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EGFR-initiated endocytosis of Wnt9a and Fzd9b is required for β-catenin signaling

Cell to cell communication through secreted ligands like those encoded by the Wnt gene family is critical for development and homeostasis during organismal life. One of the bottlenecks in the Wnt field has been identifying specific ligand/receptor pairings and decoding the mechanisms for their downstream signals. We previously discovered that the Wnt9a ligand signals through the cell surface receptors Fzd9b, LRP5/6 and EGFR to promote early proliferation of hematopoietic stem cells during development. We used this exquisitely specific ligand/receptor complex as a platform to determine if Wnt9a requires endocytosis for signaling. Using fluorescently labeled, biologically active Wnt9a and Fzd9b fusion proteins, we demonstrate here that the Wnt9a receptor complex is rapidly endocytosed within one minute of contact with Fzd9b. Following this, the Wnt9a/Fzd9b complex is trafficked through the cell to early and late endosomes, lysosomes, and the endoplasmic reticulum; it is also recycled back to the membrane. Using small molecule inhibitors, genetic and siRNA approaches, we identified that mechanistically this endocytosis requires EGFR-mediated phosphorylation of the Fzd9b tail, followed by endocytosis through a caveolin and EPS15 dependent pathway. Specific modes of endocytosis and trafficking may represent one of the ways in which Wnt/Fzd specificity is established, since other Wnt ligands do not require endocytosis for activity.