title: ERK5 activation by Gq-coupled muscarinic receptors is independent of receptor internalization and β-arrestin recruitment.
abstract: G-protein-coupled receptors (GPCRs) are known to activate both G protein- and β-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 β-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 Gαq and two novel effectors, PKCζ and MEK5. However, the possible contribution of β-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and β-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 β-arrestins. Indeed, the overexpression of Gαq, but not that of β-arrestin1 or β-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of β-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a β-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, β-arrestin recruitment or receptor phosphorylation but rather is dependent on Gαq-signalling.
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid, UAM-CSIC, Madrid, Spain ; Instituto de Investigación Sanitaria La Princesa, Madrid, Spain.
authors.forename: Guzmán
authors.initials: G
authors.lastname: Sánchez-Fernández
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid, UAM-CSIC, Madrid, Spain ; Instituto de Investigación Sanitaria La Princesa, Madrid, Spain.
authors.forename: Sofía
authors.initials: S
authors.lastname: Cabezudo
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid, UAM-CSIC, Madrid, Spain ; Instituto de Investigación Sanitaria La Princesa, Madrid, Spain.
authors.forename: Carlota
authors.initials: C
authors.lastname: García-Hoz
authors.affiliation: Medical Research Council Toxicology Unit, University of Leicester, Leicester, United Kingdom.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Tobin
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid, UAM-CSIC, Madrid, Spain ; Instituto de Investigación Sanitaria La Princesa, Madrid, Spain.
authors.forename: Federico
authors.initials: F
authors.lastname: Mayor
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid, UAM-CSIC, Madrid, Spain ; Instituto de Investigación Sanitaria La Princesa, Madrid, Spain.
authors.forename: Catalina
authors.initials: C
authors.lastname: Ribas
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D051379:Mice; D048054:Mitogen-Activated Protein Kinase 7 / Q000378:metabolism*; D010766:Phosphorylation; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015398:Signal Transduction; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D011976:Receptors, Muscarinic; D000071557:beta-Arrestins; D048054:Mitogen-Activated Protein Kinase 7; D043802:GTP-Binding Protein alpha Subunits, Gq-G11