SUBSTRATES AND INSULIN RECEPTOR ENDOCYTOSIS

底物和胰岛素受体胞吞作用

• 批准号: 6042645
• 负责人: Sonia M. Najjar
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6042645
• 关键词: DNA replication; autosomal dominant trait; clathrin; disease /disorder model; endocytosis; genetically modified animals; glycoproteins; hormone regulation /control mechanism; hyperinsulinism; insulin; insulin receptor; insulin sensitivity /resistance; intermolecular interaction; laboratory mouse; liver; membrane proteins; mutant; noninsulin dependent diabetes mellitus; phosphomonoesterases; phosphoproteins; protein sequence

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.

胰岛素作用受损或胰岛素抵抗是非胰岛素依赖性糖尿病 (NIDDM) 的一个标志。 由于胰岛素抵抗是 NIDDM 发病机制的一个主要因素，因此了解胰岛素抵抗机制对于确定改善易患 NIDDM 个体胰岛素敏感性的新方法具有潜在意义。 胰岛素与其受体结合会激活受体的酪氨酸激酶，从而引起受体和其他底物（例如肝细胞中的质膜糖蛋白ppl20）的磷酸化。在没有胰岛素的情况下，pp120 在细胞内结构域中的 Ser503 上被 cAMP 依赖性激酶磷酸化，并且这种磷酸化是胰岛素受体激酶响应胰岛素而在 Tyr488 上磷酸化所必需的。 ppl20 在胰岛素作用中的作用尚不清楚。 培养细胞中的 pp120 表达通过受体介导的内吞作用机制与培养基中胰岛素清除率的增加相关，表明 pp120 在门静脉循环胰岛素清除过程中很重要。相反，磷酸化缺陷型 pp120 亚型（截短的以及 Y488F 和 S503A 定点突变体）的表达不会增加受体介导的胰岛素内化，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 通过响应胰岛素而进行受体介导的内化来发挥其作用。 因此，pp120 似乎参与了将胰岛素受体靶向内吞囊泡的蛋白质复合物。 pp120（位于 Tyr488）和胰岛素受体（位于其近膜结构域的 Tyr960）之间的复合物形成似乎是由细胞内蛋白质介导的。 我们在此建议鉴定这些蛋白质。此外，我们建议探讨 ppl20 在体内胰岛素作用机制中的作用。 为此，我们培育了一种在肝脏中过表达磷酸化缺陷型 ppl20 S503A 亚型的转基因小鼠。 该转基因系将解决磷酸化缺陷型 pp 120 的表达是否与从门静脉循环中去除过量胰岛素的能力减弱有关，从而导致外周高胰岛素血症。 由于高胰岛素血症会导致靶组织上的受体下调，因此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。