Clathrin and AP2 are required for PtdIns(4,5)P2-mediated formation of LRP6 signalosomes.

Canonical Wnt signaling is initiated by the binding of Wnt proteins to their receptors, low-density lipoprotein-related protein 5 and 6 (LRP5/6) and frizzled proteins, leading to phosphatidylinositol (4,5)bisphosphate (PtdIns(4,5)P(2)) production, signalosome formation, and LRP phosphorylation. However, the mechanism by which PtdIns(4,5)P(2) regulates the signalosome formation remains unclear. Here we show that clathrin and adaptor protein 2 (AP2) were part of the LRP6 signalosomes. The presence of clathrin and AP2 in the LRP6 signalosomes depended on PtdIns(4,5)P(2), and both clathrin and AP2 were required for the formation of LRP6 signalosomes. In addition, WNT3A-induced LRP6 signalosomes were primarily localized at cell surfaces, and WNT3A did not induce marked LRP6 internalization. However, rapid PtdIns(4,5)P(2) hydrolysis induced artificially after WNT3A stimulation could lead to marked LRP6 internalization. Moreover, we observed WNT3A-induced LRP6 and clathrin clustering at cell surfaces using super-resolution fluorescence microscopy. Therefore, we conclude that PtdIns(4,5)P(2) promotes the assembly of LRP6 signalosomes via the recruitment of AP2 and clathrin and that LRP6 internalization may not be a prerequisite for Wnt signaling to β-catenin stabilization.