Wnt-dependent Frizzled clustering is required for Dishevelled phosphorylation but insufficient for β-catenin stabilization

Wnt–β-catenin signaling begins when Wnt ligands engage the receptors Frizzled (Fzd) and LRP5 or LRP6 (LRP5/6), leading to the recruitment and phosphorylation of the intracellular protein Dishevelled (Dvl), which is necessary for stabilization of the transcriptional coactivator β-catenin. Understanding the mechanisms by which ligand binding to Fzd activates Wnt–β-catenin signaling is crucial for rational ligand design to selectively modulate Wnt responses in the context of diseases and tissue regeneration. Here, we determined that ligand-induced Fzd clustering was the initiating event for the recruitment and phosphorylation of the downstream signaling mediator Dvl. Using synthetic, bivalent antibodies and single-molecule microscopy, we found that Wnts and bivalent Fzd-binding antibodies, but not monovalent antibodies, clustered Fzd at the plasma membrane in cells, activating Dvl independently of LRP5/6. However, β-catenin–mediated signaling required LRP5/6 recruitment as an additional step to enable inhibition of the kinase GSK3α or GSK3β and stabilization of β-catenin. This two-step mechanism may separate Fzd activation from β-catenin pathway output, underlying a mechanism by which Wnts encode signaling specificity and may inform the design of selective Wnt pathway modulators.