The Calcitonin Receptor Stimulates Shc Tyrosine Phosphorylation and Erk1/2 Activation

Abstract

While it is well established that adenylyl cyclase and phospholipase C-b 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 Ca21 was required for Erk1/2 phosphorylation, since preventing any change in cytosolic free Ca21 by chelating both cytosolic and extracellular Ca21 abolished the response. However, the change in Ca21 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 mM thapsigargin, each of which induced elevations of Ca21 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.