Regulation of the Wnt signal by receptor-mediated endocytosis

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The following observations concerning Wnt signal and endocytosis were obtained in this research.(1)Purification of Wnt proteinsApproximately 40〜60 μg of Wnt-3a and Wnt-5a were purified from 1 L of conditioned medium using column chromatographises. Purified Wnt-3a accumulated β-catenin and activated Rho-kinase in various cell lines. Purified Wnt-5a suppressed Wnt-3a-dependent Tcf transcriptional activation thorough the activation of NLK. Further, we also succeeded to establish cell lines producing Wnt-11.(2)Post-translational modification of Wnt proteinsWnt proteins has been suggested to be modified post-translationaly ; lipidation and gycosylation. However, the analyses using purified proteins have been little done. Mass spectrometory demonstarated that Wnt-5a is modified with palmitic scid. When Wnt-3a and Wnt-5a were treated with glycosidase-F, the mobility shift on the SDS-gel occurred, indicating that they are glycosylated. Palmitoylation of Wnt-5a was required for it actions. Glycosylation is necessary for the secretion but not for the actions.(3)Receptor mediated endocytosis of WntWe established an assay to observe endocytosis of Frizzled 5 and LRP6 in a Wnt-response manner. Both receptors were detected on the plasma membranes without Wnt-stimulation. Wnt-3a induced the inetrnalization of Frizzled 5 and LRP6 in 30-60 min and formed a small vesicle colocalized with EEA1, an early endocytotic marker. After 2〜3 h after stimulation, Frizzled 5 and LRP6 appeared on the plasma membranes again. A dominant negative form of Dynamin, which inhibited receptor internalization, suppressed Wnt-3a-dependent accumulation of β-catenin. The siRNA for caveolin or clathrin also suppressed Wnt-3a-dependent accumulation of β-catenin. These results indicate the receptor endocytosis of Wnt are essential for the activation of Wnt signal.