Molecular mechanism of Norrin signaling through Frizzled4 and LRP5/6

依据：异常视网膜新生血管和血视网膜屏障（BRB）完整性受损是导致 各种疾病的视力丧失。分泌型配体Norrin的信号传导指导细胞的发育和维持。 BRB通过激活Wnt/β-连环蛋白途径。在这条通路中，Norrin通过受体Frizzled起作用- 4（Fzd 4）和低密度脂蛋白受体相关蛋白5或6（LRP 5/6），导致细胞内 膜募集的转导Dishevelled（Dvl），并最终，稳定的转录 用于靶基因转录的辅激活因子β-连环蛋白。虽然这一途径已在细胞和 发育模型，对途径激活的生化理解，特别是受体如何 跨膜传递Norrin结合信号以募集Dvl，构成了知识上的空白。 目的：利用纯化的蛋白质，所提出的工作将严格表征分子间的相互作用， 配体/受体/辅助受体/转换器复合物，并确定Norrin诱导的物理基础 换能器募集。Norrin/Fzd/LRP复合物内构象变化的变构模型 将测试导致Fzd以高亲和力募集Dvl的方法。 方法：目的1定量Norrin/Fzd/LRP/Dvl复合物的组分之间的变构偶联。 将在膜环境中进行结合试验，以确定Norrin和LRP 影响Dvl.目的2鉴定Norrin/Fzd/LRP/Dvl中信号传导的结构基础 复杂. Norrin/Fzd/LRP复合物的冷冻电子显微镜（cryo-EM）结构将采用 并且没有绑定的Dvl DEP域。结构上重要的残基的功能相关性将在 基于细胞的转录报告基因测定。 结果：这些生物化学和结构研究将揭示引发事件的物理基础， Norrin/β-catenin信号传导。机械论的洞察力将1）提供一个有价值的框架， 受体激活，2）揭示影响该途径的遗传缺陷的物理基础，3）告知策略 以视网膜中的这条通路为目标。此外，这些结果将被翻译为 更广泛的Wnt/b-连环蛋白信号通路，有助于研究这种发育途径作为靶点， 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.