Cyclic AMP-dependent and Epac-mediated activation of R-Ras by G protein-coupled receptors leads to phospholipase D stimulation.
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.
De Jesús
Maider López
ML
Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany.
Stope
Matthias B
MB
Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany.
Weernink
Paschal A Oude
PAO
Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany.
Mahlke
Yvonne
Y
Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany.
Börgermann
Christof
C
Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany.
Ananaba
Viktoria N
VN
Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany.
Rimmbach
Christian
C
Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany.
Rosskopf
Dieter
D
Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany.
Michel
Martin C
MC
Department of Pharmacology and Pharmacotherapy, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.
Jakobs
Karl H
KH
Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany.
Schmidt
Martina
M
Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany; Department of Pharmacology and Pharmacotherapy, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands. Electronic address: m.schmidt@rug.nl.
D002384:Catalysis; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D010739:Phospholipase D / Q000378:metabolism*; D043587:Receptor, Muscarinic M3 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction; D018631:ras Proteins / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; D043587:Receptor, Muscarinic M3; D043562:Receptors, G-Protein-Coupled; D006153:Guanosine Diphosphate; D000242:Cyclic AMP; D010739:Phospholipase D; D020558:GTP Phosphohydrolases; C085929:RRAS protein, human; D018631:ras Proteins