Epac- and Ca2+ -controlled activation of Ras and extracellular signal-regulated kinases by Gs-coupled receptors.
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.
Keiper
Melanie
M
Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany.
Stope
Matthias B
MB
Szatkowski
Daniel
D
Böhm
Anja
A
Tysack
Karina
K
Vom Dorp
Frank
F
Saur
Oliver
O
Oude Weernink
Paschal A
PA
Evellin
Sandrine
S
Jakobs
Karl H
KH
Schmidt
Martina
M
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D054801:Phosphoinositide Phospholipase C; D010957:Plasmids / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; C491790:RASGRP1 protein, human; D000242:Cyclic AMP; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C423320:phospholipase C epsilon; D019205:GTP-Binding Protein alpha Subunits, Gs; D018631:ras Proteins; D002118:Calcium