INSULIN RECEPTOR/KINASE REGULATION IN THE INTACT CELLS

在培养的小鼠3 T3-L1细胞中，胰岛素对照 胰岛素受体/蛋白激酶的自磷酸化。 的 该反应的动力学和浓度依赖性表明， 是控制代谢的调节途径的一部分 对胰岛素的反应。 这些途径中的许多涉及其他 蛋白激酶和某种形式的信号放大。 放置 酶级联中胰岛素受体/蛋白激酶将 使其受到多层次的监管。 问题是 了解胰岛素受体蛋白的细胞调节 激酶活性 长期目标是改变 胰岛素受体/蛋白激酶， 已在体外定义的抑制剂。 体外动力学 用纯化的受体/激酶进行的实验应确定： 1.位点特异性自磷酸化对活化的影响 和底物选择;这需要纯化180 kDa的 来自3 T3-L1细胞的酸性底物。2.的机制 底物和非底物抑制和激活 多肽;这些多肽的化学和酶修饰 多肽将用于进一步改变它们的作用。3. 一个 用于鉴定细胞来源的调节剂的测定。 与受体/激酶原位自磷酸化和 随后纯化，将通过1. 肽图谱 磷酸化位点，和2. 测定基板 选择. 对于原位应用于受体/激酶，方法 将被开发以结合所选择的基底和/或 抑制剂进入细胞。 测量将由1。 “外源底物”的磷酸化，2. 受体/激酶的自磷酸化，和3.改变 生物反应。 拟议研究的结果可能直接适用于 2型糖尿病（一种“后受体缺陷”），以其他生长激素 受体/蛋白激酶，以及非转化或 细胞与病毒癌基因的转化能力。

In cultured mouse 3T3-L1 cells, insulin controls autophosphorylation of the insulin receptor/protein kinase. The kinetics and concentration dependence of this reaction suggest it is part of the regulatory pathways that control metabolic responses to insulin. Many of these pathways involve other protein kinases and some form of signal amplification. Placing the insulin receptor/protein kinase in an enzyme cascade would make it subject to multiple levels of regulation. The problem is to understand cellular regulation of insulin receptor-protein kinase activity. The long-term objective is to alter the in situ activity of the insulin receptor/protein kinase by using selective substrates and inhibitors that have been defined in vitro. In vitro kinetic experiments with purified receptor/kinase should establish: 1. The effect of site-specific autophosphorylation on activation and substrate selection; this requires purification of a 180 kDa acidic substrate from 3T3-L1 cells. 2. The mechanisms of inhibition and activation by substrate and nonsubstrate polypeptides; chemical and enzymic modifications of these polypeptides will be used to further alter their effects. 3. An assay for use in identifying regulators of cellular origin. Comparisons with receptor/kinase autophosphorylated in situ and subsequently purified, will be done by 1. peptide mapping of the phosphorylation sites, and 2. determination of substrate selection. For application to the receptor/kinase in situ, methods will be developed to incorporate selected substrates and/or inhibitors into the cells. Measurements will be made of 1. phosphorylation of the "exogenous substrate", 2. autophosphorylation of the receptor/kinase, and 3. altered biological responses. Results of the proposed studies may be directly applicable to Type 2 diabetes (a "post-receptor defect"), to other growth hormone receptor/protein kinases, and to the nontransforming or transforming abilities of cellular versus viral oncogenes.