title: ARF6 activation by Galpha q signaling: Galpha q forms molecular complexes with ARNO and ARF6.
abstract: G protein-coupled receptors (GPCRs) are widely expressed hepta-helical receptors with tightly regulated pleiotropic effects. ADP-Ribosylation Factor 6 (ARF6) plays an important role in GPCR trafficking and is the subject of intense research. However, the mechanisms underlying activation and regulation of ARF6 by GPCRs are poorly characterized. Here we report that Galpha(q) signaling leads to the activation of ARF6. Stimulation of the TPbeta receptor triggered ARF6 activation which was completely inhibited by the RGS domain of GRK2 known to specifically bind and sequester Galpha(q). Co-immunoprecipitation studies revealed that ARNO (a guanine nucleotide exchange factor for ARF6) and ARF6 formed complexes preferentially with activated Galpha(q) compared to non-activated Galpha(q). Formation of the Galpha(q) complexes with ARNO and ARF6 was detected early and was optimal after 30 min of receptor stimulation corresponding with the profile of ARF6 activation. Interestingly, binding experiments using purified proteins showed that Galpha(q) interacted directly with ARNO. Galpha(q)-dependent TPbeta receptor-mediated activation of ARF6 resulted in phosphoinositol-4,5-bisphosphate production which was potently inhibited by dominant negative mutants of ARNO and ARF6. Furthermore, our data show that the expression of ARNO and ARF6 promoted, whereas dominant negative mutants of these proteins inhibited the internalization of the TPbeta receptor. This further elucidates our previous data on the PLCbeta- and PKC-independent mechanism involved in Galpha(q)-mediated internalization of the TPbeta receptor. Taken altogether, our results support a novel model where activated Galpha(q) forms molecular complexes with ARNO and ARF6, possibly through a direct interaction with ARNO, leading to ARF6 activation.
authors.affiliation: Service de Rhumatologie, Département de Médecine, Faculté de Médecine and Centre de Recherche Clinique, Université de Sherbrooke, 3001 12e Avenue Nord, Fleurimont, Quebec, Canada.
authors.forename: Patrick
authors.initials: P
authors.lastname: Giguère
authors.forename: Moulay Driss
authors.initials: MD
authors.lastname: Rochdi
authors.forename: Geneviève
authors.initials: G
authors.lastname: Laroche
authors.forename: Emilie
authors.initials: E
authors.lastname: Dupré
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Whorton
authors.forename: Roger K
authors.initials: RK
authors.lastname: Sunahara
authors.forename: Audrey
authors.initials: A
authors.lastname: Claing
authors.forename: Gilles
authors.initials: G
authors.lastname: Dupuis
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Parent
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism* / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006801:Humans; D046912:Multiprotein Complexes / Q000378:metabolism; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: D000089982:ADP-Ribosylation Factor 6; D020690:GTPase-Activating Proteins; D046912:Multiprotein Complexes; C102474:cytohesin-2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human