PATHWAYS OF INSULIN AND IGFI RECEPTOR SIGNALING

近年来，在理解 具有内在酪氨酸的受体的分子机制 激酶活性介导其细胞效应。这些进步 主要来自细胞质蛋白的鉴定和克隆 其与活化的受体酪氨酸激酶（RTK）相互作用， 将信号从受体传递到细胞内部。的 胰岛素受体（IR）和胰岛素样生长因子I受体（IGFIR） 代表RTK的一个亚类，其介导多种效应， 包括调节细胞有丝分裂、代谢 和差异化。虽然一些特征良好的促有丝分裂 信号通路已被证明来自这些受体， 代谢和分化相关信号的性质较少 清楚我们必须充分了解 通过这些受体介导信号传导的途径。此类信息 将使人们能够深入了解II型糖尿病等疾病， 以胰岛素抵抗和某些肿瘤疾病为特征， IGFI也牵涉其中 在这份提案中，我们将提出以下基本问题 关于胰岛素和IGFI信号传导，（1）细胞内 靶点和信号通路负责介导不同的 IR和IGFIR在骨骼肌中的作用，胰岛素和IGFI- 反应组织？(2)信号蛋白是如何通过 结构相似IR和IGFIR调节这种不同的细胞效应 有丝分裂、代谢和分化（3）什么是 新的（非SH 2）磷酸酪氨酸依赖性 SHC和IRS-1与IR和IGFIR的相互作用， 识别？ 为了开始解决这些问题，我们开发了一种新颖的方法 用它可以快速鉴定和克隆蛋白质产物 与这些受体的胞质结构域直接相互作用。我们 已经成功地适应了蛋白质：蛋白质的"双杂交"测定 相互作用的酵母酿酒酵母，以证明和 表征IR和IGFIR与几种蛋白质的相互作用 已知参与胰岛素和IGFI信号传导，包括IRS-1，p85， 和SHC。该试验允许鉴定一种新的同源 SHC和IRS-1中的基序，其介导磷酸酪氨酸依赖的 与IR和IGFIR的NPEY基序相互作用。另外我们有 证明了双杂交试验在鉴定中的实用性 以及从cDNA文库中克隆已知的和新的相互作用蛋白。 这种新的方法应该能够更全面地了解 这些重要受体的基本分子机制 功能

Much progress has been made in recent years toward an understanding of the molecular mechanisms by which receptors with intrinsic tyrosine kinase activity mediate their cellular effects. These advances have come predominantly from the identification and cloning of cytoplasmic proteins which interact with the activated receptor tyrosine kinase (RTK) and transduce signals from the receptor to the interior of the cell. The insulin receptor (IR) and insulin-like growth factor I receptor (IGFIR) represent a subclass of RTKs which mediate a variety of effects in responsive tissues including regulation of cellular mitosis, metabolism and differentiation. Although a number of well-characterized mitogenic signaling pathways have been shown to emanate from these receptors, the nature of the metabolic and differentiation-related signaling is less clear. It is of great importance that we have a full understanding of the pathways which mediate signaling by these receptors. Such information will allow insight into diseases such as Type II diabetes which is characterized by insulin resistance and certain neoplastic diseases in which IGFI has been implicated. In this proposal we will ask the following fundamental questions regarding insulin and IGFI signaling, (1) What are the intracellular targets and signaling pathways responsible for mediating the diverse effects of the IR and IGFIR in skeletal muscle, an insulin- and IGFI- responsive tissue?, (2) How do signaling proteins acting through the structurally similar IR and IGFIR regulate such distinct cellular effects as mitogenesis, metabolism and differentiation?, and (3) What is the molecular nature of the novel (non-SH2) phosphotyrosine-dependent interaction of SHC and IRS-1 with the IR and IGFIR which we have identified? To begin to address these questions, we have developed a novel approach with which to rapidly identify and clone cDNAs whose protein products interact directly with the cytoplasmic domains of these receptors. We have successfully adapted the "two-hybrid" assay of protein:protein interaction in the yeast Saccharomyces cerevisiae to demonstrate and characterize the interaction of the IR and IGFIR with several proteins known to be involved in insulin and IGFI signaling including IRS-1, p85, and SHC. This assay has allowed the identification of a novel homologous motif within SHC and IRS-1 which mediates the phosphotyrosine-dependent interaction with the NPEY motif of the IR and IGFIR. In addition, we have demonstrated the utility of the two-hybrid assay in the identification and cloning of known and novel interacting proteins from a cDNA library. This new approach should allow a more complete understanding of the fundamental molecular mechanisms by which these important receptors function.