Wnt signalosome assembly is governed by conformational flexibility of Axin and by the AP2 clathrin adaptor

Wnt signal transduction relies on the direct inhibition of GSK3 by phosphorylated PPPSPxS motifs within the cytoplasmic tail of the LRP6 co-receptor. How GSK3 is recruited to LRP6 remains unclear. Here, we use nuclear magnetic resonance spectroscopy to identify the membrane-proximal PPPSPxS motif and its flanking sequences as the primary binding site for both Axin and GSK3, and an intrinsically disordered segment of Axin as its LRP6-interacting region (LIR). Co-immunoprecipitation and CRISPR-engineered mutations in endogenous Axin indicate that its docking at LRP6 is antagonized by a phospho-dependent foldback within LIR and by a PRTxR motif that allows Axin and GSK3 to form a multi-pronged interaction which favors their detachment from LRP6. Crucially, signaling by LRP6 also depends on its binding to the AP2 clathrin adaptor. We propose that the Wnt-driven clustering of LRP6 within clathrin-coated locales allows the Axin-GSK complex to dock at adjacent LRP6 molecules, while also exposing it to co-targeted kinases that change its activity in Wnt signal transduction.