SUBSTRATES AND INSULIN RECEPTOR ENDOCYTOSIS

胰岛素作用受损，或胰岛素抵抗，是非胰岛素依赖型糖尿病(NIDDM)的标志。由于胰岛素抵抗是NIDDM发病机制中的主要因素，了解胰岛素抵抗的机制对于寻找新的方法来改善NIDDM易感人群的胰岛素敏感性具有潜在的意义。胰岛素与其受体的结合激活了受体的酪氨酸激酶，导致受体和其他底物的磷酸化，例如肝细胞中的一种质膜糖蛋白ppl20。在没有胰岛素的情况下，PP120在胞内区的Ser503上被cAMP依赖的激酶磷酸化，而这种磷酸化是胰岛素受体激酶对胰岛素的反应使其在Tyr488上磷酸化所必需的。Ppl20在胰岛素作用中的作用还不是很清楚。培养细胞中pp120的表达通过受体介导的内吞作用与胰岛素从培养液中清除的速率增加相关，这表明pp120在胰岛素从门静脉循环中清除的过程中起重要作用。相反，磷酸化缺陷的pp120亚型(截短和Y488F和S503A定点突变体)的表达并没有增加受体介导的胰岛素内化，这表明p120对胰岛素内吞作用的影响取决于其磷酸化状态。免疫荧光和生物素标记研究表明，pp120通过受体介导的内化反应胰岛素发挥作用。因此，pp120似乎参与了一种蛋白质复合体，该复合体将胰岛素受体定位于内吞囊泡。位于Tyr488的pp120和位于其膜旁区域Tyr960的胰岛素受体之间的复合体的形成似乎是由细胞内蛋白介导的。在此，我们建议鉴定这些蛋白质。此外，我们建议解决ppl20在体内胰岛素作用机制中的作用。为此，我们培育了一只转基因小鼠，在肝脏中过表达磷酸化缺陷的ppl20的S503A亚型。转基因品系将解决磷酸化缺陷PP 120的表达是否与钝化从门静脉循环中清除多余胰岛素的能力有关，从而导致外周高胰岛素血症。由于高胰岛素血症导致靶组织上受体下调，因此通常与胰岛素抵抗有关。这些拟议的研究将为高胰岛素血症、胰岛素抵抗和糖尿病的潜在机制提供新的见解。

Impaired insulin action, or insulin resistance, is a hallmark of Non-Insulin-Dependent Diabetes Mellitus (NIDDM). Because insulin resistance is a major factor in the pathogenesis of NIDDM, understanding the mechanisms of insulin resistance has potential implications in identifying novel means to improve insulin sensitivity in individuals predisposed to NIDDM. Insulin binding to its receptor activates the tyrosine kinase of the receptor to cause phosphorylation of the receptor and of other substrates, such as ppl20, a plasma membrane glycoprotein in the hepatocyte. pp120 is phosphorylated on Ser503 in the intracellular domain by cAMP-dependent kinase in the absence of insulin, and this phosphorylation is required for its phosphorylation on Tyr488 by the insulin receptor kinase in response to insulin. The role of ppl20 in insulin action is not well understood. pp120 expression in cultured cells was correlated with increased rate of insulin clearance from the medium through a mechanism of receptor-mediated endocytosis, suggesting that pp120 is important in the process of insulin clearance from the portal circulation. In contrast, expression of phosphorylation-defective pp120 isoforms (truncated and the Y488F and S503A site-directed mutants) did not increase receptor-mediated insulin internalization, suggesting that the effect of p120 on insulin endocytosis depends on its phosphorylation state. Immunofluorescence and biotin-labeling studies suggested that pp120 exerts its effect by undergoing receptor-mediated internalization in response to insulin. Thus, it appears that pp120 takes part in a complex of proteins that target the insulin receptor to endocytosis vesicles. The complex formation between pp120, at Tyr488, and the insulin receptor, at Tyr960 of its juxtamembrane domain, appears to be mediated by intracellular proteins. We herein propose to identify these proteins. Additionally, we propose to address the role of ppl20 in the mechanism of insulin action in vivo. To this end, we have generated a transgenic mouse overexpressing a phosphorylation-defective S503A isoform of ppl20 in liver. The transgenic line will address whether expression of a phosphorylation-defective pp 120 is associated with a blunted ability to remove excess insulin from the portal circulation, causing peripheral hyperinsulinemia. Since hyperinsulinemia leads to receptor down-regulation on target tissues, it is usually associated with insulin resistance. These proposed studies should provide novel insights into a potential mechanism of hyperinsulinemia, insulin resistance and diabetes.