Binding of phospholipase C-related but catalytically inactive protein to phosphatidylinositol 4,5-bisphosphate via the PH domain
Binding of phospholipase C-related but catalytically inactive protein to phosphatidylinositol 4,5-bisphosphate via the PH domain
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DOI：
10.1016/j.cellsig.2009.03.008
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发表时间：
2009-07-01
影响因子：
4.8
通讯作者：
Hirata, Masato
中科院分区：
文献类型：
--
作者：
Gao, Jing;Takeuchi, Hiroshi;Hirata, Masato
调节分子相互作用的一个众所周知的蛋白质模块是Pleckstrin Homology(PH)结构域，其最具特征的配体是磷脂酰肌醇。在本研究中，我们使用各种结合分析方法，分析了与磷脂酰肌醇4，5-二磷酸[PtdIns(4，5)P-2]结合的磷脂酰肌醇4，5-二磷酸(PtdIns(4，5)P-2)与PRIP(磷脂酶C相关但催化非活性蛋白)的PH域。由PRIP-1和PRIP-2制备的PH结构域在体外与可溶性配体的结合特征与PLC-Delta 1相似，但具有PRIP-1而不是PRIP-2的PH结构域在细胞质中分布均匀，表明PRIP-1的N端延伸抑制了与存在于质膜中的PtdIns(4，5)P-2的结合。具有PRIP-1 N端延伸的PLC-Delta 1 PH结构域的嵌合分子显示出与具有N端延伸的PRIP-1 PH结构域相似的定位。与不同浓度的PtdIns(4，5)P-2脂质体的结合实验表明，PLC-Delta 1的PH结构域结合最大，即使在1.2mol%的浓度下也是如此，而PRIP-1和-2的PH结构域则随着浓度的增加而结合。我们还使用皂苷渗透的PC12细胞进行了结合实验。在三磷酸腺苷存在下，PRIP的pH域增加了与脑胞浆提取物预先孵育的细胞的结合，在此过程中可能合成了PtdIns(4，5)P-2。PH结构域与下列EF手型基序的结合表现出钙离子依赖的结合。这些结果表明，PRIP的PH结构域可能与质膜上存在的PtdIns(4，5)P-2结合，这取决于脂质配体和钙离子的浓度，提示PRIP可能在这些参数的变化中发挥生理作用，可能包括Ins(1，4，5)P-3。(C)2009 Elsevier Inc.保留所有权利。
A well-known protein module regulating molecular interactions is the pleckstrin homology (PH) domain whose best-characterised ligand is phosphoinositide. In the present study, we analysed the PH domain from PRIP (phospholipase C-related but catalytically inactive protein, comprising types 1 and 2) regarding phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P-2] binding employing a variety of binding assays. The PH domains prepared from PRIP-1 and -2 showed similar binding profiles to soluble ligands in vitro and showed similar plasma membrane localisation to that of PLC-delta 1; however, the PH domain with the N-terminal extension of PRIP-1 but not PRIP-2 showed even distribution throughout the cytoplasm, indicating that the N-terminal extension of PRIP-1 inhibited binding to PtdIns(4,5)P-2 present in the plasma membrane. A chimeric molecule of PLC-delta 1 PH domain with the N-terminal extension of PRIP-1 exhibited similar localisation to PRIP-1 PH domain with the N-terminal extension. Binding assay to liposomes containing various concentrations of PtdIns(4,5)P-2 revealed that the PH domain of PLC-delta 1 bound steeply to the maximum, even at a concentration of 1.2 mol%, whereas the PH domains from PRIP-1 and -2 bound depending on the concentration up to 5 mol%. We also performed binding experiments using saponin-permeabilised PC12 cells. PH domains from PRIP increased the binding to cells preincubated with the brain cytosol extract in the presence of ATP, during which PtdIns(4,5)P-2 were probably synthesised. The binding of PH domain with the following EF hand motifs showed Ca2+-dependent binding. These results indicate that the PH domain of PRIP binds to PtdIns(4,5)P-2 present in the plasma membrane, depending on the concentrations of the lipid ligand and Ca2+, Suggesting that PRIP might play physiological roles in events involved in the changes of these parameters, probably including Ins(1,4,5)P-3. (C) 2009 Elsevier Inc. All rights reserved.