Structural and biochemical studies of the insulin and IGF1 receptors

胰岛素和胰岛素样生长因子-1(IGF1)是密切相关的多肽激素/生长因子 调节细胞生长和代谢。胰岛素和IGF1通过与靶细胞结合，发挥其生物学效应 不同的细胞表面受体，分别是胰岛素受体(IR)和IGF1受体(IGF1R)，它们是 结构上相关的受体酪氨酸激酶。IR和IGF1R的基础活性较低，这是受刺激的 通过与胞外结构域的配体结合，导致特定酪氨酸残基的自动磷酸化 胞质酪氨酸激酶结构域。IR和IGF1R的异常信号与人类疾病有关： 胰岛素抵抗信号减少在非胰岛素依赖型糖尿病中起作用，而胰岛素抵抗信号增加在糖尿病中起作用。 IGF1R和IR与癌症有关。尽管对分离物进行了广泛的结构和生化研究 这些受体的胞外结构域和胞质结构域，全面了解信号 IR和IGF1R的信号转导机制尚不清楚。为了解决这些问题，我们已经制定了高度 全长IR和IGF1R的纯化制剂，可溶于洗涤剂，具有功能性，并对其 配基。我们将使用生化方法和单分子荧光共振能量转移 (FRET)以了解酪氨酸激酶结构域在未连接、基础和 磷酸化的、激活的状态。这些结构-功能研究将与 IR的代谢功能和IGF1R在培养细胞中的促生长作用。我们还将测试 假设细胞内的类固醇和脂质成分通过IR和IGF1R影响跨膜信号。去做 为此，我们将利用新的方法来操纵质膜的组成 活细胞。最后，我们将研究IGF1R与E-钙粘附素的一个可溶片段之间的相互作用 从癌细胞中排出的物质。以前的工作表明，sEcad激活了培养的 在癌细胞和人类癌症样本中，我们已经证明这是由于直接的相互作用 在蛋白质之间。我们将确定IGF1R和sEAD上对此很重要的结合决定因素 互动。SECAD-IGF1R相互作用为治疗干预提供了一个新的靶点。这项工作 在这项拨款申请中提出的建议将增进我们对 IR和IGF1R的跨膜信号转导。这些信息应该被证明是有价值的设计 在细胞内作用以激活IR的小分子激动剂；这种激动剂将有潜在的抗IR作用 糖尿病治疗学。此外，从对激活机制的理解来看，有可能 开发IR/IGF1R抑制剂以阻断癌细胞中的受体信号。

Insulin and insulin-like growth factor-1 (IGF1) are closely related polypeptide hormones/growth factors that regulate cell growth and metabolism. Insulin and IGF1 exert their biological effects on target cells by binding to distinct cell surface receptors, the insulin receptor (IR) and the IGF1 receptor (IGF1R), respectively, which are structurally related receptor tyrosine kinases. IR and IGF1R have low basal activity, which is stimulated through ligand binding to the ectodomains, resulting in autophosphorylation of specific tyrosine residues in the cytoplasmic tyrosine kinase domains. Aberrant signaling by IR and IGF1R is relevant to human disease: decreased signaling of IR plays a role in non-insulin-dependent diabetes mellitus, while increased signaling of IGF1R and IR is implicated in cancer. Despite extensive structural and biochemical studies of the separate ectodomains and cytoplasmic domains of these receptors, a comprehensive understanding of the signal transduction mechanism for IR and IGF1R is still lacking. To address these issues, we have produced highly purified preparations of full-length IR and IGF1R that are detergent-soluble, functional, and responsive to their ligands. We will use biochemical approaches and single-molecule fluorescence resonance energy transfer (FRET) to understand the relative positioning of the tyrosine kinase domains in the unliganded, basal and phosphorylated, activated states. These structure-function studies will be correlated with studies of the metabolic functions of IR and the growth-promoting effects of IGF1R in cultured cells. We will also test the hypothesis that cellular sterol and lipid composition affects transmembrane signaling by IR and IGF1R. To do this, we will take advantage of new methodology to manipulate the composition of the plasma membrane in living cells. Finally, we will characterize the interaction between IGF1R and a soluble fragment of E-cadherin (sEcad) that is shed from cancer cells. Previous work showed that sEcad activates IGF1R signaling in cultured cancer cells and in human cancer specimens, and we have shown that this is due to a direct interaction between the proteins. We will identify binding determinants on IGF1R and on sEcad that are important for this interaction. The sEcad-IGF1R interaction represents a novel target for therapeutic intervention. The work proposed in this grant application will advance our knowledge of the molecular mechanisms involved in transmembrane signaling by IR and IGF1R. This information should prove to be valuable in the design of small-molecule agonists that act intracellularly to activate IR; such agonists would have potential use as anti- diabetic therapeutics. In addition, from an understanding of the activation mechanism, it might be possible to develop IR/IGF1R inhibitors to block receptor signaling in cancer cells.