Structural and Functional Studies of HER Receptors

HER 受体的结构和功能研究

• 批准号: 10523114
• 负责人: Natalia Jura
• 金额: $ 40.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10523114
• 关键词: Affect; Biological; Cell membrane; Cells; Complex; Coupled; Cryoelectron Microscopy; Data Set; Development; Dimerization; Disease; ERBB2 gene; ERBB3 gene; Elements; Epidermal Growth Factor Receptor; ErbB4 gene; Extracellular Domain; Family; Future; Goals; Growth Factor; Human; Human Genome; Knowledge; Length; Ligand Binding; Ligands; Malignant Neoplasms; Measurement; Membrane; Modeling; Molecular; Molecular Conformation; Motion; Mutation; Negative Staining; Outcome; Phosphorylation; Phosphotransferases; Process; Proliferating; Proteins; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Recombinants; Resolution; Role; Sampling; Series; Side; Signal Transduction; Structure; System; Testing; Transmembrane Domain; Work; X-Ray Crystallography; cell growth; combat; combinatorial; dimer; extracellular; human disease; innovation; member; receptor; response; therapeutic target

PROJECT SUMMARY/ABSTRACT Receptor tyrosine kinases (RTKs) are activated by extracellular ligands to transduce the bulk of the signals that control cellular growth, proliferation and survival. The canonical model of RTK activation defines the role of ligands as dimerizing agents that bring receptors into close proximity to activate the intracellular kinase domains. However, many RTKs form dimers in the absence of ligands and their activation is dependent on the proper association of domains on both sides of the plasma membrane. The molecular mechanisms governing such allosteric effects remain unknown due to the lack of full-length receptor structures. The main goal of this proposal is to understand how these mechanisms operate in the family of human epidermal growth factor (EGFR/HER) receptors by obtaining their high-resolution full-length structures. HERs are unique RTKs because in contrast to other RTKs, their kinase domains are not activated by trans-phosphorylation but by the formation of an asymmetric kinase dimer in which one kinase domain becomes an allosteric activator of another. Through structural work on portions of these receptors, we and others have shown that the asymmetric kinase domain module of HER kinases is coupled to conformation of the adjacent juxtamembrane and transmembrane domains, and those in turn are affected by the orientation of the extracellular domain modules. These relative structures are additionally modulated by different HER receptor ligands which have been shown to elicit different biological outcomes. How all these elements come together at the cell membrane is unknown. The inability to purify stable complexes of HER receptors has impeded full-length structural studies. We have now developed a robust system for expressing and purifying recombinant, nearly full-length HER receptors and routinely collect negative stain EM (NS-EM) and cryo-EM data sets on these samples. Using this pipeline, we will focus on obtaining high resolution structures of full length HER receptors in their inactive and ligand-bound active states. We will focus on three members of the HER receptor family: HER2, HER3 and HER4 which engage in a range of heterodimeric complexes in response to a spectrum of ligands. We hypothesize that the combinatorial power of receptor interactions starts at the level of active complex formation. Using cryo-EM, X-ray crystallography, enzymatic measurements and cell-based testing of structurally-derived models, we will focus on answering the following questions: 1. How do ligand-induced conformational changes propagate in the receptor across the plasma membrane? 2. What are the differences in mechanism between different receptor heterodimers? 3. How is the mechanism of activation fine-tuned by different ligands and by disease mutations? While our studies will be focused on HER receptors, the developed innovative experimental approaches will be applicable to the entire RTK family and other single-pass proteins, thus paving the way for many future discoveries. The biological knowledge we will acquire in the process will also contribute to the development of innovative therapeutics that target selected HER receptor complexes in human diseases.

项目摘要/摘要 受体酪氨酸激酶(RTK)由细胞外配体激活，转导大部分信号。 控制细胞的生长、增殖和存活。RTK激活的规范模型定义了 配体作为二聚体，使受体接近，激活细胞内的激酶域。 然而，许多RTK在没有配体的情况下形成二聚体，它们的激活依赖于适当的 质膜两侧结构域的联结。控制这一现象的分子机制 由于缺乏全长受体结构，变构效应仍不清楚。这项提案的主要目标是 是为了了解这些机制是如何在人表皮生长因子(EGFR/HER)家族中发挥作用的 通过获得高分辨率的全长结构来获得受体。她的RTK是独一无二的，因为与 其他RTK的激活域不是通过反式磷酸化激活的，而是通过形成一个 不对称的激酶二聚体，其中一个激活域成为另一个的变构激活剂。穿过 对这些受体部分的结构研究，我们和其他人已经证明了不对称激酶域 她的激酶模块与相邻的膜旁和跨膜结构域的构象相连， 而这些又受到细胞外域模块的取向的影响。这些相对结构 另外还受到不同的HER受体配体的调节，这些配体已经被证明可以引起不同的生物学效应 结果。所有这些元素是如何在细胞膜上聚集在一起的还不得而知。不能净化稳定 她的受体复合体阻碍了全长结构研究。我们现在已经开发了一个强大的系统 用于表达和纯化重组的、几乎全长的HER受体，并常规收集阴性染色 EM(NS-EM)和低温EM数据集。利用这条管道，我们将专注于获得高 全长HER受体处于非活性和配体结合活性状态的分辨结构。我们将专注于 关于HER受体家族的三个成员：HER2，HER3和HER4，它们参与一系列异二聚体 络合物对一系列配体的反应。我们假设受体的组合能力 相互作用始于活性络合物的形成水平。使用低温电子显微镜，X射线结晶学，酶促 结构派生模型的测量和基于单元的测试，我们将重点回答以下问题 问题：1.配体诱导的构象变化是如何通过血浆在受体中传播的 膜？2.不同受体异源二聚体的作用机制有何不同？ 不同配体和疾病突变微调激活的机制？而我们的学习将会是 以她的受体为中心，开发的创新实验方法将适用于整个 RTK家族和其他单程蛋白，从而为未来的许多发现铺平了道路。生物学的 我们在这一过程中获得的知识也将有助于创新疗法的发展， 靶点选择了人类疾病中的HER受体复合体。