Structure and Function of Insulin Receptor, its Gene Expression and its Signal Transduction Mechanism

1)以磷脂酰肌醇3-激酶作为胰岛素受体酪氨酸激酶的体外底物。胰岛素导致过量表达人胰岛素受体的细胞的抗磷酸酪氨酸免疫沉淀物中的磷脂酰肌醇3-激酶活性急剧而迅速地增加。因此，该酶可能是胰岛素信号转导的介体之一。至少有两个问题仍有待阐明：i)磷脂酰肌醇3-激酶是否是胰岛素受体酪氨酸激酶的直接底物，ii)胰岛素受体激酶对磷脂酰肌醇3-激酶的酪氨酸磷酸化是否改变了特定的酶活性，或者磷脂酰肌醇3-激酶的酪氨酸磷酸化形式的量是否增加而比活性没有变化。我们报告了高纯度的磷脂酰肌醇3-激酶的85 kDa亚基在体外被激活的正常胰岛素受体在酪氨酸残基上被磷酸化的证据，但…此外，我们使用FPLC Mono Q柱，将酪氨酸磷酸化形式与非磷酸化形式的酶部分分离。胰岛素刺激的磷脂酰肌醇3-激酶活性主要在含有几乎所有酪氨酸磷酸化形式的部分中检测到。这些体外和体内实验的结果表明，磷脂酰肌醇3-激酶是胰岛素受体酪氨酸激酶的直接底物，并且受体激酶对磷脂酰肌醇3-激酶的酪氨酸磷酸化可能会增加比活性。2)高效表达人胰岛素基因所需的四个Sp1结合位点的簇。将含有hIR基因启动子不同缺失和插入的氯霉素乙酰转移酶嵌合表达载体分别导入CHO和COS细胞，结果表明-629~-1(启动子ATG为+1)区域具有较高的启动子活性。通过低背景pSVOOCAT载体系统体内检测，4个G-C盒(593~-618)簇的DNA元件促进了转录。使用部分纯化的LacZ-Sp1的DNase I足迹和凝胶延迟实验可以结合到启动子的四个G-C盒的簇上。因此，人胰岛素受体基因的高效表达可能需要转录因子Sp1与位于ATG翻译起始密码子上游-593至-618碱基对的四个G-c盒结合。较少

1) Phosphatidylinositol 3-Kinase as a Substrate for Insulin Receptor Tyrosine Kinase In Vitro. Insulin causes a dramatic and rapid increase in phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immunoprecipitates of cells overexpressing the human insulin receptor. Therefore, this enzyme may be one mediator of insulin signal transduction. At least, two questions remain to be elucidated ; i) whether or not the phosphatidylinositol 3-kinase is a direct substrate for the insulin receptor tyrosine kinase, ii) whether tyrosine phosphorylation of phosphatidylinositol 3-kinase by the insulin receptor kinase alters the specific enzyme activity, or whether the amount of the tyrosine-phosphorylated form of the phosphatidylinositol 3-kinase increases with no change in the specific activity. We report here evidence that the 85-kDa subunit of the highly purified phosphatidylinositol 3-kinase is phosphorylated on the tyrosine residue by the activated normal insulin receptor, in vitro, but … More not by the mutant insulin receptor which lacks the tyrosine kinase activity.In addition, we partially separated the tyrosine phosphorylated form from the unphosphorylated one of the enzyme, using a FPLC Mono Q column. The insulin-stimulated phosphatidylinositol 3-kinase activity was mainly detected in the fraction which contained almost all of the tyrosine-phosphorylated form. These results of in vitro and in vivo experiments show that the phosphatidylinositol 3-kinase is a direct substrate for the insulin receptor tyrosine kinase and that tyrosine phosphorylation of the phosphatidylinositol 3-kinase by the receptor kinase probably increases the specific activity.2) A Cluster of Four Sp1 Binding Sites Required for Efficient Expression of the Human Insulin Gene. Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions and insertions of the promoter of HIR gene into CHO and COS cells indicated that the region between-629 and-1 (initiator ATG is+1) is sufficient for maximal promoter activity. The DNA element of the cluster of four G-C boxes (593 to-618) enhanced the transcription, examined by the low background pSVOOCAT vector system in vivo. DNase I footprinting and gel retardation experiments using partially purified LacZ-Sp1 can bind to the cluster of the four G-C boxes of the promoter. Thus, the efficient expression of the human insulin receptor gene possibly requires the binding of transcriptional factor Sp1 to four G-c boxes located-593 to-618 base pairs upstream of the ATG translation initiation codon. Less