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.

项目概要/摘要 受体酪氨酸激酶 (RTK) 被细胞外配体激活以转导大量信号， 控制细胞生长、增殖和存活。 RTK 激活的规范模型定义了以下角色： 配体作为二聚剂，使受体紧密结合以激活细胞内激酶结构域。 然而，许多 RTK 在没有配体的情况下形成二聚体，并且它们的激活依赖于适当的 质膜两侧结构域的关联。控制这种现象的分子机制 由于缺乏全长受体结构，变构效应仍然未知。本提案的主要目标 的目的是了解这些机制如何在人类表皮生长因子 (EGFR/HER) 家族中发挥作用 通过获得其高分辨率全长结构来识别受体。 HER 是独特的 RTK，因为与 其他 RTK，它们的激酶结构域不是通过反式磷酸化激活的，而是通过形成 不对称激酶二聚体，其中一个激酶结构域成为另一个激酶结构域的变构激活剂。通过 通过对这些受体部分的结构研究，我们和其他人已经证明，不对称激酶结构域 HER 激酶的模块与相邻的近膜和跨膜结构域的构象耦合， 这些反过来又受到细胞外结构域模块方向的影响。这些相对结构 另外还受到不同 HER 受体配体的调节，这些配体已被证明可引发不同的生物活性 结果。所有这些元素如何在细胞膜上聚集在一起尚不清楚。无法纯化稳定 HER 受体复合物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 用于表达和纯化几乎全长的重组 HER 受体并定期收集阴性染色 这些样品的 EM (NS-EM) 和冷冻电镜数据集。利用这条管道，我们将专注于获得高 全长 HER 受体非活性状态和配体结合活性状态的解析结构。我们将重点 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 配合物响应一系列配体。我们假设受体的组合能力 相互作用始于活性复合物形成的水平。使用冷冻电镜、X 射线晶体学、酶法 结构衍生模型的测量和基于细胞的测试，我们将重点回答以下问题 问题： 1.配体诱导的构象变化如何在受体中跨血浆传播 膜？ 2. 不同受体异二聚体的作用机制有何差异？ 3. 怎么样 通过不同配体和疾病突变微调的激活机制？虽然我们的学习将 专注于 HER 受体，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，从而为许多未来的发现铺平了道路。生物的 我们在此过程中获得的知识也将有助于创新疗法的开发 靶向人类疾病中选定的 HER 受体复合物。