Cyclic AMP-dependent and Epac-mediated Activation of R-Ras by G Protein-coupled Receptors Leads to Phospholipase D Stimulation

• Published in: The Journal of biological chemistry Vol. 281; no. 31; pp. 21837 - 21847
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.08.2006; American Society for Biochemistry and Molecular Biology
• Subjects: Catalysis; Cell Line; Cyclic AMP - metabolism; GTP Phosphohydrolases - metabolism; Guanine Nucleotide Exchange Factors - metabolism; Guanosine Diphosphate - metabolism; Humans; Phospholipase D - metabolism; ras Proteins - metabolism; Receptor, Muscarinic M3 - metabolism; Receptors, G-Protein-Coupled - metabolism; Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M604156200

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 Gs-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.