The calcitonin receptor stimulates Shc tyrosine phosphorylation and Erk1/2 activation. Involvement of Gi, protein kinase C, and calcium.
While it is well established that adenylyl cyclase and phospholipase C-beta are two proximal signal effectors for the calcitonin receptor, the more distal signaling pathways are less well characterized. G protein-coupled receptors can activate Erk1/2 by Gs-, Gi-, or Gq-dependent signaling pathways, depending on the specific receptor and cell type examined. Since the calcitonin receptor can couple to all three of these G proteins, the ability of calcitonin to activate Erk1/2 was investigated. Calcitonin induced time- and concentration-dependent increases in Shc tyrosine phosphorylation, Shc-Grb2 association and Erk1/2 phosphorylation and activation in a HEK 293 cell line that stably expresses the rabbit calcitonin receptor C1a isoform. Pertussis toxin, which inactivates Gi, and calphostin C, a protein kinase C inhibitor, each partially inhibited calcitonin-induced Shc tyrosine phosphorylation, Shc-Grb2 association, and Erk1/2 phosphorylation. In contrast, neither forskolin nor H89, a protein kinase A inhibitor, had a significant effect on basal or calcitonin-stimulated Erk1/2 phosphorylation. Our results suggest that the calcitonin receptor induces Shc phosphorylation and Erk1/2 activation in HEK293 cells by parallel Gi- and PKC-dependent mechanisms. The calcitonin-induced elevation of cytosolic free Ca2+ was required for Erk1/2 phosphorylation, since preventing any change in cytosolic free Ca2+ by chelating both cytosolic and extracellular Ca2+ abolished the response. However, the change in Ca2+ that is induced by calcitonin is not sufficient to account for the calcitonin-induced Erk1/2 phosphorylation, since treatment with 100 nM ionomycin or 10 microM thapsigargin, each of which induced elevations of Ca2+ comparable to those induced by calcitonin, induced significantly less Erk1/2 phosphorylation than that induced by calcitonin. Erk1/2 may have important roles as downstream effectors mediating cellular responses to calcitonin stimulation.
Chen
Y
Y
Departments of Cell Biology and Orthopedics and the Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Shyu
J F
JF
Santhanagopal
A
A
Inoue
D
D
David
J P
JP
Dixon
S J
SJ
Horne
W C
WC
Baron
R
R
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D037361:Adenylate Cyclase Toxin; D000818:Animals; D002116:Calcitonin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051380:GRB2 Adaptor Protein; D019204:GTP-Binding Proteins / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D037342:Pertussis Toxin; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D018003:Receptors, Calcitonin / Q000502:physiology*; D055765:Shc Signaling Adaptor Proteins; D014443:Tyrosine / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D037361:Adenylate Cyclase Toxin; D004791:Enzyme Inhibitors; D051380:GRB2 Adaptor Protein; D011506:Proteins; D018003:Receptors, Calcitonin; D055765:Shc Signaling Adaptor Proteins; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D019000:Phosphotyrosine; D014443:Tyrosine; D015759:Ionomycin; D002116:Calcitonin; D037342:Pertussis Toxin; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D002118:Calcium