AT2receptors recruit c-Src, SHP-1 and FAK upon activation by Ang II in PND15 rat hindbrain.

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 85kDa 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.

Purkinje cells express Angiotensin II AT(2) receptors at different developmental stages.

Angiotensin II (Ang II) binds and activates two major receptors subtypes, namely AT(1) and AT(2). In the fetus, AT(2) receptors predominate in all tissues and decline shortly after birth, being restricted to a few organs including brain. Interpretation of the function of Ang II in the cerebellum requires a thorough understanding of the localization of Ang II receptors. The aim of the present paper is to evaluate the localization of Ang II AT(2) receptors in the Purkinje cell (PC) layer during development. By binding autoradiography, a clear complementary pattern of AT(1) and AT(2) binding labeled by [(125)I] Ang II was observed in young rats within the cerebellar cortex. This pattern was present at the stages P8 and P15, but not at P30 and P60, where AT(2) binding appears low and superimposed with AT(1) binding. We demonstrate that AT(2) antibodies recognized postmitotic Purkinje cells, labeling the somata of these cells at all the stages studied, from P8 to P60, suggesting that PCs express these receptors from early stages of development until adulthood. In P8 and P15 animals, we observed a clear correspondence between immunolabeling and the well-defined layer observed by binding autoradiography. Confocal analysis allowed us to discard the co-localization of AT(2) receptors with glial fibrillary acidic protein (GFAP), a glial marker. Double immunolabeling allowed us to demonstrate the co-localization of Ang II AT(2) receptors with zebrin II, a specific PC marker. Since PCs are the sole output signal from the cerebellar cortex and considering the role of cerebellum in movement control, the specific receptor localization suggests a potential role for Ang II AT(2) receptors in the cerebellar function.

Involvement of c-Src tyrosine kinase in SHP-1 phosphatase activation by Ang II AT2 receptors in rat fetal tissues.

Angiotensin II (Ang II) AT(2) receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10(-7) M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT(2) antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT(2) receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT(2) or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT(2) or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT(2) receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT(2) receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT(2) subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.