Molecular mechanism of Norrin signaling through Frizzled4 and LRP5/6

理论基础：视网膜新生血管异常和血视网膜屏障(BRB)完整性受损的原因 各种疾病导致的视力丧失。由分泌配体Norrin发出的信号指导细胞的发育和维持 通过激活Wnt/b-catenin途径激活BRB。在这一途径中，Norrin通过Frizzled型受体发挥作用。 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 转导分子的膜招募紊乱(DVL)，最终，转录的稳定 目的基因转录的辅活化子b-连环蛋白。虽然这一途径已经在细胞和 发育模型，对途径激活的生化理解，特别是受体如何 通过跨膜传递Norrin结合信号来招募DVL，构成了知识上的鸿沟。 目的：使用纯化的蛋白质，这项拟议的工作将严格表征 配体/受体/辅受体/转导复合体及确定去甲肾上腺素诱导的物理基础 换能器招募。Norrin/Fzd/LRp复合体中构象变化的变构模型 使FZD招募具有高亲和力的DVL将受到考验。 方法：目标1定量Norrin/Fzd/LRP/DVL复合体各组分之间的变构偶联。 结合分析将在膜环境中进行，以确定Norrin和LRP在多大程度上 影响DVL的FZD招募。目标2确定Norrin/FZD/LRP/DVL中信号的结构基础 很复杂。将用低温电子显微镜(Cryo-EM)研究Norrin/FZD/LRP复合体的结构 并且没有结合的DVL DEP结构域。结构上重要残基的功能相关性将在 基于细胞的转录报告分析。 结果：这些生化和结构研究将揭示 Norrin/β-连环蛋白信号转导。机械论的洞察力将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)通知策略 在视网膜中特定和合理地靶向这条通路。此外，这些结果将可翻译为 更广泛的Wnt/b-catenin信号通路，有助于将这一发育途径作为研究的靶点 再生医学和肿瘤学。

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.