Abstract
The molecular basis of Wnt-Frizzled specificity is a central question in developmental biology. Reck, a multi-domain and multi-functional glycosylphosphatidylinositol-anchored protein, specifically enhances beta-catenin signaling by Wnt7a and Wnt7b in cooperation with the 7-transmembrane protein Gpr124. Among amino acids that distinguish Wnt7a and Wnt7b from other Wnts, two clusters are essential for signaling in a Reck- and Gpr124-dependent manner. Both clusters are far from the site of Frizzled binding: one resides at the amino terminus and the second resides in a protruding loop. Within Reck, the fourth of five tandem repeats of an unusual domain with six-cysteines (the CC domain) is essential for Wnt7a stimulation: substitutions P256A and W261A in CC4 eliminate this activity without changing protein abundance or surface localization. Mouse embryos carrying ReckP256A,W261A have severe defects in forebrain angiogenesis, providing the strongest evidence to date that Reck promotes CNS angiogenesis by specifically stimulating Wnt7a and Wnt7b signaling.
Highlights
Vascularization of the brain and retina requires beta-catenin signaling in vascular endothelial cells (ECs) (Xu et al, 2004; Stenman et al, 2008; Daneman et al, 2009)
Fz4, Fz5, Fz6, and Fz8 cysteine-rich domain (CRD)-Myc-GPI proteins accumulate at the cell surface of living cells to approximately the same level, and they bind Wnt7a-1D4 with comparable efficiencies — as shown, respectively, by anti-Myc and anti-1D4 binding to intact cells — but only Fz5 and Fz8 CRDs confer high levels of Reck(CC1-5)-alkaline phosphatase (AP) binding (Figure 1B)
The Reck(CC1-5)-AP binding signals reflect a specificity for particular Fz CRD sequences rather than differences in the abundances of cell-surface Wnt7a1D4 or FzCRD-Myc-GPI
Summary
Vascularization of the brain and retina requires beta-catenin (i.e. canonical Wnt) signaling in vascular endothelial cells (ECs) (Xu et al, 2004; Stenman et al, 2008; Daneman et al, 2009). Later in development and in the mature CNS, beta-catenin signaling is required to generate and maintain the blood-brain barrier (BBB) and its retinal counterpart, the blood-retina barrier (BRB) (Liebner et al, 2008; Stenman et al, 2008; Daneman et al, 2009) In both of these contexts, ligands Wnt7a, Wnt7b, and/or Norrin are produced by CNS glia and/or neurons to activate Frizzled (Fz) receptors and Lrp5/6 co-receptors on ECs (reviewed in Sun and Smith, 2018; Wang et al, 2019). CRISPR/Cas9-mediated alanine substitutions at two critical amino acids in Reck cause a severe defect in CNS angiogenesis and likely represents a clean elimination of Wnt7a/7b stimulation without affecting the structure or function of other Reck domains
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