Molecular mechanism of Norrin signaling through Frizzled4 and LRP5/6

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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.