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G蛋白信号调节因子2(RGS2)和RGS4在活细胞中与蛋白酶激活受体1(PAR1)形成不同的G蛋白依赖性复合物。

Regulator of G protein signaling 2 (RGS2) and RGS4 form distinct G protein-dependent complexes with protease activated-receptor 1 (PAR1) in live cells.

作者信息

Ghil Sungho, McCoy Kelly L, Hepler John R

机构信息

Department of Life Science, Kyonggi University, Suwon, Republic of Korea.

Department of Pharmacology, Rollins Research center, Emory University School of Medicine, Atlanta, Georgia, United States of America.

出版信息

PLoS One. 2014 Apr 17;9(4):e95355. doi: 10.1371/journal.pone.0095355. eCollection 2014.

Abstract

Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor (GPCR) that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to Gi, Gq and G12 to activate linked signaling pathways. Regulators of G protein signaling (RGS) proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET), we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven) and either RGS2-Luciferase (RGS2-Luc) or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gαq/11, and PAR1-RGS4 by Gαo, but not by other Gα subunits. Gαq/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR) stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A) that eliminates PAR1-Gq/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.

摘要

蛋白酶激活受体1(PAR1)是一种G蛋白偶联受体(GPCR),可被天然蛋白酶激活以调节多种生理活动。我们之前报道过PAR1与Gi、Gq和G12偶联以激活相关的信号通路。G蛋白信号调节(RGS)蛋白作为GTP酶激活蛋白来抑制GPCR/G蛋白信号传导。一些RGS蛋白直接与某些GPCR相互作用以调节其信号,尽管决定选择性RGS/GPCR偶联的细胞机制尚不清楚。在这里,我们使用生物发光共振能量转移(BRET)技术,测试了RGS2和RGS4是否在活的COS-7细胞中与PAR1结合以调节PAR1/Gα介导的信号传导。我们报告PAR1以G蛋白依赖性方式选择性地与RGS2或RGS4相互作用。在没有Gα的情况下,在PAR1-维纳斯(PAR1-Ven)与RGS2-荧光素酶(RGS2-Luc)或RGS4-Luc之间几乎观察不到BRET活性。然而,在存在特定Gα亚基的情况下,Gαq/11使PAR1与RGS2之间的BRET活性显著增强,Gαo使PAR1与RGS4之间的BRET活性显著增强,但其他Gα亚基则无此作用。PAR1可促进Gαq/11-YFP/RGS2-Luc的BRET活性,并且激动剂(TFLLR)刺激可使其显著增强。然而,PAR1-Ven/RGS-Luc的BRET活性被消除PAR1-Gq/11偶联的PAR1突变体(R205A)阻断。纯化的PAR1细胞内第三环直接与纯化的His-RGS2或His-RGS4结合。在细胞中,RGS2和RGS4分别抑制PAR1/Gα介导的钙信号和MAPK/ERK信号,但不抑制RhoA信号。我们的研究结果表明,RGS2和RGS4以Gα依赖性方式直接与PAR1相互作用以调节PAR1/Gα介导的信号传导,并突出了一种选择性GPCR/G蛋白/RGS偶联的细胞机制。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/03de/3990635/e0f7c933ebe7/pone.0095355.g001.jpg

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