Kavran J M, Klein D E, Lee A, Falasca M, Isakoff S J, Skolnik E Y, Lemmon M A
Department of Biochemistry and Biophysics, and Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6089, USA.
J Biol Chem. 1998 Nov 13;273(46):30497-508. doi: 10.1074/jbc.273.46.30497.
Pleckstrin homology (PH) domains are small protein modules involved in recruitment of signaling molecules to cellular membranes, in some cases by binding specific phosphoinositides. We describe use of a convenient "dot-blot" approach to screen 10 different PH domains for those that recognize particular phosphoinositides. Each PH domain bound phosphoinositides in the assay, but only two (from phospholipase C-delta1 and Grp1) showed clear specificity for a single species. Using soluble inositol phosphates, we show that the Grp1 PH domain (originally cloned on the basis of its phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) binding) binds specifically to D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (the PtdIns(3,4,5)P3 headgroup) with KD = 27.3 nM, but binds D-myo-inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) or D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) over 80-fold more weakly. We show that this specificity allows localization of the Grp1 PH domain to the plasma membrane of mammalian cells only when phosphatidylinositol 3-kinase (PI 3-K) is activated. The presence of three adjacent equatorial phosphate groups was critical for inositol phosphate binding by the Grp1 PH domain. By contrast, another PH domain capable of PI 3-K-dependent membrane recruitment (encoded by EST684797) does not distinguish Ins(1,3,4)P3 from Ins(1,3,4,5)P3 (binding both with very high affinity), despite selecting strongly against Ins(1,4,5)P3. The remaining PH domains tested appear significantly less specific for particular phosphoinositides. Together with data presented in the literature, our results suggest that many PH domains bind similarly to multiple phosphoinositides (and in some cases phosphatidylserine), and are likely to be regulated in vivo by the most abundant species to which they bind. Thus, using the same simple approach to study several PH domains simultaneously, our studies suggest that highly specific phosphoinositide binding is a characteristic of relatively few cases.
普列克底物蛋白同源（PH）结构域是小的蛋白质模块，在某些情况下通过结合特定的磷酸肌醇参与将信号分子募集到细胞膜上。我们描述了使用一种便捷的“点杂交”方法来筛选10种不同的PH结构域，以找出那些能识别特定磷酸肌醇的结构域。在该检测中，每个PH结构域都能结合磷酸肌醇，但只有两个（来自磷脂酶C-δ1和Grp1）对单一类型的磷酸肌醇表现出明显的特异性。使用可溶性肌醇磷酸，我们发现Grp1 PH结构域（最初基于其对磷脂酰肌醇3,4,5-三磷酸（PtdIns(3,4,5)P3）的结合而克隆）与D-肌醇1,3,4,5-四磷酸（Ins(1,3,4,5)P4）（PtdIns(3,4,5)P3的头部基团）特异性结合，解离常数KD = 27.3 nM，但与D-肌醇1,3,4-三磷酸（Ins(1,3,4)P3）或D-肌醇1,4,5-三磷酸（Ins(1,4,5)P3）的结合亲和力要弱80多倍。我们发现，只有当磷脂酰肌醇3-激酶（PI 3-K）被激活时，这种特异性才使得Grp1 PH结构域定位于哺乳动物细胞的质膜。三个相邻的赤道磷酸基团的存在对于Grp1 PH结构域结合肌醇磷酸至关重要。相比之下，另一个能够依赖PI 3-K进行膜募集的PH结构域（由EST684797编码）不能区分Ins(1,3,4)P3和Ins(1,3,4,5)P3（对两者的结合亲和力都非常高），尽管它对Ins(1,4,5)P3有很强的排斥。所测试其余的PH结构域对特定磷酸肌醇的特异性明显较低。结合文献中的数据，我们的结果表明，许多PH结构域与多种磷酸肌醇（在某些情况下还与磷脂酰丝氨酸）的结合方式相似，并且在体内可能受其结合的最丰富类型的调节。因此，通过使用相同的简单方法同时研究多个PH结构域，我们的研究表明，高度特异性的磷酸肌醇结合是相对较少情况下的特征。
