Giguère Patrick, Rochdi Moulay Driss, Laroche Geneviève, Dupré Emilie, Whorton Matthew R, Sunahara Roger K, Claing Audrey, Dupuis Gilles, Parent Jean-Luc
Service de Rhumatologie, Département de Médecine, Faculté de Médecine and Centre de Recherche Clinique, Université de Sherbrooke, 3001 12e Avenue Nord, Fleurimont, Quebec, Canada.
Cell Signal. 2006 Nov;18(11):1988-94. doi: 10.1016/j.cellsig.2006.03.003. Epub 2006 Mar 14.
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.
G蛋白偶联受体（GPCRs）是广泛表达的七螺旋受体，具有严格调控的多效性作用。ADP-核糖基化因子6（ARF6）在GPCR转运中起重要作用，是深入研究的对象。然而，GPCRs激活和调节ARF6的潜在机制仍不清楚。在此我们报告，Gαq信号传导导致ARF6的激活。刺激TPβ受体触发ARF6激活，已知能特异性结合并隔离Gαq的GRK2的RGS结构域可完全抑制该激活。免疫共沉淀研究表明，与未激活的Gαq相比，ARNO（一种ARF6的鸟嘌呤核苷酸交换因子）和ARF6优先与激活的Gαq形成复合物。在受体刺激30分钟后，早期检测到Gαq与ARNO和ARF6形成复合物，且此时复合物形成最为理想，这与ARF6激活的情况相符。有趣的是，使用纯化蛋白进行的结合实验表明，Gαq直接与ARNO相互作用。Gαq依赖的TPβ受体介导的ARF6激活导致磷酸肌醇-4,5-二磷酸的产生，ARNO和ARF6的显性负突变体可有效抑制该产生。此外，我们的数据表明，ARNO和ARF6的表达促进了TPβ受体的内化，而这些蛋白的显性负突变体则抑制了TPβ受体的内化。这进一步阐明了我们之前关于Gαq介导的TPβ受体内化中涉及的不依赖PLCβ和PKC的机制的数据。综上所述，我们的结果支持一种新模型，即激活的Gαq可能通过与ARNO直接相互作用，与ARNO和ARF6形成分子复合物，从而导致ARF6激活。
