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

1)磷脂酰肌醇3-激酶作为胰岛素受体酪氨酸激酶的底物。胰岛素引起过表达人胰岛素受体的细胞抗磷酸酪氨酸免疫沉淀物中磷脂酰肌醇3-激酶活性的急剧和快速增加。因此，该酶可能是胰岛素信号转导的一种介质。至少，还有两个问题有待阐明；1)磷脂酰肌醇3-激酶是否为胰岛素受体酪氨酸激酶的直接底物，2)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酶的活性，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特定活性不变。我们在此报告了高纯度磷脂酰肌醇3-激酶的85-kDa亚基在酪氨酸残基上被激活的正常胰岛素受体磷酸化的证据，而不是被缺乏酪氨酸激酶活性的突变胰岛素受体磷酸化。此外，我们使用FPLC Mono Q柱将酪氨酸磷酸化形式与未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，酪氨酸被受体激酶磷酸化可能会增加磷脂酰肌醇3-激酶的比活性。2)人类胰岛素基因高效表达所需的四个Sp1结合位点。将含有HIR基因启动子的各种缺失和插入的嵌合氯霉素乙酰转移酶质粒转染CHO和COS细胞，表明启动子活性在-629和-1（启动子ATG为+1）之间的区域是最大的。通过低背景pSVOOCAT载体系统在体内检测，4个G-C盒（593 ~ 618）簇的DNA元件增强了转录。使用部分纯化的LacZ-Sp1进行DNase I足迹和凝胶阻滞实验，可以结合启动子的四个G-C盒簇。因此，人类胰岛素受体基因的有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 -618个碱基对的4个G-c盒结合。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