De Jesús Maider López, Stope Matthias B, Weernink Paschal A Oude, Mahlke Yvonne, Börgermann Christof, Ananaba Viktoria N, Rimmbach Christian, Rosskopf Dieter, Michel Martin C, Jakobs Karl H, Schmidt Martina
Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany.
Department of Pharmacology and Pharmacotherapy, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
J Biol Chem. 2006 Aug 4;281(31):21837-21847. doi: 10.1074/jbc.M604156200. Epub 2006 Jun 5.
The activation of the Ras-related GTPase R-Ras, which has been implicated in the regulation of various cellular functions, by G protein-coupled receptors (GPCRs) was studied in HEK-293 cells stably expressing the M3 muscarinic acetylcholine receptor (mAChR), which can couple to several types of heterotrimeric G proteins. Activation of the receptor induced a very rapid and transient activation of R-Ras. Studies with inhibitors and activators of various signaling pathways indicated that R-Ras activation by the M3 mAChR is dependent on cyclic AMP formation but is independent of protein kinase A. Similar to the rather promiscuous M3 mAChR, two typical G(s)-coupled receptors also induced R-Ras activation. The receptor actions were mimicked by an Epac-specific cyclic AMP analog and suppressed by depletion of endogenous Epac1 by small interfering RNAs, as well as expression of a cyclic AMP binding-deficient Epac1 mutant, but not by expression of dominant negative Rap GTPases. In vitro studies demonstrated that Epac1 directly interacts with R-Ras and catalyzes GDP/GTP exchange at this GTPase. Finally, it is shown that the cyclic AMP- and Epac-activated R-Ras plays a major role in the M3 mAChR-mediated stimulation of phospholipase D but not phospholipase C. Collectively, our data indicate that GPCRs rapidly activate R-Ras, that R-Ras activation by the GPCRs is apparently directly induced by cyclic AMP-regulated Epac proteins, and that activated R-Ras specifically controls GPCR-mediated phospholipase D stimulation.
在稳定表达可与多种异源三聚体G蛋白偶联的M3毒蕈碱型乙酰胆碱受体（mAChR）的HEK-293细胞中，研究了G蛋白偶联受体（GPCRs）对与多种细胞功能调节有关的Ras相关GTP酶R-Ras的激活作用。该受体的激活诱导了R-Ras非常快速且短暂的激活。对各种信号通路抑制剂和激活剂的研究表明，M3 mAChR对R-Ras的激活依赖于环磷酸腺苷（cAMP）的形成，但不依赖于蛋白激酶A。与相当混杂的M3 mAChR类似，两种典型的G(s)偶联受体也诱导了R-Ras的激活。受体的作用可被Epac特异性的cAMP类似物模拟，并被小干扰RNA对内源Epac1的耗尽以及cAMP结合缺陷型Epac1突变体的表达所抑制，但不受显性负性Rap GTP酶表达的影响。体外研究表明，Epac1直接与R-Ras相互作用，并催化该GTP酶的GDP/GTP交换。最后，研究表明，cAMP和Epac激活的R-Ras在M3 mAChR介导的磷脂酶D刺激中起主要作用，但在磷脂酶C刺激中不起作用。总体而言，我们的数据表明，GPCRs可快速激活R-Ras，GPCRs对R-Ras的激活显然是由cAMP调节的Epac蛋白直接诱导的，并且激活的R-Ras特异性地控制GPCR介导的磷脂酶D刺激。
