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: 10350608
• 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/10350608
• 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; 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; Problem Solving; 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; base; 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)是一类细胞表面受体。两者都是胰岛素 受体(IR)和1型胰岛素样生长因子受体(IGF1R)是发挥作用的II型RTK 在控制葡萄糖代谢、细胞生长、增殖、分化和 迁移。IR或IGF1R信号异常会导致许多人类疾病，包括 糖尿病和癌症，这使它们成为有希望的治疗目标。尽管功能强大 与疾病的重要性和密切联系，确切的配体诱导激活机制 由于缺乏全长的高分辨率结构，IR和IGF1R的结构尚不清楚 受体/配体复合体。该项目的目标是揭示配体(胰岛素和IGF1， 分别与这两种不同类型的受体结合，以及配体结合如何导致 受体激活。尽管IR和IGF1R都有很高的序列同源性和结构 相似性，它们已被证明在配体结合性质上表现出很大的多样性， 这表明了不同的激活机制。始终如一地，我们的初步结构工作 结果表明，完全连接的IR二聚体与4个胰岛素分子在两种不同类型的胰岛素上结合 位点-众所周知的位点1和我们新发现的位点2；而只有一个IGF1分子 结合不对称的IGF1R二聚体，意味着IGF1与IGF1R的结合涉及负性 协作性。我们将结合低温电子显微镜(Cryo-EM)、生物物理、生化和 基于细胞的方法了解胰岛素结合位点1和2在IR激活中的作用 解释IGF1与IGF1R结合中负协同性的来源，揭示IGF1与IGF1R结合的原因 IR和IGF1R跨膜区在受体激活中的作用目标1将是 重点对活性状态下的全长IR进行了功能和结构分析。这些研究 将揭示胰岛素与新的IR位点2的详细结合方式，并鉴定 每种类型的胰岛素结合的关键作用。此外，我们将测试IRS是否与不同的 结合的胰岛素数量表现出不同水平的活性，并触发不同的下游 发信号。目标2将重点放在全长IGF1R的生化和结构分析上 活动状态。我们将确定负合作约束的结构要求 IGF1到IGF1R，并解释了负合作对IGF1R的功能重要性 激活。目标3将专注于TM-TM相互作用的直接可视化 IR和IGF1R的状态解释为什么TM在IR和IGF1R的活性状态下二聚化 对受体的激活很重要。