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

我们分离到一个新的肌醇1，4，5-三磷酸[INS(1，4，5)P3]结合蛋白，其相对分子质量为130 kDa(P130)。分子克隆研究表明，该蛋白类似于磷脂酶C-Delta_1(PLC-Delta_1)，但对磷脂酰肌醇没有催化活性，因此其生物学功能尚不清楚。为了更深入地了解这一功能，本研究开展了以下研究。与包括PLC-Delta_1在内的多种类型的PLC相比，p130中最有可能的催化结构域存在几个单一的氨基酸替换，称为X-和Y-结构域。不久将进行大肠杆菌的转基因和随后的PLC活性检测。构建了编码p130的基因与新霉素抗性基因融合在一起的表达载体。将该质粒导入COS-1细胞，在抗生素存在下进一步培养，获得稳定表达p130的细胞。通过与未转基因细胞进行比较，将很快检测到对缓激肽或生长因子刺激的细胞功能的改变。构建了含有p130基因但缺少p130不同区域的表达载体，并将其导入COS-1细胞，以定位负责与INS(1，4，5)P3结合的区域。缺失p130的Ph区的细胞提取液不能结合，表明Ph区参与了结合。P130的PH域与INS(1，4，5，5)P3和INS(1，4，5，6)P4结合，而PLC-Delta_1只与INS(1，4，5)P3结合。基于这些结果，我们提出了与PH结构域相关的生理配体应该是肌醇磷酸盐。

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.