Protease-activated receptor 1 (PAR1) coupling to Gq/11 but not to Gi/o or G12/13 is mediated by discrete amino acids within the receptor second intracellular loop
Protease-activated receptor 1 (PAR1) coupling to Gq/11 but not to Gi/o or G12/13 is mediated by discrete amino acids within the receptor second intracellular loop
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DOI：
10.1016/j.cellsig.2012.01.011
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发表时间：
2012-06-01
影响因子：
4.8
通讯作者：
Hepler, John R.
中科院分区：
文献类型：
--
作者：
McCoy, Kelly L.;Gyoneva, Stetka;Hepler, John R.
蛋白酶激活受体1（PAR 1）是一种不常见的G蛋白受体，它与G（q/11）、G（i/o）和G（12/13）等多个G蛋白亚家族及其相关的信号通路相互作用，调节广泛的病理生理过程。然而，PAR 1与多种G蛋白相互作用的分子机制还不清楚。PAR 1是否在相同、不同或重叠的结合位点与各种G蛋白相互作用尚不清楚。在这里，我们研究了PAR 1和G（q/11），G（i/o）和G（12/13）亚家族的代表性成员之间的功能和特异性结合相互作用。我们报告，G（q/11）的物理和功能相互作用与特定的氨基酸在第二个细胞内（i2）环的PAR 1。我们鉴定了PAR 1 i2环内的五个氨基酸，当单独突变时，每个氨基酸都显著降低了转染的COS-7细胞（功能性PAR 1缺失细胞）中连接的磷酸肌醇形成的PAR 1活化。在这些突变中，只有R205 A完全消除了直接G（q/11），与PAR 1的结合，以及在COS-7细胞和PAR 1-/-原代星形胶质细胞中PAR 1指导的磷酸肌醇和钙动员。与此形成鲜明对比的是，PAR 112环突变中没有一个破坏PAR 1与G（o）或G（12）的直接结合，或者它们分别与连接的百日咳毒素敏感性ERK磷酸化和C3毒素敏感性Rho活化的功能偶联。在星形胶质细胞中，我们的研究结果表明，PAR 1介导的钙信号转导涉及一个新的G（q/11）-PLC信号通路。总之，我们已经确定了PAR 1与G（q/11）相互作用的关键分子决定因素，我们的研究结果支持了G（q/11），G（i/o）或G（12/13）各自与PAR 1胞质区域内的不同位点结合的模型。(C)2012爱思唯尔公司All rights reserved.
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-/- primary astrocytes. In stark contrast, none of the PAR1 i2 loop mutations disrupted direct PAR1 binding to either G(o) or G(12), 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 epsilon 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. (C) 2012 Elsevier Inc. All rights reserved.