FAK is involved in Angiotensin II AT2 receptor signaling pathway.

MANZUR, MJ; SEGUIN, LR; CIUFFO, GM

Mérida

Congreso; Cell Signalling Networks 2011; 2011

13th IUBMB Conference, Soc. Mexicana de Bioqca.

Angiotensin II (Ang II), initially described as a primary vasoconstrictor peptide, has been proposed as an important growth modulating factor. Ang II exerts its biological effects by activating two distinct membrane receptors, AT1 and AT2. The functional role of AT2 receptors is unclear, it activates unconventional signaling pathways which vary between different cell lines and models where it was studied. In the present study we compared the signaling pathways of AT2 receptors in two different models: a) ex vivo preparations from PND15 rat hindbrain, which represent a physiological developmental condition; b) HeLa cells expressing AT2 receptors. Previously, we demonstrated the participation of PTPase SHP-1 and c-Src family kinase members (SFK) in the signaling pathway of AT2 receptors. PND15 rat hindbrain membrane preparations were stimulated with Ang II (10-7M) for different times, solubilized, immunoprecipitated with anti-AT2 or anti-SHP-1 antibodies and analyzed by western blot. PND15 hindbrain membrane preparations exhibit two different FAK forms (p125FAK and p85FAK). p85FAK associated to AT2 receptors in a time-dependent manner after Ang II induction. Immunocomplexes obtained with anti-AT2 did not contain p125FAK.  The participation of c-Src was evidenced by using SFK inhibitor PP2. PTPase inhibitor Na3VO4 induced un-coupling the p85FAK from AT2 receptors. The association of FAK to SHP-1 was shown in immunocomplexes obtained with anti-SHP-1, where both FAK forms were present. The participation of FAK in Ang II signaling mechanism suggests a role in cell migration or apoptosis. To further explore this mechanism we studied apoptosis mediated by Ang II AT2 receptors in HeLa cells over-expressing AT2 receptors, considering the participation of RhoA. HeLa cells co-expressing AT2 receptors and EGFP-RhoA WT or its mutants (the constitutively active RhoA V14) or the dominant negative (RhoA N19), were morphologically studied after Ang II stimulation for 0, 30 and 240 minutes. Apoptosis evaluated by confocal microscopy evidenced increased apoptotic features at earlier times in HeLa-AT2 cells expressing RhoA V14, while cells expressing RhoA N19, showed a significantly reduced nuclear condensation. Similarly, HeLa-AT2 cells expressing RhoA V14 exhibited increased caspase 3/7 activation. Participation of other signaling molecules such as JNK and FAK was assayed in this signaling mechanism. Our results suggest that AT2 receptors modulate JNK phosphorylation and FAK cleavage in response to Ang II stimulation, a mechanism where RhoA plays a central role. Taken together, these results demonstrate an inducible signaling pathway of Ang II AT2 receptors which involves FAK, suggesting a potential role in a critical stage of rat hindbrain development either in cell migration or cellular apoptosis.