Wnt5a regulates multiple intracellular signalling cascades, but how Wnt5a determines the specificity of these pathways is not well understood. This study examined whether the internalization of Wnt receptors affects the ability of Wnt5a to regulate its signalling pathways. Wnt5a activated Rac in the b-catenin-independent pathway, and Frizzled2 (Fz2) and Ror1 or Ror2 were required for this action. Fz2 was internalized through a clathrin-mediated route in response to Wnt5a, and inhibition of clathrindependent internalization suppressed the ability of Wnt5a to activate Rac. As another action of Wnt5a, it inhibited Wnt3a-dependent lipoprotein receptor-related protein 6 (LRP6) phosphorylation and b-catenin accumulation. Wnt3a-dependent phosphorylation of LRP6 was enhanced in Wnt5a knockout embryonic fibroblasts. Fz2 was also required for the Wnt3a-dependent accumulation of b-catenin, and Wnt5a competed with Wnt3a for binding to Fz2 in vitro and in intact cells, thereby inhibiting the b-catenin pathway. This inhibitory action of Wnt5a was not affected by the impairment of clathrin-dependent internalization. These results suggest that Wnt5a regulates distinct pathways through receptor internalizationdependent and -independent mechanisms.

Wnt and Dickkopf (Dkk) regulate the stabilization of beta-catenin antagonistically in the Wnt signaling pathway; however, the molecular mechanism is not clear. In this study, we found that Wnt3a acts in parallel to induce the caveolin-dependent internalization of low-density-lipoprotein receptor-related protein 6 (LRP6), as well as the phosphorylation of LRP6 and the recruitment of Axin to LRP6 on the cell surface membrane. The phosphorylation and internalization of LRP6 occurred independently of one another, and both were necessary for the accumulation of beta-catenin. In contrast, Dkk1, which inhibits Wnt3a-dependent stabilization of beta-catenin, induced the internalization of LRP6 with clathrin. Knockdown of clathrin suppressed the Dkk1-dependent inhibition of the Wnt3a response. Furthermore, Dkk1 reduced the distribution of LRP6 in the lipid raft fraction where caveolin is associated. These results indicate that Wnt3a and Dkk1 shunt LRP6 to distinct internalization pathways in order to activate and inhibit the beta-catenin signaling, respectively.

Summary-catenin-mediated Wnt signaling is crucial in animal development and tumor progression. The phosphorylation of low-density lipoprotein receptor-related protein 6 (LRP6), a single-span transmembrane Wnt receptor, plays a vital role in this signaling. Dickkopf1 (Dkk1) has been shown to inhibit the Wnt--catenin pathway, but the mechanism is not yet clear. Here, evidence is presented that Wnt3a-dependent phosphorylation of LRP6 occurs in the lipid raft and that Dkk1 inhibits the formation of a complex between LRP6 and casein kinase 1 (CK1) by removing LRP6 from the lipid raft. Dkk1 internalized LRP6 in a Rab5-dependent mechanism to prevent phosphorylation mediated by CK1. The internalized LRP6 was recycled back in a Rab11-dependent mechanism to the cell-surface membrane, and the recycled LRP6 again responded to Wnt3a and Dkk1. Internalized Dkk1 was trafficked in a Rab7-mediated route and degraded in the lysosome. These results suggest that Dkk1 induces the internalization of LRP6 to suppress its phosphorylation in the lipid raft and allows subsequent recycling of LRP6 so that it can be reused for signaling.Key words: Dkk1, LRP6, Internalization, Recycling, Rab5 Journal of Cell Science Trafficking of Dkk1 and LRP6HEK293T cells were fractionated in the presence of Triton X-100 to separate them into detergent-resistant microdomains (DRMs) and non-DRMs. Although DRMs do not necessarily correspond to the lipid raft in cells, detergent insolubility is a valuable and widely used method to identify proteins that are localized to the lipid raft and probably function there. Endogenous LRP6 was observed in both DRMs and non-DRMs, and Wnt3a did not affect the distribution of LRP6 between the two fractions (Fig. 1A). Although Wnt3a bound to LRP6 in both fractions with a similar efficiency (Fig. 1B), Wnt3a induced the phosphorylation of LRP6 at Ser1490 and Thr1479, which are known to be phosphorylated by GSK-3 and CK1, respectively, only in DRMs (Fig. 1C). It has been shown that caveolin plays a role in the Wnt3a-dependent internalization of LRP6 and accumulation of -catenin (Yamamoto et al., 2006;Yamamoto et al., 2008). Knockdown of caveolin1 affected neither the distribution of LRP6 between DRMs and non-DRMs nor the Wnt3a-dependent phosphorylation of LRP6, but it inhibited the accumulation of -catenin (Fig. 1D,E). Nystatin, which binds to cholesterol and disrupts the lipid raft, inhibited Wnt3a-dependent LRP6 phosphorylation and -catenin accumulation (Fig. 1E). These results suggested that Wnt3a induces the phosphorylation of LRP6 in the lipid raft, but that this phosphorylation is not sufficient for the stabilization of -catenin. Moreover, these findings also indicated that the presence of lipid rafts is important for Wnt3a signaling.Dvl and CK1 are important components in the Wnt--catenin pathway (Knippschild et al., 2005;Wharton, 2003). Endogenous Dvl was distributed mainly into non-DRMs and partly into DRMs ( Fig. 2A). Wnt3a induced a mobility shift of Dvl in an SDSpolyacrylamide gel in both microdomains ...

Wnt5a is a representative ligand that activates the β-catenin-independent pathway in Wnt signaling. It was reported that the expression of Wnt5a in human gastric cancer is associated with aggressiveness and poor prognosis and that knockdown of Wnt5a reduces the ability of gastric cancer cells to metastasize in nude mice. Therefore, Wnt5a and its signaling pathway might be important targets for the therapy of gastric cancer. The aim of this study was to examine whether an anti-Wnt5a antibody affects metastasis of gastric cancer cells. One anti-Wnt5a polyclonal antibody (pAb5a-5) inhibited migration and invasion activities in vitro of gastric cancer cells with a high expression level of Wnt5a. Previously, it was shown that Wnt5a induces the internalization of receptors, which is required for Wnt5a-dependent activation of Rac1. pAb5a-5 inhibited Wnt5a-dependent internalization of receptors, thereby suppressed Wnt5a-dependent activation of Rac1. Laminin γ2 is one of target genes of Wnt5a signaling and Rac1 was involved in its expression. pAb5a-5 also inhibited Wnt5a-dependent expression of laminin γ2. In an experimental liver metastasis assay, gastric cancer cells were introduced into the spleens of nude mice. Laminin γ2 was required for liver metastatic ability of gastric cancer cells in vivo. Furthermore, intraperitoneal injection of pAb5a-5 inhibited the metastatic ability of gastric cancer cells. These results suggest that an anti-Wnt5a antibody was capable of suppressing Wnt5a-dependent internalization of receptors, resulting in the prevention of metastasis of gastric cancer cells by inhibiting the activation of Rac1 and the expression of laminin γ2.