Implication of G Beta-Gamma Proteins and c-SRC Tyrosine Kinase in PTH-Induced Signal Transduction in Rat Intestinal Cells

GENTILI CLAUDIA; BOLAND, RICARDO; RUSSO DE BOLAND ANA

Nashville, Tennessee

Congreso; 27th Annual Meeting ASBMR (American Society for Bone and Mineral Research); 2005

American Society for Bone and Mineral Research

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 demonstrated 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 sec (+120 %), while the second phase progressively increases up to  5 min (+220%). The hormone activates c-Src in intestinal cells through fast changes in the tyrosine phosphorylation of the enzyme. The first event in the activation of c-Src is the dephosphorylation of Tyr 527 (that happens at few seconds of PTH treatment), followed by a second event of activation with phosphorylation at Tyr 416 (+2 fold , 5 min). Removal of external Ca2+ (EGTA 0.5 mM) and chelation of intracellular Ca2+ with BAPTA (5mM) suppressed Tyr 416 phophorylation and Tyr 527 dephosphorylation, indicating that Ca2+ is an upstream activator of c-Src in enterocytes stimulated with PTH. The G-protein subunits, Gas and Gb, are associated with c-Src in basal conditions and this association increases two-three fold in cells treated with PTH. Sequestration of Gb subunits abolished hormone-dependent c-Src Tyr 416 phosphorylation and ERK1/ERK2 activation. The results of this work show 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 Gbg-c-Src to the phosphorylation of the MAP kinases ERK1 and ERK2. <!--[if !supportEmptyParas]--> <!--[endif]-->