Keeping Wnt Signalosome in Check by Vesicular Traffic

Wingless (Wg, or Wnt in mammals) belongs to a family of cysteine-rich glyco- and lipoproteins functioning as paracrine and autocrine ligands (Willert et al., 2003; Willert and Nusse, 2012). Initiation of Wnt signaling is triggered by binding of Wg/ Wnt to their various 7-pass transmembrane Frizzled receptors (Fzd) (Wu and Nusse, 2002; Schulte and Bryja, 2007; Schulte, 2010). Wnt-conjugated Fzd recruits cytoplasmic protein Dishevelled (Dvl) forming Fzd-Dvl complex (Wong et al., 2003). This complex, in association with the low-density lipoprotein receptor-related protein 5 and 6 (LRP5/6; or Arrow in Drosophila) induces a signal relay, often referred to as the “canonical” pathway, driving specific gene expression in a β-catenin dependent fashion (Tamai et al., 2000; Wehrli et al., 2000; He et al., 2004; MacDonald and He, 2012). The same complex is also responsible for a separate signaling cascade namely the noncanonical pathway activating the functions of small GTPases of the Rho subfamily (Eaton et al., 1996; Strutt et al., 1997; Boutros and Mlodzik, 1999; Fanto et al., 2000; Habas et al., 2001; Habas et al., 2003; Wallingford and Habas, 2005). The canonical Wnt/β-catenin pathway in particular plays fundamental roles during fetal development and adult tissue homeostasis; inappropriate activation of this pathway in human diseases has highlighted its profound influences on cellular behavior (Reya and Clevers, 2005; Nusse et al., 2008; Angers and Moon, 2009; MacDonald et al., 2009; Clevers and Nusse, 2012). Healthy cells must employ various mechanisms to regulate Wnt pathway activity (Nusse, 2012). That is to balance efficient signal transduction and avoidance of overactivation. Ligand-induced receptor trafficking is one of the most important regulatory events in many signaling pathways (Sorkin and von, 2009). Internalizations of cell surface cargos, for example the various ligand-receptor complexes, through clathrin-dependent or clathrin-independent pathways are collectively known as endocytosis (Marsh and McMahon, 1999; Rappoport, 2008). In clathrin-dependent process, plasma membrane-bound cargos are first recognized by clathrin adaptor proteins (APs), by which the coat proteins—clathrins are recruited to the cargo-concentrated membrane microdomains. Clathrin adaptor complexes include AP-1, AP-2, and AP-3, which show distinct functional and structural features (Conner and Schmid, 2003; Touz et al., 2004; Gupta et al., 2006). Each AP complex is a heterotetramer consisting two large adaptin subunits, one medium, and one small subunit. By linking cargo to clathrin and other regulatory proteins, APs play key roles in coat assembly and protein trafficking (Kirchhausen, 1999; Owen et al., 2004). For endocytosis, the AP-2 complex (with α1, β1, μ2, and σ1 subunits) is particularly important for linking clathrin to plasma membrane and for cargo selection and recruitment into coated pits (Traub, 2009; Kirchhausen, 2012). Once recruited to plasma membrane, clathrin deforms the membrane into clathrin-coated pits using its specialized 3-interlocked spiral shaped structure (Marsh and McMahon, 1999). At neck of the pits, a GTPase called dynamin binds and mediates membrane fission for vesicle release (Marsh and McMahon, 1999; Praefcke and McMahon, 2004). In clathrin-independent pathway, endocytosis is mediated through lipid rafts and caveolin family proteins (Razani et al., 2002b; Lajoie and Nabi, 2010). Lipid rafts are cholesterol and glycosphingolipid enriched membrane microdomains (Simons and Toomre, 2000; Simons and Ehehalt, 2002), where caveolin inserts hairpin loop structures to facilitate the formation of caveolae –flask-shaped membrane invaginations, roughly 50 nm in size, enriched with lipid rafts (Anderson, 1998; Parton and Simons, 2007). Membrane fission and vesicle release in caveolae-mediated endocytosis also require dynamin (Henley et al., 1999). Various classes of signaling molecules contain caveolin-binding motifs therefore this pathway may facilitate the concentration of signaling molecules. Once internalized, cargo proteins travel to membrane-bound vesicles known as early endosomes, where they are sorted: some recycled back to plasma membrane for reuse while others delivered to late endosome and lysosome for degradation (Sorkin and von, 2009; Wolfe and Trejo, 2007). Upon vesicular budding into the cytosol, coat proteins (clathrin and caveolin) quickly reassembled for recycling. Here, we gather considerable amount of evidences to support the view that Wnt signal receiving cells utilize highly flexible and sophisticated membrane trafficking systems to balance the signal transduction. Same signaling molecules (e.g., Fzd or LRP6), in a highly context-dependent manner, can serve as regulatory hub for various signaling purposes: amplification, maintenance, inhibition, and termination. As Wnt-induced signalosome is, in our view, one of the most important pathway elements, we center our discussions on how vesicular traffic may influence signalosome function via various mechanisms. Based on current literatures, we provide updates on the vesicular trafficking mechanisms underlying three major ligand-receptor complexes that potently control Wnt signaling: Wnt-Fzd-LRP6, Dkk1-Kremen-LRP6, and R-spondin-LGR5-RNF43. Contrasting and converging views are discussed with emphasis given to major merits of prevailing models. We tentatively conclude that Wnt engagement with surface receptors triggers a rapid biochemical response potentially within certain membrane microdomain, which, in concert with subsequent internalization of signalosome, may contribute to full and sustained pathway activation. Such pathway activation is balanced by degradation of excessive ligands and clearance of surface receptors via key endocytotic machineries and mechanisms that downplay the signal strength.