1. Epac- and Ca2+ -controlled activation of Ras and extracellular signal-regulated kinases by Gs-coupled receptors.
- Author
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Keiper M, Stope MB, Szatkowski D, Böhm A, Tysack K, Vom Dorp F, Saur O, Oude Weernink PA, Evellin S, Jakobs KH, and Schmidt M
- Subjects
- Animals, Cell Line, Cell Line, Tumor, Cyclic AMP metabolism, DNA-Binding Proteins metabolism, Dose-Response Relationship, Drug, Enzyme Activation, Guanine Nucleotide Exchange Factors metabolism, Humans, Immunoblotting, MAP Kinase Signaling System, Phosphoinositide Phospholipase C, Plasmids metabolism, Rats, Time Factors, Transfection, Type C Phospholipases metabolism, Calcium metabolism, GTP-Binding Protein alpha Subunits, Gs metabolism, Guanine Nucleotide Exchange Factors physiology, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 metabolism, ras Proteins metabolism
- Abstract
We have recently reported that two typical Gs-coupled receptors, the beta2-adrenergic receptor and the receptor for prostaglandin E1, stimulate phospholipase C-epsilon (PLC-epsilon) and increase intracellular Ca2+ concentration ([Ca2+]i) in HEK-293 cells and N1E-115 neuroblastoma cells, respectively, by a pathway involving Epac1, a cAMP-activated and Rap-specific guanine nucleotide exchange factor (GEF), and the GTPase Rap2B. Here we have demonstrated that these Gs-coupled receptors use this pathway to activate H-Ras and the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Specifically, agonist activation of the receptors resulted in activation of H-Ras and ERK1/2. The latter action was suppressed by dominant negative H-Ras, but not Rap1A. The receptor actions were independent of protein kinase A but fully mimicked by an Epac-specific cAMP analog as well as by a constitutively active Rap2B mutant. On the other hand, a cAMP-binding-deficient Epac1 mutant, the Rap GTPase-activating proteinII, and a dominant negative Rap2B mutant suppressed receptor- and Epac-mediated activation of H-Ras and ERK1/2. Finally, we have demonstrated that activation of H-Ras and ERK1/2 requires the lipase activity of PLC-epsilon and the subsequent [Ca2+]i increase, suggesting that H-Ras activation is mediated by a Ca2+ -activated GEF. In line with this hypothesis, receptor-mediated activation of H-Ras and ERK1/2 was strongly enhanced by expression of RasGRP1, a Ca2+ -regulated Ras-GEF. Collectively, our data indicated that Gs-coupled receptors can activate H-Ras and subsequently the mitogen-activated protein kinases ERK1/2 by a Ca2+ -activated Ras-GEF, possibly RasGRP1, mediated by cAMP-activated Epac proteins, which then lead via Rap2B and PLC-epsilon stimulation to [Ca2+]i increase.
- Published
- 2004
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