Protease-activated receptor 1 (PAR1) coupling to G_q/11 but not to G_i/o or G_12/13 is mediated by discrete amino acids within the receptor second intracellular loop

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• 作者：Kelly L. McCoy^a ; Stefka Gyoneva^a ; Christopher P. Vellano^a ; Alan V. Smrcka^b ; Stephen F. Traynelis^a ; John R. Hepler^a ; ^{jhepler@emory.edu}
• 关键词：BBB ; blood&ndash ; brain barrier ; BSA ; bovine serum albumin ; DMEM ; Dulbecco's modified Eagle's medium ; ECL ; enhanced chemiluminescence ; ERK ; extracellular signal-regulated kinase ; GPCR ; G protein-coupled receptor ; GRK2 ; G protein-coupled receptor kinase-2
• 刊名：Cellular Signalling
• 出版年：2012
• 期刊代码：42_08986568
• 类别：bio
• 出版时间：June, 2012
• 卷：24
• 期：6
• 页码：1351-1360
• 文件大小：1103 K

摘要

Protease-activated receptor 1 (PAR1) is an unusual GPCR that interacts with multiple G protein subfamilies (G_q/11, G_i/o, and G_12/13) and their linked signaling pathways to regulate a broad range of pathophysiological processes. However, the molecular mechanisms whereby PAR1 interacts with multiple G proteins are not well understood. Whether PAR1 interacts with various G proteins at the same, different, or overlapping binding sites is not known. Here we investigated the functional and specific binding interactions between PAR1 and representative members of the G_q/11, G_i/o, and G_12/13 subfamilies. We report that G_q/11 physically and functionally interacts with specific amino acids within the second intracellular (i2) loop of PAR1. We identified five amino acids within the PAR1 i2 loop that, when mutated individually, each markedly reduced PAR1 activation of linked inositol phosphate formation in transfected COS-7 cells (functional PAR1-null cells). Among these mutations, only R205A completely abolished direct G_q/11 binding to PAR1 and also PAR1-directed inositol phosphate and calcium mobilization in COS-7 cells and PAR1鈭?鈭?primary astrocytes. In stark contrast, none of the PAR1 i2 loop mutations disrupted direct PAR1 binding to either G_o or G₁₂, or their functional coupling to linked pertussis toxin-sensitive ERK phosphorylation and C3 toxin-sensitive Rho activation, respectively. In astrocytes, our findings suggest that PAR1-directed calcium signaling involves a newly appreciated G_q/11-PLC蔚 pathway. In summary, we have identified key molecular determinants for PAR1 interactions with G_q/11, and our findings support a model where G_q/11, G_i/o or G_12/13 each bind to distinct sites within the cytoplasmic regions of PAR1.