Implication of G proteins and c-SRC tyrosine kinase in parathyroid hormone-induced signal transduction in rat enterocytes
نویسندگان
چکیده
Parathyroid hormone (PTH) interacts in target tissues with a G protein-coupled receptor (GPCR) localized in the plasma membrane. Although activation of GPCR can elicit rapid stimulation of cellular protein tyrosine phosphorylation, the mechanism by which G proteins activate protein-tyrosine kinases is not completely understood. In the present work, we demonstrate that PTH rapidly increases the activity of non-receptor tyrosine kinase c-Src in rat intestinal cells (enterocytes). The response is biphasic, the early phase is fast and transient, peaking at 30 s (+120%), while the second phase progressively increases up to 5 min (+220%). The hormone activates c-Src in intestinal cells through fast changes in tyrosine phosphorylation of the enzyme. The first event in the activation of c-Src is the dephosphorylation of Tyr527 (which happens after a few seconds of PTH treatment), followed by a second event of activation with phosphorylation at Tyr416 (+twofold, 5 min). Removal of external Ca (EGTA, 0·5 mM) and chelation of intracellular Ca with 1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetracetic acid acetoxymethyl ester (BAPTA) (5 μM) suppressed Tyr527 dephosphorylation and Tyr416 phosphorylation, indicating that Ca is an upstream activator of c-Src in enterocytes stimulated with PTH. The G protein subunits, G s and G , are associated with c-Src in basal conditions and this association increases twoto threefold in cells treated with PTH. Blocking of G subunits by preincubation of cells with a G antibody abolished hormone-dependent c-Src Tyr416 phosphorylation and ERK1/ERK2 activation. The results of this work indicate that PTH activates c-Src in intestinal cells through conformational changes via G proteins and calcium-dependent modulation of tyrosine phosphorylation of the enzyme, and that PTH receptor activation leads via G –c-Src to the phosphorylation of the MAP kinases, ERK1 and ERK2. Journal of Endocrinology (2006) 188, 69–78
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