Molecular mechanism of Norrin signaling through Frizzled4 and LRP5/6

Norrin 通过 Frizzled4 和 LRP5/6 信号传导的分子机制

基本信息

批准号：
10456949
负责人：
Elise Bruguera
金额：
$ 4.06万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-04 至 2023-08-03
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10456949
关键词：
Advanced Development Affect Affinity Age related macular degeneration Binding Binding Proteins Biochemical Biological Assay Blindness Blood Vessels Blood-Retinal Barrier Cell Differentiation process Cell Proliferation Cells Cellular biology Communication Complex Controlled Environment Coupled Coupling Cryoelectron Microscopy Data Development Diabetic Retinopathy Disease Dissection Electrons Environment Event Exposure to Foundations G-Protein-Coupled Receptors Genes Genetic Transcription Goals Growth Interferometry Intracellular Membranes Knowledge LDL-Receptor Related Protein 1 Ligand Binding Ligands Lipids Luciferases Maintenance Measures Mediating Membrane Membrane Proteins Microscopy Modeling Molecular Molecular Conformation Mutagenesis Mutation Natural regeneration Neural Retina Norrie&apos s disease Oncology Outcome Pathway interactions Process Protein Biochemistry Proteins Rare Diseases Receptor Activation Regenerative Medicine Reporter Reporter Genes Research Personnel Resolution Retina Retinal Neovascularization Role Signal Pathway Signal Transduction Structure System Testing Tissues Training Transcription Coactivator Transducers Vascularization Vision Work base beta catenin insight lipoprotein receptor related protein 5 member migration preservation receptor reconstitution recruit small molecule therapeutic development therapeutic target

项目摘要

Rationale: Aberrant retinal neovascularization and compromised blood-retina barrier (BRB) integrity causes vision loss in various diseases. Signaling by the secreted ligand Norrin directs development and maintenance of the BRB through activation of the Wnt/b-catenin pathway. In this pathway, Norrin acts through receptors Frizzled- 4 (Fzd4) and Low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to the intracellular membrane recruitment of the transducer Dishevelled (Dvl) and ultimately, the stabilization of transcriptional coactivator b-catenin for transcription of target genes. While this pathway has been studied in cellular and developmental models, a biochemical understanding of pathway activation, and specifically how receptors transmit the Norrin-binding signal across the membrane to recruit Dvl, constitutes a gap in knowledge. Objective: Using purified proteins, the proposed work will rigorously characterize molecular interactions within the ligand/receptor/coreceptor/transducer complex and determine the physical basis for Norrin-induced transducer recruitment. An allosteric model in which conformational changes within the Norrin/Fzd/LRP complex cause Fzd to recruit Dvl with high affinity will be tested. Approach: Aim 1 quantifies allosteric coupling between components of the Norrin/Fzd/LRP/Dvl complex. Binding assays will be conducted in a membrane environment to determine the extent to which Norrin and LRP influence Fzd recruitment of Dvl. Aim 2 identifies to structural basis of signaling in the Norrin/Fzd/LRP/Dvl complex. Cryo-electron microscopy (cryo-EM) structures of the Norrin/Fzd/LRP complex will be pursued with and without bound Dvl DEP domain. The functional relevance of structurally important residues will be tested in cell-based transcriptional reporter assays. Outcomes: These biochemical and structural studies will uncover the physical basis of the initiating events in Norrin/β-catenin signaling. Mechanistic insight will 1) provide a valuable framework from which to understand receptor activation, 2) reveal the physical basis of genetic defects affecting this pathway, and 3) inform strategies to specifically and rationally target this pathway in the retina. Additionally, these results will be translatable to the broader Wnt/b-catenin signaling pathway, contributing to studies of this developmental pathway as a target in regenerative medicine and oncology.

理由：异常的视网膜新生血管形成和血视网膜屏障 (BRB) 完整性受损各种疾病中的视力丧失由分泌配体 Norrin 发出的信号指导视力的发育和维持。通过激活 Wnt/b-连环蛋白途径来调节 BRB 在该途径中，Norrin 通过受体 Frizzled- 发挥作用。 4 (Fzd4) 和低密度脂蛋白受体相关蛋白 5 或 6 (LRP5/6)，导致细胞内 Disheveled (Dvl) 传感器的膜募集，最终稳定转录共激活剂 b-连环蛋白用于靶基因的转录，而该途径已在细胞和细胞中进行了研究。发育模型，对通路激活的生化理解，特别是受体如何跨膜传递 Norrin 结合信号以招募 Dvl，构成了知识空白。目的：使用纯化的蛋白质，拟议的工作将严格表征内部的分子相互作用配体/受体/共受体/转导复合物并确定 Norrin 诱导的物理基础 Norrin/Fzd/LRP 复合物内构象发生变化的变构模型。导致Fzd以高亲和力招募Dvl将被测试。方法：目标 1 量化 Norrin/Fzd/LRP/Dvl 复合物组分之间的变构耦合。结合测定将在膜环境中进行，以确定 Norrin 和 LRP 的结合程度影响 Dvl 的 Fzd 募集确定 Norrin/Fzd/LRP/Dvl 中信号传导的结构基础。 Norrin/Fzd/LRP 复合物的冷冻电子显微镜 (cryo-EM) 结构将被研究。且没有结合的Dvl DEP结构域。结构上重要的残基的功能相关性将在中进行测试。基于细胞的转录测定记者。结果：这些生化和结构研究将揭示引发事件的物理基础 Norrin/β-连环蛋白信号传导机制的见解将 1) 提供一个有价值的框架来理解。受体激活，2) 揭示影响该途径的遗传缺陷的物理基础，3) 提供策略信息此外，这些结果将可转化为专门且合理地针对视网膜中的这条通路。更广泛的 Wnt/b-catenin 信号通路，有助于将该发育通路作为靶点的研究再生医学和肿瘤学。