WNT7A/B assemble a GPR124-RECK-LRP5/6 coreceptor complex to activate β-catenin signaling in brain endothelial cells.

WNT7A and WNT7B, secreted by neural cells, are essential regulators of developmental brain angiogenesis and blood-brain barrier integrity. In brain endothelial cells, WNT7 proteins activate β-catenin signaling through the ligand-specific receptor complex GPR124-RECK and classical WNT receptors of the FZD and LRP families. Previous studies suggested that WNT7 isoforms assemble a GPR124-RECK-FZD-LRP5/6 multireceptor complex for signaling. However, direct biochemical evidence for this complex and its signaling mechanisms remains elusive. Here, we investigated the formation and signaling mechanisms of WNT7 coreceptor complexes in brain endothelial cells using CRISPR/Cas9, biochemical analyses, and cell-based assays. Unexpectedly, cells with knockout of all FZD isoforms retained ∼25% of WNT7 responsiveness, whereas classical WNT3A signaling was completely abolished. Similarly, knockout of all Dvl paralogs, key mediators of FZD signaling, preserved ∼50% of WNT7 signaling activity but fully blocked WNT3A responses. In contrast, knockout of Gpr124, Reck, or Lrp5/6 completely abrogated WNT7 signaling. Although both WNT7A and WNT3A triggered LRP6 phosphorylation, only WNT3A induced DVL2 phosphorylation. Biochemical analyses revealed WNT7-dependent recruitment of LRP5/6, but not FZD, to the GPR124-RECK heterodimer, forming a GPR124-RECK-WNT7-LRP5/6 complex. In GPR124-deficient cells, WNT7 proteins still assembled a RECK-WNT7-LRP5/6 core complex, yet this complex lacked signaling activity and LRP6 phosphorylation. Clustering of RECK-WNT7-LRP5/6 complexes with recombinant dimeric GPR124 ectodomain or a RECK antibody partially restored signaling, suggesting that GPR124 mediates formation of higher-order complexes. Our findings indicate that WNT7 signaling in brain endothelial cells is driven by distinct coreceptor complexes: a FZD-independent GPR124-RECK-LRP5/6 complex and FZD-dependent complexes that likely act in synergy.