Studies on A Novel Ins (1,4,5) P_3 Binding Proteins

这个130 kDa的蛋白是从大鼠脑中分离得到的一种新的D-Ins(1，4，5)P3结合蛋白，最近发现它类似于磷脂酶C-Delta_1。在本实验中，我们用编码全长分子的基因及其缺失突变体在COS-1细胞中表达了蛋白质，以定位D-Ins(1，4，5)P3的结合域。N端232个残基的截断完全取消了D-[^~3H]Ins(1，4，5)P_3的结合活性，而N端115个残基的截断则保持了与全长分子相似的亲和力。C末端数百个残基的缺失不影响结合。这些数据表明，D-ins(1，4，5)P3的结合域位于116-232残基区域，对应于该分子的Pleckstrin同源区域(PH域)。从细菌表达系统中分离得到的PH结构域(残基95-232)能够与D[~(3 H)]Ins(1，4，5)P3结合。利用磷酸肌醇位置异构体和D-Ins(1，4，5)P3类似物，通过C端缺失的结构和分离的PH结构域检测结合特异性，以探讨D-ins(1，4，5)P3被130 kDa蛋白的PH结构域识别所需的结构要求。肌醇上的4，5-二磷酸似乎是识别所必需的，额外的1-磷酸增加了亲和力。3位羟基似乎提供了氢键作用。

The 130-kDa protein was isolated as a novel inositol 1,4,5-trisphosphate (D-Ins (1,4,5) P_3) binding protein from rat brain and was recently found to be similar to phospholipase C-delta_1. Here we expressed proteins in COS-1 cells by transfection with genes encoding an entire-length of the molecule and its deletion mutants to locate the binding domain for D-Ins (1,4,5) P_3. The cytosol fraction of COS-1 cells transfected with a gene encoding a full-length of the molecule exhibited an D- [^3H] Ins (1,4,5) P_3 binding activity. Truncation of 232 residues from the N-terminus completely abolished the binding activity of D- [^3H] Ins (1,4,5) P_3, while truncation of N-terminal 115 residues kept the binding activity with a similar affinity as a full-length molecule. Deletion of several hundred residues from the C-terminus did not affect the binding. These data indicate that the binding domain of D-Ins (1,4,5) P_3 resides in the region of residues from 116 to 232, which corresponds to the pleckstrin homology omain (PH domain) of the molecule. The PH domain (residues 95-232) isolated from a bacterial expression system was capable to bind-D [^3H] Ins (1,4,5) P_3. Binding specificity was examined with a construct deleted at C-terminals and isolated PH domain, using inositol phosphate positional isomers and D-Ins (1,4,5) P_3 analogues to explore the structural requirement of D-Ins (1,4,5) P_3 for recognition by the PH domain of the 130-kDa protein. The 4,5-bisphosphates on myo-inositol appeared to be required for the recognition and an additional 1-phosphate increased the affinity. Hydroxyl group at 3-position seemed to provide hydrogen bonding interaction.