INSULIN ACTION AND MUTANT INSULIN RECEPTORS

本申请中提出的研究将检验这些影响 人类胰岛素受体在胰岛素上的特定突变 行动。每一种突变产生各种胰岛素的能力 胰岛素靶细胞(脂肪细胞)的反应将是 通过细胞转染实验进行检测。以下是 胰岛素受体β亚基的突变将是 研究：(1)Phe取代Tyr-960，(2)删除 氨基酸序列，Ala-954至Ala-965，(3)43的缺失 β亚基COOH末端的氨基酸，开始 在ALA-1301，(4)Phe取代Tyr-1146，(5)取代 Phe取代Tyr-1316，(6)Phe取代Tyr-1322。 这些突变是根据先前的信息选择的 表明了REACH区域在信号生成中的作用 受体。目前，前三种突变可用于 研究，而最后三个将在赠款期间建造 句号。 包含突变的cdna构建物将被放置在 真核细胞表达载体。此向量将用于 转染小鼠前脂肪细胞成纤维细胞(3T3-1) F442a)和中国仓鼠胚胎(CHEF/18)细胞系。单元格 表达野生型和突变型人胰岛素受体 将被选择、克隆并用于胰岛素作用的研究，两者 在细胞被驱动分化为 脂肪细胞。 将检查的胰岛素反应包括激活 对葡萄糖的运输和代谢，降脂作用和 调节基因表达水平变化的能力 对于c-fos和c-myc。各种突变型胰岛素的能力 引发胰岛素这些作用的受体将与任何 受体或受体的自动磷酸化的变化 内源底物的磷酸化。 这些研究的结果应该会提供更多关于 胰岛素受体的结构特征 产生胰岛素反应。这些研究还将有助于 确定胰岛素是否调节不同的细胞过程 需要来自胰岛素受体的相同或不同的信号。 总体而言，在制定更有效的 糖尿病和其他已改变状态的管理计划 胰岛素的作用。

The studies proposed in this application will examine the effects of specific mutations in the human insulin receptor on insulin action. The ability of each mutation to evoke various insulin responses in an insulin-target cell (the adipocyte) will be examined by cell transfection experiments. The following mutations in the beta subunit of the insulin receptor will be studied: (1) Substitution of Phe for Tyr-960, (2) Deletion of the amino acid sequence, Ala-954 through Ala-965, (3) Deletion of 43 amino acids at the COOH terminus of the beta subunit, beginning at Ala-1301, (4) Substitution of Phe for Tyr-1146, (5) Substitution of Phe for Tyr-1316, (6) Substitution of Phe for Tyr-1322. These mutations were selected based on prior information that indicated a role for reach region in signal generation by the receptor. At present, the first three mutations are available for study, whereas the last three will be constructed during the grant period. The cDNA constructs that contain the mutations will be placed in an eukaroyte expression vector. This vector will be used to transfect preadipocyte fibroblasts from established mouse (3T3- F442A) and Chinese hampster embryo (CHEF/18) cell lines. Cells that express the wild-type and mutant human insulin receptors will be selected, cloned and used in studies of insulin action, both before and after the cells have been driven to differentiate to adipocytes. The insulin responses that will be examined include the activation of glucose transport and metabolism, the antilipolytic effect and the ability to mediate changes int he expression of mRNA levels for c-fos and c-myc. The ability of the various mutant insulin receptors to elicit these actions of insulin will be compared to any changes noted in the autophosphorylation of the receptors or phosphorylation of endogenous substrates. Results from these studies should provide more information of the structural features of the insulin receptor necessary for generating an insulin response. These studies will also help to determine if the diverse cellular processes regulated by insulin require the same or different signals from the insulin receptor. Overall, such information is needed in devising a more effective management program for diabetes and other states of altered insulin action.