ARF6 activation by Gα q signaling: Gα 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 Gα q signaling leads to the activation of ARF6. Stimulation of the TPβ receptor triggered ARF6 activation which was completely inhibited by the RGS domain of GRK2 known to specifically bind and sequester Gα q. Co-immunoprecipitation studies revealed that ARNO (a guanine nucleotide exchange factor for ARF6) and ARF6 formed complexes preferentially with activated Gα q compared to non-activated Gα q. Formation of the Gα 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 Gα q interacted directly with ARNO. Gα q-dependent TPβ 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 TPβ receptor. This further elucidates our previous data on the PLCβ- and PKC-independent mechanism involved in Gα q-mediated internalization of the TPβ receptor. Taken altogether, our results support a novel model where activated Gα q forms molecular complexes with ARNO and ARF6, possibly through a direct interaction with ARNO, leading to ARF6 activation.