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

新型Ins(1,4,5)P_3结合蛋白的研究

• 批准号: 07044278
• 负责人: HIRATA Masato
• 金额: $ 1.41万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044278/
• 关键词: Inositol phosphates; Calcium ion; Pleckstrin homology domain; Cellular signalling; Receptor

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.

130-kDa蛋白是从大鼠脑中分离到的一种新的1，4，5-三磷酸肌醇（D-Ins（1，4，5）P_3）结合蛋白，最近发现它与磷脂酶C-δ_1相似。我们将编码D-Ins（1，4，5）P_3结合域的全长及其缺失突变体的基因转染COS-1细胞，在COS-1细胞中表达D-Ins（1，4，5）P_3结合域。用编码该分子全长的基因转染COS-1细胞，其胞浆组分显示出D- [^3H] Ins（1，4，5）P_3结合活性。N端232个残基的截断完全消除了D- [^3H] Ins（1，4，5）P_3的结合活性，而N端115个残基的截断保持了与全长分子相似的结合活性。从C-末端缺失几百个残基不影响结合。这些数据表明D-Ins（1，4，5）P_3的结合结构域位于116 - 232位残基的区域，对应于分子的pleckstrin同源域（PH结构域）。从细菌表达系统中分离的PH结构域（残基95-232）能够结合-D [^3H] Ins（1，4，5）P_3。用C-末端缺失和分离的PH结构域构建了一个130-kDa蛋白的PH结构域，并使用磷酸肌醇位置异构体和D-Ins（1，4，5）P_3类似物研究了D-Ins（1，4，5）P_3的结构要求。肌醇上的4，5-二磷酸似乎是识别所需的，另外的1-磷酸增加了亲和力。3-位的羟基似乎提供氢键相互作用。