AT 2 receptors recruit c-Src, SHP-1 and FAK upon activation by Ang II in PND15 rat hindbrain

Abstract

The functional role of AT 2 receptors is unclear and it activates unconventional signaling pathways, which in general do not involve a classical activation of a G-protein. In the present study, we aimed to investigate the transduction mechanism of AT 2 Ang II receptors in PND15 rat hindbrain membrane preparations, which represents a physiological developmental condition. To determine whether Ang II AT 2 receptors induced association to SHP-1 in rat hindbrain, co-immunoprecipitation assays were performed. Stimulation of Ang II AT 2 receptors induced both a transient tyr-phosphorylation and activation of SHP-1. The possible participation of c-Src in Ang II-mediated SHP-1 activation, we demonstrated by recruitment of c-Src in immunocomplexes obtained with anti AT 2 or anti-SHP-1 antibodies. The association of SHP-1 to c-Src was inhibited by PD123319 and the c-Src inhibitor PP2. Similarly, SHP-1 activity determined in AT 2-immunocomplexes was inhibited by PD123319 and the c-Src inhibitor PP2. Following stimulation with Ang II, AT 2 receptors recruit c-Src, which was responsible for SHP-1 tyr-phosphorylation and activation. Since AT 2 receptors are involved in neuron migration, we tested the presence of FAK in immunocomplexes. Surprisingly, AT 2-immunocomplexes contained mainly the 85 kDa fragment of FAK. Besides, p125FAK associated to SHP-1. In summary, we demonstrated the presence of an active signal transduction mechanism in PND15 rat hindbrain, a developmental stage critical for cerebellar development. In this model, we showed a complex containing AT 2/SHP-1/c-Src/p85FAK, suggesting a potential role of Ang II AT 2 receptors in cerebellar development and neuronal differentiation.