Abstract
Beta-catenin (i.e., canonical Wnt) signaling controls CNS angiogenesis and the blood-brain and blood-retina barriers. To explore the role of the Discs large/membrane-associated guanylate kinase (Dlg/MAGUK) family of scaffolding proteins in beta-catenin signaling, we studied vascular endothelial cell (EC)-specific knockout of Dlg1/SAP97. EC-specific loss of Dlg1 produces a retinal vascular phenotype that closely matches the phenotype associated with reduced beta-catenin signaling, synergizes with genetically-directed reductions in beta-catenin signaling components, and can be rescued by stabilizing beta-catenin in ECs. In reporter cells with CRISPR/Cas9-mediated inactivation of Dlg1, transfection of Dlg1 enhances beta-catenin signaling ~4 fold. Surprisingly, Frizzled4, which contains a C-terminal PDZ-binding motif that can bind to Dlg1 PDZ domains, appears to function independently of Dlg1 in vivo. These data expand the repertoire of Dlg/MAGUK family functions to include a role in beta-catenin signaling, and they suggest that proteins other than Frizzled receptors interact with Dlg1 to enhance beta-catenin signaling.
Highlights
The mammalian retina depends on two distinct vascular networks: the choroidal and intra-retinal circulations, which supply the outer third and inner two-thirds of the retina, respectively (Sun and Smith, 2018)
The experiments described above provide strong evidence that the scaffolding protein Dlg1 enhances beta-catenin signaling in endothelial cell (EC) in the context of central nervous system (CNS) angiogenesis and blood-brain barrier (BBB)/blood-retina barrier (BRB) development and maintenance (Figure 8—figure supplement 2)
This conclusion is based on: (i) the similarity in the retinal vascular phenotypes associated with EC-specific loss of Dlg1 and EC-specific reductions in beta-catenin signaling, (ii) the phenotypic synergy produced by the combination of EC-specific loss of Dlg1 and loss-of-function mutations in Fz4 or Tspan12 and Ndp or Tspan12, (iii) the nearly complete rescue of EC-specific loss of Dlg1 by EC-specific stabilization of beta-catenin, and (iv) the 3–4 fold enhancement of beta-catenin signaling stimulated by multiple Wnts or Norrin in Dlg1 knockout cell lines in response to Dlg1 transfection
Summary
The mammalian retina depends on two distinct vascular networks: the choroidal and intra-retinal circulations, which supply the outer third and inner two-thirds of the retina, respectively (Sun and Smith, 2018). Wnt7a and Wnt7b are produced predominantly by glia (Wang et al, 2018a) These ligands act on ECs through a Frizzled (Fz) receptor, Lrp5/6 co-receptor, and two Wnt7a/b-specific cell-surface coactivators, Reck and Gpr124 (Zhou and Nathans, 2014; Posokhova et al, 2015; Vanhollebeke et al, 2015; Cho et al, 2017; Eubelen et al, 2018; Vallon et al, 2018). Earlier studies showed that Discs large homologue 1 [Dlg; called Dlgh or synapse-associated protein 97 (SAP97)], an intracellular scaffolding protein with PDZ and guanylate kinase domains, can bind to a PDZ-binding motif present at the C-terminus of several Frizzleds, including Fz4 (Hering and Sheng, 2002) These data suggest that Dlg or other Dlg family members could play a role in beta-catenin signaling, presumably via an effect on trafficking or localization of Frizzled receptors. The first two PDZ domains of Dlg bind in vitro to a consensus PDZ-binding motif at the Fz4 C-terminus, genetic epistasis analyses of the EC-specific Dlg knockout and a CRISPR/Cas9-generated mutation of the Fz4 PDZ-binding motif argues that these two proteins function independently in the context of beta-catenin signaling
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