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Wnt/β-catenin signaling is an ancient pathway that regulates key aspects of embryonic development, cell differentiation, proliferation, and adult stem cell homeostasis. Work from different laboratories has shed light on the molecular mechanisms underlying the Wnt pathway, including structural details of ligand-receptor interactions. One key aspect that has emerged from multiple studies is that endocytosis of the receptor complex plays a crucial role in fine-tuning Wnt/β-catenin signaling. Endocytosis is a key process involved in both activation as well as attenuation of Wnt signaling, but how this is regulated is still poorly understood. Importantly, recent findings show that Wnt also regulates central metabolic pathways such as the acquisition of nutrients through actin-driven endocytic mechanisms. In this review, we propose that the Wnt pathway displays diverse characteristics that go beyond the regulation of gene expression, through a connection with the endocytic machinery.
Figure 1, General model of Wnt/β-catenin signaling. In absence of Wnt ligands, a destruction complex formed by Axin, APC, GSK3, and CK1 actively promotes β-catenin protein turnover through a proteasome-dependent mechanism, maintaining Wnt signaling in an OFF state. Conversely, in the Wnt ON state, a Wnt ligand binds to its cognate receptors, Fzd and Lrp5/6, inducing the formation of a multiprotein complex known as signalosome and inhibiting the destruction complex activity. The signalosome is subsequently endocytosed into early endosomes (EE), which later mature into multivesicular bodies (MVBs). Signalosome endocytosis is required to transduce the Wnt signal. Consequently, β-catenin protein is stabilized and translocates into the nucleus, where together with TCF/LEF it activates the transcription of Wnt target genes. Among genes activated by β-catenin/TCF, the transmembrane E3 ligases RNF43/ZNRF3 represent elements of an important negative feedback mechanism, which attenuate Wnt signaling by ubiquitination and degradation of the Fzd/Lrp5/6 receptor complex via the endolysosomal system. Note that, while Lrp6 stability is also regulated by RNF43/ZNRF3, it is still unclear whether it is directly ubiquitinated by the transmembrane E3 ligases
Figure 2. Crystal structure of the Wnt-Frizzled complex. Wnt adopts a “thumb and index” structure that contacts Frizzled ectodomain on two opposite sides. (a) Ribbon models of xWnt8 (red) and Frizzled 8 cysteine rich domain (yellow). The palmitoleic acid moiety (PAM) and asparagine-linked glycans are drawn as sticks, with the following atom color code: green = carbon; red = oxygen; blue = nitrogen. The black arrowhead points at the appended PAM extending in a zigzag pattern from Ser187 of the xWnt8 N-terminal domain. Black arrows indicate Asn104-linked glycan (two N-acetylglucosamine and two mannose residues), Asn263-linked glycan (two N-acetylglucosamine residues and one mannose residue) on xWnt8; and Asn49-linked glycan (two N-acetylglucosamine residues) on Frizzled CRD. (b) Surface representation of the Wnt-Fzd complex, ‘face-on’ and ‘side-on’ (after a 90 degrees rotation). Note how the palmitoleic acid (arrowheads) fits into the hydrophobic groove of Frizzled CRD. The images were created by uploading the crystallographic data of xWnt8-Fzd8 complex deposited on Protein Data Bank (PDB ID 4F0A; https://www.rcsb.org/) (Janda et al., 2012), to the web-based online tool EzMol (http://www.sbg.bio.ic.ac.uk/ezmol/) (Reynolds et al., 2018)
Figre 3. Cellular mechanisms of endocytosis. The diagram shows clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis, all of which have pertinence to Wnt signaling. The major regulatory proteins for the different endocytic processes are indicated. Although these pathways proceed through different mechanisms, they converge at the level of the late endosomal/lysosomal system. Note that macropinocytosis is a receptor-independent pathway, different from clathrin- and caveolin-dependent endocytosis
Figure 4. Endocytosis regulates Wnt signaling. Upon activation of the Wnt pathway, the signalosome (formed by the ligand–receptor complex, and associated proteins) is rapidly internalized through caveolar endocytosis. Early endosome (EE) vesicles containing the signalosome fuse and mature into multivesicular bodies (MVB), dragging GSK3 and Axin into the intraluminal vesicles of the MVB. Several endosomal proteins, such as the ESCRT machinery (including HRS and VPS4), are required for this process. Prmt1 and arginine methylation are also key regulators of GSK3 sequestration into MVBs. On the other hand, endocytosis can also be utilized to dampen Wnt signal. An example is Dkk1 binding to Lrp6 and the transmembrane protein Kremen, which promote Lrp6 clearance from the plasma membrane through clathrin-mediated endocytosis. Wnt can also mediate nutrient uptake, by regulating macropinocytosis. GSK3 and Axin normally repress micropinocytosis; however, when Wnt signaling is turned on, their sequestration into MVBs allows for sustained macropinocytic activity
