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Structural and biochemical studies of the insulin and IGF1 receptors

胰岛素和 IGF1 受体的结构和生化研究

基本信息

批准号：
10477232
负责人：
W Todd MILLER
金额：
--
依托单位：
NORTHPORT VA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-01-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10477232
关键词：
Address Adhesions Affect Affinity Agonist Amino Acid Sequence Antidiabetic Drugs Applications Grants Binding Biochemical Biological Cancer Model Cell Surface Receptors Cell membrane Cells Cellular Metabolic Process Cholesterol Collaborations Crystallization Cultured Cells Cyclodextrins Cytoplasmic Tail Dependence Detergents Diabetes Mellitus Dimerization Disease Disulfides Down-Regulation E-Cadherin ERBB2 gene Epitopes Extracellular Domain Family Fluorescence Growth Hepatocyte Homeostasis Hormone Receptor Hormones Human IGF1 gene In Vitro Individual Insulin Insulin Receptor Insulin-Like Growth Factor I Insulin-Like-Growth Factor I Receptor Kinetics Knowledge Laboratories Length Ligand Binding Ligands Link Lipids Malignant Neoplasms Mammalian Cell Measurement Membrane Membrane Lipids Methodology Molecular Molecular Conformation Monitor Mus Mutagenesis Non-Insulin-Dependent Diabetes Mellitus Phospholipids Phosphorylation Phosphotransferases Photoaffinity Labels Plasma Exchange Play Polypeptide Hormones Positioning Attribute Preparation Prognosis Protein Tyrosine Kinase Proteins Receptor Activation Receptor Protein-Tyrosine Kinases Receptor Signaling Regulation Resistance Role Signal Transduction Site Skin Cancer Specimen Sphingolipids Sterols Structure System Techniques Testing Therapeutic Therapeutic Intervention Transmembrane Domain Tyrosine Tyrosine Kinase Domain Western Blotting Work cancer cell cancer invasiveness cancer therapy cell growth cell type design dimer experimental study growth hormone regulating factor human disease inhibitor insight insulin regulation insulin signaling malignant breast neoplasm member metabolic abnormality assessment migration new therapeutic target novel novel therapeutics receptor receptor function receptor structure function single-molecule FRET small molecule tumor unilamellar vesicle

项目摘要

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.

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，调节细胞生长和新陈代谢。胰岛素和 IGF1 通过结合到靶细胞上发挥其生物学作用不同的细胞表面受体分别为胰岛素受体 (IR) 和 IGF1 受体 (IGF1R) 结构相关的受体酪氨酸激酶。 IR和IGF1R基础活性低，受到刺激通过配体与胞外域结合，导致特定酪氨酸残基的自磷酸化细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 信号传导减少在非胰岛素依赖型糖尿病中发挥作用，而 IR 信号传导增加在非胰岛素依赖型糖尿病中发挥作用。 IGF1R 和 IR 与癌症有关。尽管对单独的结构和生化进行了广泛的研究这些受体的胞外域和胞质域，全面了解信号 IR和IGF1R的转导机制仍然缺乏。为了解决这些问题，我们制作了高度全长 IR 和 IGF1R 的纯化制剂，可溶于洗涤剂、具有功能性且对其响应敏感配体。我们将使用生化方法和单分子荧光共振能量转移 (FRET) 了解酪氨酸激酶结构域在未配体、基础和磷酸化、激活状态。这些结构-功能研究将与以下研究相关： IR 的代谢功能和 IGF1R 在培养细胞中的生长促进作用。我们还将测试假设细胞甾醇和脂质成分影响 IR 和 IGF1R 的跨膜信号传导。要做的事为此，我们将利用新的方法来操纵质膜的组成活细胞。最后，我们将表征 IGF1R 和 E-钙粘蛋白可溶性片段之间的相互作用 (sEcad) 从癌细胞中脱落。先前的研究表明 sEcad 在培养物中激活 IGF1R 信号传导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蛋白质之间。我们将确定 IGF1R 和 sEcad 上对此很重要的结合决定簇相互作用。 sEcad-IGF1R 相互作用代表了治疗干预的新靶点。工作本拨款申请中提出的建议将增进我们对涉及的分子机制的了解 IR 和 IGF1R 的跨膜信号传导。这些信息在设计中应该被证明是有价值的在细胞内发挥作用以激活 IR 的小分子激动剂；此类激动剂有可能用作抗糖尿病治疗。此外，根据对激活机制的理解，也许可以开发 IR/IGF1R 抑制剂来阻断癌细胞中的受体信号传导。