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

我们分离到一个新的1，4，5-三磷酸肌醇[Ins（1，4，5）P_3]结合蛋白（p130），分子量为130-kDa。分子克隆研究表明，该蛋白与磷脂酶C-δ_1（PLC-delta_1）相似，但对磷酸肌醇没有催化活性，其生物学功能尚不清楚。为了深入了解这一功能，本研究进行了以下研究.与包括PLC-delta_1在内的许多类型的PLC相比，在p130中最可能的催化结构域（称为X结构域和Y结构域）中有几个单个氨基酸替换。构建了表达p130的突变体质粒，其中X-和/或Y-结构域被PLC-δ_1的X-和/或Y-结构域取代。大肠杆菌的转染和随后的PLC活性测定将很快完成。构建质粒，其中编码p130的基因与提供新霉素抗性的基因一起掺入。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。与未转染的细胞相比，细胞功能对缓激肽或生长因子刺激的反应的改变将很快被检查。构建了含有p130基因但缺少p130不同区域的质粒，并将每个质粒转染COS-1细胞，以定位负责结合Ins（1，4，5）P_3的区域。缺乏p130的普列克底物蛋白同源结构域（PH结构域）的细胞提取物不能结合，表明PH结构域参与结合。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，我们提出了生理相关的配体PH域将肌醇磷酸。

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.