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

点击翻译按钮获取中文摘要

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