Structural and biochemical studies of the insulin and IGF1 receptors

胰岛素和胰岛素样生长因子-1（IGF 1）是密切相关的多肽激素/生长因子， 调节细胞生长和新陈代谢。胰岛素和IGF1通过结合靶细胞发挥其生物学作用， 不同的细胞表面受体，胰岛素受体（IR）和IGF1受体（IGF1R），分别是 结构上相关的受体酪氨酸激酶。IR和IGF1R具有低的基础活性， 通过配体与胞外域结合，导致细胞内特定酪氨酸残基的自磷酸化， 胞质酪氨酸激酶结构域。IR和IGF1R的异常信号传导与人类疾病相关： IR信号的减少在非胰岛素依赖型糖尿病中起作用，而IR信号的增加在非胰岛素依赖型糖尿病中起作用。 IGF1R和IR与癌症有关。尽管有大量的结构和生物化学研究， 这些受体的胞外域和胞质结构域，信号的全面理解 IR和IGF1R的转导机制尚不清楚。为了解决这些问题，我们制定了高度 全长IR和IGF1R的纯化制剂，其是洗涤剂可溶的、功能性的并且对其响应。 配体。我们将使用生物化学方法和单分子荧光共振能量转移 （FRET）来理解酪氨酸激酶结构域在未配体的、基底的和 磷酸化活化状态这些结构-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 假设细胞固醇和脂质组成影响IR和IGF1R的跨膜信号传导。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，我们将描述IGF1R和E-钙粘蛋白可溶性片段之间的相互作用 （sEcad）从癌细胞脱落。以前的研究表明，sEcad激活IGF1R信号转导在培养的细胞中， 在癌细胞和人类癌症标本中，我们已经证明这是由于直接相互作用， 在蛋白质之间。我们将确定IGF1R和sEcad上的结合决定簇，这些决定簇对此很重要。 互动sEcad-IGF1R相互作用代表了治疗干预的新靶点。工作 在这项拨款申请中提出的将推进我们对参与的分子机制的认识， 通过IR和IGF1R的跨膜信号传导。这一信息应证明是有价值的设计 小分子激动剂，其在细胞内起作用以激活IR;这样的激动剂将具有作为抗IR的潜在用途。 糖尿病治疗学此外，通过对激活机制的理解， 开发IR/IGF1R抑制剂以阻断癌细胞中的受体信号传导。

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.