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）的标志。 由于胰岛素抵抗是NIDDM发病机理的主要因素，因此了解胰岛素抵抗的机制在识别新型手段以提高易感NIDDM的个体的胰岛素敏感性方面具有潜在的影响。 胰岛素与其受体结合激活受体的酪氨酸激酶，引起受体和其他底物的磷酸化，例如PPL20，肝细胞中的质膜糖蛋白。在没有胰岛素的情况下，通过cAMP依赖性激酶在Ser503上磷酸化的PP120，并且这种磷酸化是胰岛素响应于胰岛素的胰岛素受体激酶在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PP120的表达与受体介导的内吞作用机理的胰岛素清除率增加相关，这表明PP120在门户循环中胰岛素清除率的过程中很重要。相反，磷酸化缺陷PP120同工型的表达（截短和Y488F和S503A站点定向的突变体）没有增加受体介导的胰岛素内在化，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的研究表明，PP120通过响应胰岛素的受体介导的内在化来发挥其作用。 因此，看来PP120参与了靶向胰岛素受体的蛋白质复合物，以促胰岛素受体到内吞作用囊泡。 PP120，Tyr488和胰岛素受体之间的复合物形成，在其近去膜结构域的Tyr960上似乎是由细胞内蛋白介导的。 我们在这里建议识别这些蛋白质。此外，我们建议解决PPL20在体内胰岛素作用机理中的作用。 为此，我们产生了一种过表达肝脏中PPL20磷酸化缺陷的S503A同工型的转基因小鼠。 转基因线将解决磷酸化缺陷PP 120的表达是否与从门户循环中去除过量胰岛素的钝能力有关，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的受体下调，因此通常与胰岛素抵抗有关。 这些提出的研究应提供有关高胰岛素血症，胰岛素抵抗和糖尿病的潜在机制的新见解。