INSULIN RECEPTOR/KINASE REGULATION IN THE INTACT CELLS

在培养的小鼠3T3-L1细胞中，胰岛素对照 胰岛素受体/蛋白激酶的自动磷酸化。这个 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到其他 蛋白激酶和某种形式的信号放大。放置 胰岛素受体/蛋白激酶在酶的级联反应中 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控 激活酶活性。 长期目标是改变细胞的原位活动。 胰岛素受体/蛋白激酶选择性底物和 已经在体外定义的抑制剂。体外动力学 用纯化的受体/激酶进行的实验应该确定： 1.位点特异性自磷酸化对激活的影响 和底物选择；这需要纯化180 kDa 3T3-L1细胞的酸性底物。2.药物的作用机制 底物和非底物的抑制和激活 多肽；这些多肽的化学和酶修饰 多肽将被用来进一步改变它们的效果。3.安邦 用于鉴定细胞来源的调节因子的化验。 受体/激酶原位自磷酸化与原位磷酸化的比较 随后进行纯化，将通过1.肽图谱的 磷酸化位点；2.底物测定 选择。为了应用于原位受体/激酶，方法 将被开发成结合选定的衬底和/或 抑制剂进入细胞。测量值将为1。 “外源底物”的磷酸化； 受体/激酶的自动磷酸化，以及3.改变 生物反应。 建议的研究结果可能直接适用于类型 糖尿病(一种“受体后缺陷”)，与其他生长激素有关 受体/蛋白激酶，以及对非转化或 细胞癌基因对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.