ARF6 activation by Galpha q signaling: Galpha q forms molecular complexes with ARNO and ARF6.
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.
Giguère
Patrick
P
Service de Rhumatologie, Département de Médecine, Faculté de Médecine and Centre de Recherche Clinique, Université de Sherbrooke, 3001 12e Avenue Nord, Fleurimont, Quebec, Canada.
Rochdi
Moulay Driss
MD
Laroche
Geneviève
G
Dupré
Emilie
E
Whorton
Matthew R
MR
Sunahara
Roger K
RK
Claing
Audrey
A
Dupuis
Gilles
G
Parent
Jean-Luc
JL
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
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