Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs
Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs
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DOI：
10.1074/jbc.m002435200
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发表时间：
2000-10-20
影响因子：
4.8
通讯作者：
Czech, MP
中科院分区：
文献类型：
--
作者：
Klarlund, JK;Tsiaras, W;Czech, MP
GRP1和相关蛋白ARNO和cytohesin-1是含有pleckstrin同源（PH）结构域的ARF交换因子，被认为通过结合多磷酸肌醇将这些蛋白靶向细胞膜。这里我们发现，这三种蛋白的PH结构域对二辛酸磷脂酰肌醇3,4,5-三磷酸（PtdIns(3,4,5)P-3）表现出相对较高的亲和力，GRP1、ARNO和cytohesin-1的K-D值分别为0.05、1.6和1.0 muM。然而，在这些蛋白中，GRP1的PH结构域是独一无二的，它对PtdIns(3,4,5)P-3具有显著的选择性，而对磷脂酰肌醇4,5-二磷酸（PtdIns(4,5)P-2）具有大约650倍的表观亲和力。在GRP1的Gly(274)-Gly（275）基序中添加甘氨酸可大大增加其对PtdIns(4,5)P-2的结合亲和力，但对其与PtdIns(3,4,5)P-3的结合影响不大，而在ARNO PH结构域相应的甘油三酯基序中删除单个甘氨酸可显著降低其对PtdIns(4,5)P-2的结合亲和力，但对PtdIns(3,4,5)P-3没有影响。在完整的细胞中，在胰岛素的作用下，全长GRP1和cytohesin-1、血凝素表位标记的GRP1的PH结构域被募集到细胞表面的皱褶中，而细胞hesin-1的PH结构域则没有。这些数据表明，GRP1 PH域中独特的二甘氨酸基序，与ARNO和cytohesin-1中的三甘氨酸相反，指导了其显著的PtdIns(3,4,5)P-3结合选择性。
GRP1 and the related proteins ARNO and cytohesin-1 are ARF exchange factors that contain a pleckstrin homology (PH) domain thought to target these proteins to cell membranes through binding polyphosphoinositides, Here we show the PH domains of all three proteins exhibit relatively high affinity for dioctanoyl phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P-3), with K-D values of 0.05, 1.6 and 1.0 muM for GRP1, ARNO, and cytohesin-1, respectively. However, the GRP1 PH domain was unique among these proteins in its striking selectivity for PtdIns(3,4,5)P-3 versus phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P-2), for which it exhibits about 650-fold lower apparent affinity. Addition of a glycine to the Gly(274)-Gly(275) motif in GRP1 greatly increased its binding affinity for PtdIns(4,5)P-2 with little effect on its binding to PtdIns(3,4,5)P-3, while deletion of a single glycine in the corresponding triglycine motif of the ARNO PH domain markedly reduced its binding affinity for PtdIns(4,5)P-2 but not for PtdIns(3,4,5)P-3. In intact cells, the hemagglutinin epitope-tagged PH domain of GRP1 was recruited to ruffles in the cell surface in response to insulin, as were full-length GRP1 and cytohesin-1, but the PH domain of cytohesin-1 was not. These data indicate that the unique diglycine motif in the GRP1 PH domain, as opposed to the triglycine in ARNO and cytohesin-1, directs its remarkable PtdIns(3,4,5)P-3 binding selectivity.