Klein Stéphanie, Franco Michel, Chardin Pierre, Luton Frédéric
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR 6097, 660, route des Lucioles, 06560 Valbonne, France.
J Biol Chem. 2006 May 5;281(18):12352-61. doi: 10.1074/jbc.M601021200. Epub 2006 Mar 9.
We have analyzed both biochemically and functionally a series of Arf6 mutants, providing new insights into the molecular mode of action of the small G protein Arf6. First, by comparing a fast-cycling mutant (Arf6(T157N)) and a GTPase-deficient mutant (Arf6(Q67L)), we established the necessity for completion of the Arf6 GDP/GTP cycle for recycling of major histocompatibility complex molecules to the plasma membrane. Second, we found that aluminum fluoride (AlF), known for inducing membrane protrusion in cells expressing exogenous wild-type Arf6, stabilized a functional wild-type Arf6.AlF(x) . GTPase-activating protein (GAP) complex in vitro and in vivo. We also found that the tandem mutation Q37E/S38I prevented the binding of two Arf GAPs, but not the effector ARHGAP10, and blocked the formation of membrane protrusion and actin reorganization. Together, our results with AlF(x) and Arf6(Q37E/S38I) demonstrate the critical role of the Arf6 GAPs as effectors for Arf6-regulated actin cytoskeleton remodeling. Finally, competition experiments conducted in vivo suggest the existence of a membrane receptor for GDP-bound Arf6.
我们对一系列Arf6突变体进行了生化和功能分析，为小G蛋白Arf6的分子作用模式提供了新的见解。首先，通过比较快速循环突变体（Arf6(T157N)）和GTP酶缺陷突变体（Arf6(Q67L)），我们确定了Arf6 GDP/GTP循环的完成对于主要组织相容性复合体分子循环至质膜的必要性。其次，我们发现，以在表达外源性野生型Arf6的细胞中诱导膜突出而闻名的氟化铝（AlF），在体外和体内稳定了功能性野生型Arf6·AlF(x)·GTP酶激活蛋白（GAP）复合物。我们还发现，串联突变Q37E/S38I阻止了两种Arf GAP的结合，但不阻止效应器ARHGAP10的结合，并阻断了膜突出的形成和肌动蛋白重组。总之，我们对AlF(x)和Arf6(Q37E/S38I)的研究结果证明了Arf6 GAP作为Arf6调节的肌动蛋白细胞骨架重塑效应器的关键作用。最后，在体内进行的竞争实验表明存在一种与GDP结合的Arf6的膜受体。
