Cell-to-cell communication through secreted Wnt ligands that bind to members of the Frizzled (Fzd) family of transmembrane receptors is critical for development and homeostasis. Wnt9a signals through Fzd9b, the co-receptor LRP5 or LRP6 (LRP5/6), and the epidermal growth factor receptor (EGFR) to promote early proliferation of zebrafish and human hematopoietic stem cells during development. Here, we developed fluorescently labeled, biologically active Wnt9a and Fzd9b fusion proteins to demonstrate that EGFR-dependent endocytosis of the ligand-receptor complex was required for signaling. In human cells, the Wnt9a-Fzd9b complex was rapidly endocytosed and trafficked through early and late endosomes, lysosomes, and the endoplasmic reticulum. Using small-molecule inhibitors and genetic and knockdown approaches, we found that Wnt9a-Fzd9b endocytosis required EGFR-mediated phosphorylation of the Fzd9b tail, caveolin, and the scaffolding protein EGFR protein substrate 15 (EPS15). LRP5/6 and the downstream signaling component AXIN were required for Wnt9a-Fzd9b signaling but not for endocytosis. Knockdown or loss of EPS15 impaired hematopoietic stem cell development in zebrafish. Other Wnt ligands do not require endocytosis for signaling activity, implying that specific modes of endocytosis and trafficking may represent a method by which Wnt-Fzd specificity is established.

中文翻译：

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Wnt9a 和 Fzd9b 的 EGFR 依赖性内吞作用促进斑马鱼造血干细胞发育过程中的 β-catenin 信号传导

通过与跨膜受体卷曲 (Fzd) 家族成员结合的分泌型 Wnt 配体进行的细胞间通讯对于发育和体内平衡至关重要。 Wnt9a 通过 Fzd9b、辅助受体 LRP5 或 LRP6 (LRP5/6) 以及表皮生长因子受体 (EGFR) 发出信号，促进斑马鱼和人类造血干细胞在发育过程中的早期增殖。在这里，我们开发了荧光标记的、具有生物活性的 Wnt9a 和 Fzd9b 融合蛋白，以证明配体-受体复合物的 EGFR 依赖性内吞作用是信号传导所必需的。在人类细胞中，Wnt9a-Fzd9b 复合物被快速内吞并通过早期和晚期内体、溶酶体和内质网运输。使用小分子抑制剂以及遗传和敲低方法，我们发现 Wnt9a-Fzd9b 内吞作用需要 EGFR 介导的 Fzd9b 尾部、小窝蛋白和支架蛋白 EGFR 蛋白底物 15 (EPS15) 的磷酸化。 Wnt9a-Fzd9b 信号传导需要 LRP5/6 和下游信号传导组件 AXIN，但内吞作用不需要。 EPS15 的敲低或缺失会损害斑马鱼的造血干细胞发育。其他 Wnt 配体不需要内吞作用来发挥信号活性，这意味着特定的内吞作用和运输模式可能代表了建立 Wnt-Fzd 特异性的方法。