Molecular Biology of Insulin- Receptor Interactions

描述(申请人提供)：糖尿病在美国是一个主要的健康问题，II型疾病很快就会达到流行的程度。I型是由于绝对胰岛素缺乏，而II型是由于胰岛素作用受损。在任何一种情况下，了解胰岛素作用的初始阶段、受体结合和酪氨酸激酶激活的分子基础，对于开发改进的治疗这种疾病的药物都是至关重要的。然而，尽管进行了超过25年的密集研究，这一点仍然难以捉摸。因此，这项建议的长期目标是阐明胰岛素受体复合体形成的分子机制。大量证据表明，胰岛素和受体都有两个不同的结合部位。这导致了许多假想模型的形成，以解释胰岛素-受体相互作用的复杂性。本实验室使用系统突变分析的研究已经确定并表征了其中一个受体结合位点的功能表位。这一策略现在将被用来定位和表征另一个配体结合位点的功能表位，也将被用来评估所提出的胰岛素结合的假设模型。 拟议研究的具体目标是： 1)鉴定与第二受体配体结合部位有关的受体结构域和亚结构域； 2)丙氨酸扫描突变定位第二配体结合部位的功能表位； 3)评价胰岛素和类似物与功能表位决定簇的丙氨酸突变体的结合动力学； 4)评估丙氨酸受体突变对胰岛素结合负协同作用的影响；5)比较胰岛素受体A和B亚型第二配体结合位点的功能表位。

DESCRIPTION (provided by applicant): Diabetes mellitus is a major health problem in the U.S. and the Type II disease will soon reach epidemic proportions. Type I is due to absolute insulin deficiency, whereas Type II is due to impairment of insulin action. In either case an understanding of the molecular basis of the initial stages of insulin action, receptor binding and tyrosine kinase activation, will be essential for the development of improved agents for the treatment of the disease. However, despite more than 25 years intensive study, this remains elusive. Thus the longterm objective of this proposal is to elucidate the molecular mechanisms underlying the formation of the insulin-receptor complex. The weight of evidence suggests that both insulin and the receptor have two distinct binding sites. This has lead to the formulation of a number of hypothetical models to explain complexities of the insulin-receptor interaction. Studies in this laboratory using systematic, mutational analysis have identified and characterized the functional epitope of one of these receptor binding sites. This strategy will now be used to localize and characterize the functional epitope of the other ligand binding site and also to evaluate proposed hypothetical models of insulin binding. The Specific Aims of the proposed studies are: 1) Identification of receptor domains and sub-domains contributing to the second receptor ligand binding site; 2) Localization of the functional epitope of the second ligand binding site by alanine scanning mutagenesis; 3) Evaluation of the kinetics of insulin and analog binding to alanine mutants of determinants of the functional epitope; 4) Evaluation of the effects of alanine receptor mutations on negative cooperativity of insulin binding; and 5) Comparison of the functional epitopes of the second ligand binding sites of A and B isoforms of the insulin receptor.