title: Implication of Gbetagamma proteins and c-SRC tyrosine kinase in parathyroid hormone-induced signal transduction in rat enterocytes.
abstract: 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(2+) (EGTA, 0.5 mM) and chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid acetoxymethyl ester (BAPTA) (5 microM) suppressed Tyr527 dephosphorylation and Tyr416 phosphorylation, indicating that Ca(2+) is an upstream activator of c-Src in enterocytes stimulated with PTH. The G protein subunits, Galphas and Gbeta, are associated with c-Src in basal conditions and this association increases two- to threefold in cells treated with PTH. Blocking of Gbeta subunits by preincubation of cells with a Gbeta 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 Gbetagamma-c-Src to the phosphorylation of the MAP kinases, ERK1 and ERK2.
authors.affiliation: Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahia Blanca, Argentina.
authors.forename: Claudia
authors.initials: C
authors.lastname: Gentili
authors.forename: Ricardo
authors.initials: R
authors.lastname: Boland
authors.forename: Ana
authors.initials: A
authors.lastname: Russo de Boland
mesh_terms: D000262:Adenylyl Cyclases / Q000032:analysis / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004386:Duodenum; D020895:Enterocytes / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015151:Immunoblotting; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010766:Phosphorylation; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects*; D019061:src-Family Kinases / Q000032:analysis / Q000378:metabolism*
chemical_list: D010281:Parathyroid Hormone; D021122:Protein Subunits; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D002118:Calcium