry-based binding assay. FLAG-tagged GPR40 protein was expressed in Sf9 cells, solubilized, immobilized on immunomagnetic beads, and labeled with the fluorescent probe C1-BODIPY-C12. Flow cytometry analysis showed that C1-BODIPY-C12 specifically labels a single class of binding site in a saturable and reversible manner with an apparent dissociation constant of similar to 3 mu M. The FFAs that activate GPR40 competed with C1-BODIPY-C12 binding; thus, medium-to long-chain FFAs could compete, whereas short-chain FFAs and methyl linoleate had no inhibitory effect. Furthermore, ligands that are known to activate GPR40 competed for binding in a concentration-dependent manner. All the ligands that inhibited the binding promoted phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in human embryonic kidney (HEK) 293 cells that expressed GPR40 and [Ca2+](i) responses in mouse insulinoma (MIN6) cells that natively express GPR40; however, pioglitazone, a thiazolidinedione that failed to compete for the binding, did not activate ERK or [Ca2+](i) response. This study showed that a flow cytometry-based binding assay can successfully identify direct interactions between GPR40 and its ligands. This approach would be of value in studying the pharmacology of GPCRs.