SIGNALING PATHWAY OF AT2 RECEPTORS IN DEVELOPING RAT HINDBRAIN. INVOLVEMENT OF c-SRC (SFK) IN THE ACTIVATION OF SHP-1

LEONARDO R. SEGUIN AND GLADYS M. CIUFFO

Buzios, Brasil

Congreso; I CONGRESSO IBRO/LARC DE NEUROCIÊNCIAS DA AMÉRICA LATINA, CARIBE E; 2008

IBRO-LARC

Objectives: The aim of the present study was to examine the involvement of c-Src and SHP-1 in AT2 receptor signaling pathway in developing rat hindbrain (PDN15).  Methods: The study was conducted in rat hindbrain membrane preparations. Aliquots of membranes were stimulated with Ang II in presence or absence of different competitors: Losartan, PD123319 (Ang II antagonists), the c-Src inhibitor (PP2) or its inactive analog (PP3). Proteins were solubilized and immunocomplexes were obtained with anti-AT2 or anti-SHP-1 antibodies. SHP-1 activity and the interaction with c-Src were measured in immunocomplexes and protein interaction determined by co-immunoprecipitation. Results: Following Ang II- stimulation (10-7M) we observed SHP-1 association to AT2 receptors but not to AT1 subtype, detected by co-immunoprecipitation. In Ang II-stimulated preparations, immunocomplexes obtained with either anti-AT2 or anti-SHP-1 antibodies exhibited PTPase activity, which correlate with induction of SHP-1 tyr-phosphorylation. The AT2 antagonist PD123319 blocked SHP-1 tyr-phosphorylation as well as activity. Thus, we can correlate the activation of SHP-1 with tyr-phosphorylation, mediated by AT2 receptors. In order to test the participation of c-Src in this mechanism we tested the association of c-Src with AT2 receptors and SHP-1 from immunocomplexes obtained with either anti-AT2 or anti-SHP-1 antibodies. PP2 blocked the tyr-phosphorylation and activation of SHP-1 as well as its association to c-Src. Conclusions: The present results suggest an activation of SHP-1 by tyr-phosphorylation mediated by Ang II AT2 receptors and c-Src involvement in this mechanism. These data support a physiological role of AT2 receptors in the hindbrain development.