Abstract 1031: Angiotensin II Type 2 Receptor Activation Induces SHP-1-dependent RhoA Inhibition Through Phosphorylation of its Serine 188 in Vascular Smooth Muscle Cells
Angiotensin II (Ang II) plays important roles in the control of blood pressure. Evidence indicates that Ang II type 2 receptor (AT2R) antagonizes the effects of type 1 receptor (AT1R) and inactivation of RhoA signaling has been suggested to be involved in this action. As phosphorylation of Ser188 of RhoA inhibits its activity, we analyzed the effect of Ang II on RhoA phosphorylation. In rat aortic smooth muscle cells (SMC), Ang II (0.1 μM) induced serine-phosphorylation of RhoA. This effect was abolished by the AT2R antagonist PD123319 and mimicked by the AT2R agonist CGP42112A. Additional pharmacological approach indicated that AT2-induced RhoA phosphorylation did not involve NO synthase, cAMP- or cGMP-dependent protein kinase. Phosphoresistant (S188A)- or phosphomimetic (S188E)-RhoA mutants expressed in SMC are not phosphorylated by AT2R stimulation. Transfection with siRNA duplex targeting the tyrosine phosphatase SHP-1 or catalytically inactive SHP-1 mutant prevented AT2R-mediated RhoA phosphorylation. On the other hand, inhibition of tyrosine phosphorylation by the non-selective inhibitor genistein (25 μM), the Src inhibitor SU6656 (5 μM) or siRNA-targeting Src induced RhoA phosphorylation. This effect was not additive with AT2R-mediated RhoA phosphorylation and was inhibited by siRNA-mediated SHP-1 silencing. These results indicate that SHP-1 and regulation of tyrosine phosphorylation plays a critical role in AT2R-induced RhoA phosphorylation. Co-immunoprecipitation experiments indicated that AT2R-mediated phosphorylation of RhoA was associated with an increased binding to the cytosolic guanidine dissociation inhibitor GDI, leading to inactivation of RhoA/Rho kinase signaling. In vivo, inhibition of AT1R up-regulated AT2R expression. Ser188 phosphorylation of RhoA was strongly increased in aorta and pulmonary artery of Wistar and spontaneously hypertensive rats treated with the AT1R inhibitor candesartan (2wks, 2 mg/kg/d). Our results thus indicate that AT2R stimulation induces RhoA inhibition via SHP-1-dependent phosphorylation of RhoA on its Ser188. In vivo experiments suggest that AT2R-mediated phosphorylation and inhibition of RhoA may contribute to the antihypertensive effect of AT1R inhibitors.