Structural and Functional Analyses of the Full-length Insulin Receptor (IR) and Type 1 Insulin-like Growth Factor Receptor (IGF1R) in the Liganded Active State

配体活性状态下全长胰岛素受体 (IR) 和 1 型胰岛素样生长因子受体 (IGF1R) 的结构和功能分析

• 批准号: 10574524
• 负责人: Xiaochen Bai
• 金额: $ 35.26万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-15 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10574524
• 关键词: Adopted; Affinity; Binding; Binding Sites; Biochemical; Biological Assay; Biophysics; Cell Surface Receptors; Cell membrane; Cellular Assay; Classification; Complex; Cryoelectron Microscopy; Data; Diabetes Mellitus; Dimerization; Disease; Elements; Environment; Exhibits; Extracellular Domain; Family; Goals; Growth Factor Receptors; IGF1 gene; IGF2 gene; In Vitro; Insulin; Insulin Receptor; Insulin-Like Growth Factor Receptor; Insulin-Like-Growth Factor I Receptor; Kinetics; Lead; Length; Ligand Binding; Ligands; Link; Lipid Bilayers; Lipids; Malignant Neoplasms; Maps; Methods; Modeling; Molecular Conformation; Mutagenesis; Mutation Analysis; Outcome; Phosphorylation; Play; Positioning Attribute; Proliferating; Property; Protein Family; Proteins; Protomer; Receptor Activation; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Resolution; Role; Series; Signal Transduction; Site; Source; Specificity; Structural Models; Structure; Techniques; Testing; Transmembrane Domain; Visualization; Work; blood glucose regulation; cell growth; design; dimer; disulfide bond; flexibility; glucose metabolism; human disease; improved; migration; mutant; novel; receptor; reconstitution; response; therapeutic target; Adopted; Affinity; Binding; Binding Sites; Biochemical; Biological Assay; Biophysics; Cell Surface Receptors; Cell membrane; Cellular Assay; Classification; Complex; Cryoelectron Microscopy; Data; Diabetes Mellitus; Dimerization; Disease; Elements; Environment; Exhibits; Extracellular Domain; Family; Goals; Growth Factor Receptors; IGF1 gene; IGF2 gene; In Vitro; Insulin; Insulin Receptor; Insulin-Like Growth Factor Receptor; Insulin-Like-Growth Factor I Receptor; Kinetics; Lead; Length; Ligand Binding; Ligands; Link; Lipid Bilayers; Lipids; Malignant Neoplasms; Maps; Methods; Modeling; Molecular Conformation; Mutagenesis; Mutation Analysis; Outcome; Phosphorylation; Play; Positioning Attribute; Proliferating; Property; Protein Family; Proteins; Protomer; Receptor Activation; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Resolution; Role; Series; Signal Transduction; Site; Source; Specificity; Structural Models; Structure; Techniques; Testing; Transmembrane Domain; Visualization; Work; blood glucose regulation; cell growth; design; dimer; disulfide bond; flexibility; glucose metabolism; human disease; improved; migration; mutant; novel; receptor; reconstitution; response; therapeutic target

Receptor tyrosine kinases (RTKs) represent a family of cell surface receptors. Both of insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) are type II RTKs that play essential roles in controlling glucose metabolism, cellular growth, proliferation, differentiation, and migration. Aberrant IR or IGF1R signaling causes a number of human diseases, including diabetes and cancers, which makes them promising therapeutic targets. Despite the functional importance and strong connections with diseases, the exact ligand induced activation mechanism of IR and IGF1R are still unknown, due to the lack of high resolution structures of full-length receptors/ligands complex. The goal of this project is to reveal how ligands (insulin and IGF1, respectively) binds to these two different types of receptors and how ligand binding leads to the receptor activation. Although both the IR and IGF1R share high sequence identity and structural similarity, they have been shown to exhibit great diversification in the ligand binding properties, which is indicative of diverse activation mechanism. Consistently, our preliminary structural work showed that the fully liganded IR dimer is bound by 4 insulin molecules at two distinct types of sites—the well-known site 1 and our newly discovered site 2; whereas, only one IGF1 molecule binds the asymmetric IGF1R dimer, meaning that the binding of IGF1 to IGF1R involves negative cooperativity. We will combine cryo-electron microscopy (cryo-EM), biophysical, biochemical and cellular based approaches to understand the roles of site 1 and 2 insulin binding for IR activation, explain the origin of the negative cooperativity in the binding of IGF1 to IGF1R, and reveal the function of transmembrane domains (TM) of IR and IGF1R in receptor activation. Aim 1 will be focused on the functional and structural analyses of full-length IR in the active state. These studies will reveal the detailed binding mode between insulin and the novel IR site 2, and identify the critical role of each type of insulin binding. In addition, we will test whether IRs with different number of insulins bound exhibit different levels of activities and trigger distinct downstream signaling. Aim 2 will be focused on biochemical and structural analyses of full-length IGF1R in the active state. We will identify the structural requirements for the negative cooperative binding of IGF1 to IGF1R, and explain the functional importance of the negative cooperative for IGF1R activation. Aim 3 will be focused on the direct visualization of the TM-TM interaction in the active state of IR and IGF1R to understand why the TM dimerization in the active state of IR and IGF1R is important for receptor activation.

受体酪氨酸激酶（RTK）代表细胞表面受体家族。两种胰岛素 受体（IR）和1型胰岛素样生长因子受体（IGF1R）是播放的II型RTK 控制葡萄糖代谢，细胞生长，增殖，分化和 迁移。异常IR或IGF1R信号传导引起许多人类疾病，包括 糖尿病和癌症，使它们成为有希望的治疗靶标。尽管有功能 重要性和与疾病的牢固联系，确切的配体诱导活化机制 由于缺乏全长的高分辨率结构，IR和IGF1R仍然未知 接收器/配体综合体。该项目的目的是揭示配体（胰岛素和IGF1， （分别）与这两种不同类型的接收器结合以及配体结合如何导致 受体激活。尽管IR和IGF1R都具有高序列身份和结构 相似性，它们已被证明在配体结合特性中表现出巨大的多样化， 这表明潜水员激活机制。一贯，我们的初步结构工作 表明完全配体的IR二聚体在两种不同类型的类型的4个胰岛素分子结合 网站 - 著名的站点1和我们新发现的网站2；而只有一个IGF1分子 结合不对称的IGF1R二聚体，这意味着IGF1与IGF1R的结合涉及负 合作。我们将结合冷冻电子显微镜（Cryo-EM），生物物理，生化和 基于细胞的方法，以了解位点1和2胰岛素结合在IR激活中的作用， 解释IGF1与IGF1R的结合中负协调的起源，并揭示 IR和IGF1R的跨膜结构域（TM）在受体激活中的功能。目标1将是 专注于活性状态的全长IR的功能和结构分析。这些研究 将揭示胰岛素和新型IR位点2之间的详细结合模式，并确定 每种类型的胰岛素结合的关键作用。此外，我们将测试IRS是否不同 胰岛素的数量表现出不同的活动水平，并触发下游独特的活动 信号。 AIM 2将集中于全长IGF1R的生化和结构分析 活跃状态。我们将确定负面合作结合的结构要求 IGF1到IGF1R，并解释负合作对IGF1R的功能重要性 激活。 AIM 3将集中于活动中TM-TM相互作用的直接可视化 IR和IGF1R状态以了解为什么在IR和IGF1R的活动状态中的TM二聚化 对于受体激活很重要。