Sanchez-Fernandez_2013_PLoS.One_8_e84174

G-protein-coupled receptors (GPCRs) are known to activate both G protein- and beta-arrestin-dependent signalling cascades. The initiation of mitogen-activated protein kinase (MAPK) pathways is a key downstream event in the control of cellular functions including proliferation, differentiation, migration and apoptosis. Both G proteins and beta-arrestins have been reported to mediate context-specific activation of ERK1/2, p38 and JNK MAPKs. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has been described to involve a direct interaction between Galphaq and two novel effectors, PKCzeta and MEK5. However, the possible contribution of beta-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and beta-arrestin recruitment in the activation of ERK5 by Gq-coupled GPCRs. Our results show that ERK5 activation is independent of M1 or M3 muscarinic receptor internalization. Furthermore, we demonstrate that phosphorylation-deficient muscarinic M1 and M3 receptors are still able to fully activate the ERK5 pathway, despite their reported inability to recruit beta-arrestins. Indeed, the overexpression of Galphaq, but not that of beta-arrestin1 or beta-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of beta-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a beta-arrestin-biased mutant form of angiotensin II (SII; Sar1-Ile4-Ile8 AngII) failed to promote ERK5 phosphorylation in primary cardiac fibroblasts, as compared to the natural ligand. Overall, this study shows that the activation of ERK5 MAPK by model Gq-coupled GPCRs does not depend on receptor internalization, beta-arrestin recruitment or receptor phosphorylation but rather is dependent on Galphaq-signalling.