Studies on ANovel Ins (1,4,5)P_3 Binding Protein Function

新型Ins(1,4,5)P_3结合蛋白功能的研究

• 批准号: 08044301
• 负责人: HIRATA Masato
• 金额: $ 1.66万
• 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: 1996
• 资助国家: 日本
• 起止时间: 1996 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08044301/
• 关键词: Inositol phosphates; Pleckstrin homology domain; Phospholipase; Inositol 1,4,5-trisphosphate; Phosphoinositides

We isolated a new inositol 1,4,5-trisphosphate [Ins (1,4,5) P_3] binding protein with a molecular mass of 130-kDa (p130). Molecular cloning studies revealed that the protein is similar to phospholipase C-delta_1 (PLC-delta_1) but has no catalytic activity to phosphoinositides, and Thus the biological function is not yet known. To gain the insight for the function, the following studies were carried out in the present study.1. There are several single amino acid replacements in the most likely catalytic domains, named X-and Y-domains in p130, when they compared to those of many types of PLC including PLC-delta_1. Mutant plasmids for expressing p130, in which the X-and/or Y-domains were replaced with those of PLC-delta_1, were constructed. Transfection of E.coli and subsequent assay for PLC activity will be done soon.2. A plasmid was constructed, in which the genes encoding p130 was incorporated together with the gene for affording neomycin-resistancy. COS-1 cells were transfected with the plasmid and further cultivation in the presence of the antibiotics produced the cells which stably express p130. Modification of the cell function in response to the stimulation with bradykinin or growth factor will be examined soon, comparing non-transfected cells.3. Plasmids containing p130 gene, but lacking various region of p130 were constructed and each plasmid was transfected into COS-1 cells in order to localize the region responsible for binding Ins (1,4,5) P_3. Cell extract lacking the pleckstrin homology domain (PH domain) of p130 failed in the binding, indicating that the PH domain is involved in the binding. The PH domain of p130 bound both Ins (1,4,5) P_3 and Ins (1,4,5,6) P_4, while that of PLC-delta_1 bound Ins (1,4,5) P_3 alone. Based on these results, we have proposed that the physiologically relevant ligands for PH domains would be inositol phosphates.

我们分离出一种新的肌醇 1,4,5-三磷酸 [Ins (1,4,5) P_3] 结合蛋白，分子量为 130-kDa (p130)。分子克隆研究表明，该蛋白与磷脂酶C-delta_1（PLC-delta_1）相似，但对磷酸肌醇没有催化活性，因此其生物学功能尚不清楚。为了深入了解该功能，本研究进行了以下研究： 1．当与包括 PLC-delta_1 在内的许多类型的 PLC 进行比较时，在 p130 中最可能的催化结构域（称为 X 和 Y 结构域）中存在多个单个氨基酸替换。构建了用于表达p130的突变质粒，其中X和/或Y结构域被PLC-delta_1的结构域替换。大肠杆菌的转染和随后的PLC活性测定将很快完成。2．构建了质粒，其中编码p130的基因与提供新霉素抗性的基因掺入在一起。用该质粒转染COS-1细胞并在抗生素存在下进一步培养产生稳定表达p130的细胞。很快将通过比较未转染的细胞来检查细胞功能响应缓激肽或生长因子刺激的变化。3．构建含有p130基因但缺乏p130的各个区域的质粒，并将每个质粒转染至COS-1细胞中，以定位负责结合Ins(1,4,5)P_3的区域。缺乏p130 pleckstrin 同源结构域（PH 结构域）的细胞提取物未能结合，表明 PH 结构域参与了结合。 p130 的 PH 结构域结合 Ins (1,4,5) P_3 和 Ins (1,4,5,6) P_4，而 PLC-delta_1 的 PH 结构域仅结合 Ins (1,4,5) P_3。基于这些结果，我们提出 PH 结构域的生理相关配体是肌醇磷酸盐。

项目成果

Takeuchi H.et al: "Localization of a high affinity inositol 1,4,5-trisphosphate/inositol 1,4,5,6-tetrakisphospahte binding to the pleckstrin homology module of a new 130-kDa protein : characterization of the determinants of structural specificity" Biochem

Takeuchi H.等人：“高亲和力肌醇 1,4,5-三磷酸/肌醇 1,4,5,6-四磷酸与新 130-kDa 蛋白的普莱克斯特林同源模块结合的定位：决定因素的表征

Hirata, M.et al: "Intrinsic inhibitor of inositol 1,4,5-trisphosphate binding" Mol.Cell. Biochem.(in press). (1997)

Hirata, M.et al：“肌醇 1,4,5-三磷酸结合的内在抑制剂”Mol.Cell。

Watanabe,Y.: "Synthesis of phosphorofluoridate analogues of myo-inositol 1,4,5-tris (phosphate) and their biological activity" J.Chem.Soc.,Chem.Commun.1996(15). 1815-1816 (1996)

Watanabe,Y.：“肌醇 1,4,5-tris（磷酸盐）的氟磷酸酯类似物的合成及其生物活性”J.Chem.Soc.，Chem.Commun.1996（15）。