Carbachol activates ERK2 in isolated gastric parietal cells via multiple signaling pathways.
We previously reported that both carbachol and epidermal growth factor (EGF) are potent inducers of the extracellular signal-regulated protein kinases (ERKs) in isolated gastric canine parietal cells and that induction of these kinases leads to acute inhibitory and chronic stimulatory effects on gastric acid secretion. In this study we investigated the molecular mechanisms responsible for these effects. Both carbachol (100 microM) and EGF (10 nM) induced Ras activation. The role of Ras in ERK2 induction was examined by transfecting parietal cells with a vector expressing hemoagglutinin (HA)-tagged ERK2 (HA-ERK2) together with a dominantly expressed mutant (inactive) ras gene. HA-ERK2 activity was quantitated by in-gel kinase assays. Dominant negative Ras reduced carbachol induction of HA-ERK2 activity by 60% and completely inhibited the stimulatory effect of EGF. Since Ras activation requires the assembly of a multiprotein complex, we examined the effect of carbachol and EGF on tyrosyl phosphorylation of Shc and its association with Grb2 and the guanine nucleotide exchange factor Sos. Western blot analysis of anti-Shc immunoprecipitates with an anti-phosphotyrosine antibody demonstrated that both carbachol and EGF induced tyrosyl phosphorylation of a major 52-kDa shc isoform. Grb2 association with Shc was demonstrated by blotting Grb2 immunoprecipitates with an anti-Shc antibody. Probing of anti-Sos immunoprecipitates with an anti-Grb2 antibody revealed that Sos was constitutively bound to Grb2. To examine the functional role of Sos in ERK2 activation, we transfected parietal cells with the HA-ERK2 vector together with a dominantly expressed mutant (inactive) sos gene. Dominant negative Sos did not affect carbachol stimulation of HA-ERK2 but inhibited the stimulatory effect of EGF by 60%. We then investigated the role of betagamma-subunits in carbachol induction of HA-ERK2. Parietal cells were transfected with the HA-ERK2 vector together with a vector expressing the carboxy terminus of the beta-adrenergic receptor kinase 1, known to block signaling mediated by betagamma-subunits. In the presence of this vector, carbachol induction of HA-ERK2 was inhibited by 40%. Together these data suggest that, in the gastric parietal cells, carbachol activates the ERKs through Ras- and betagamma-dependent mechanisms that require guanine nucleotide exchange factors other than Sos.
Takeuchi
Y
Y
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
Pausawasdi
N
N
Todisco
A
A
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002217:Carbachol / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002478:Cells, Cultured; D004285:Dogs; D004789:Enzyme Activation / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D051380:GRB2 Adaptor Protein; D005786:Gene Expression Regulation / Q000502:physiology; D005799:Genes, Dominant / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1; D010295:Parietal Cells, Gastric / Q000187:drug effects* / Q000201:enzymology*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000502:physiology*; D020664:Son of Sevenless Proteins; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051380:GRB2 Adaptor Protein; D008565:Membrane Proteins; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; D020664:Son of Sevenless Proteins; D014443:Tyrosine; D004815:Epidermal Growth Factor; D002217:Carbachol; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D018631:ras Proteins