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发病机制的一个主要因素，了解胰岛素抵抗的机制对于寻找改善NIDDM易感个体胰岛素敏感性的新方法具有潜在的意义。胰岛素与其受体的结合激活受体的酪氨酸激酶，导致受体和其他底物的磷酸化，如肝细胞中的质膜糖蛋白ppl20。在没有胰岛素的情况下，pp120在细胞内结构域的Ser503上被camp依赖性激酶磷酸化，而这种磷酸化是胰岛素受体激酶在胰岛素反应中磷酸化Tyr488所必需的。ppl20在胰岛素作用中的作用尚不清楚。通过受体介导的内吞作用机制，Pp120在培养细胞中的表达与培养基中胰岛素清除率的增加相关，这表明Pp120在门静脉循环中清除胰岛素的过程中很重要。相比之下，磷酸化缺陷的pp120亚型（截断型和Y488F和S503A位点定向突变型）的表达并没有增加受体介导的胰岛素内化，这表明p120对胰岛素内噬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来响应胰岛素发挥其作用。因此，pp120似乎参与了一个蛋白复合物，该蛋白复合物将胰岛素受体靶向内吞囊泡。pp120 （Tyr488）和胰岛素受体（近膜结构域Tyr960）之间的复合物形成似乎是由细胞内蛋白介导的。我们在此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，我们在肝脏中培养了过表达ppl20磷酸化缺陷的S503A亚型的转基因小鼠。转基因品系将研究磷酸化缺陷pp120的表达是否与从门静脉循环中去除多余胰岛素的能力减弱有关，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受体下调，通常与胰岛素抵抗有关。这些拟议的研究应该为高胰岛素血症、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.