Macia E, Paris S, Chabre M
CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, F-06560 Valbonne, France.
Biochemistry. 2000 May 16;39(19):5893-901. doi: 10.1021/bi992795w.
The activity on ARF of the guanine nucleotide exchange factor ARNO depends on its membrane recruitment, induced by binding of its PH domain to phosphoinositides. A polycationic C-terminal extension to the PH domain might also contribute to its specific binding to phosphatidylinositol 4,5-bisphosphate [(4,5)PIP2] and to phosphatidylinositol 3,4,5-trisphosphate [(3,4,5)PIP3], and to ionic binding to other acidic lipids. We have analyzed in vitro the relative contributions to phospholipid binding of the PH domain and C-terminal extension by cosedimentation of "PH+C domain" and "nominal PH domain" protein constructs including or not including the polycationic C-terminus, with sucrose-loaded unilamellar vesicles made of equal proportions of the neutral lipids phosphatidylcholine and phosphatidylethanolamine, and supplemented or not with 30% acidic phosphatidylserine (PS) and 2% of various phosphoinositides. Binding was measured as a function of the vesicle concentration and of the medium ionic strength. Both proteins bound with higher affinity to (3,4,5)PIP3 than to (4,5)PIP2, the selectivity for (3,4,5)PIP3 being highest for the nominal PH domain. We observed also a clear selectivity of (3,4,5)PIP3 over (4,5)PIP2 for stimulating the activity of ARNO on ARF with vesicles containing 10% PS and 1% PIP2 or PIP3. Our data suggest that the PH domain provides the specific phosphoinositide binding site and some unspecific ionic interaction with acidic PS, whereas the polybasic C domain contributes to binding mainly by unspecific ionic interactions vith PS. Phosphorylation by protein kinase C of a serine in the C domain reduces the ionic affinity of the PH+C domain for PS, but does not affect the phosphoinositide specificity.
鸟嘌呤核苷酸交换因子ARNO对ARF的活性取决于其膜募集，这是由其PH结构域与磷酸肌醇结合所诱导的。PH结构域的多阳离子C末端延伸也可能有助于其与磷脂酰肌醇4,5-二磷酸[（4,5）PIP2]和磷脂酰肌醇3,4,5-三磷酸[（3,4,5）PIP3]的特异性结合，以及与其他酸性脂质的离子结合。我们通过“PH + C结构域”和“标称PH结构域”蛋白质构建体（包括或不包括多阳离子C末端）与由等量中性脂质磷脂酰胆碱和磷脂酰乙醇胺制成的蔗糖负载单层囊泡共沉降，分析了体外PH结构域和C末端延伸对磷脂结合的相对贡献，这些囊泡补充或不补充30%的酸性磷脂酰丝氨酸（PS）和2%的各种磷酸肌醇。结合量作为囊泡浓度和介质离子强度的函数进行测量。两种蛋白质与（3,4,5）PIP3的结合亲和力均高于与（4,5）PIP2的结合亲和力，标称PH结构域对（3,4,5）PIP3的选择性最高。我们还观察到，对于含有10% PS和1% PIP2或PIP3的囊泡，（3,4,5）PIP3对（4,5）PIP2在刺激ARNO对ARF的活性方面具有明显的选择性。我们的数据表明，PH结构域提供了特异性磷酸肌醇结合位点以及与酸性PS的一些非特异性离子相互作用，而多碱性C结构域主要通过与PS的非特异性离子相互作用来促进结合。C结构域中丝氨酸的蛋白激酶C磷酸化降低了PH + C结构域对PS的离子亲和力，但不影响磷酸肌醇特异性。
