Regulation of the Wnt signal by receptor-mediated endocytosis

在本研究中获得了以下关于Wnt信号和内吞作用的观察结果。（1）Wnt蛋白的纯化使用柱色谱法从1 L条件培养基中纯化约40 μg ~ 60 μg的Wnt-3a和Wnt-5a。纯化的Wnt-3a在各种细胞系中积累β-连环蛋白并激活Rho激酶。纯化的Wnt-5a通过激活NLK抑制Wnt-3a依赖的Tcf转录激活。此外，我们还成功地建立了产生Wnt-11的细胞系。（2）Wnt蛋白的翻译后修饰Wnt蛋白的翻译后修饰主要包括脂化和糖基化。然而，使用纯化的蛋白质的分析已经做得很少。质谱分析表明，Wnt-5a被棕榈酸修饰。当Wnt-3a和Wnt-5a用糖苷酶-F处理时，SDS-凝胶上的迁移率发生变化，表明它们是糖基化的。Wnt-5a的棕榈酰化是其作用所必需的。糖基化是分泌所必需的，但不是作用所必需的。（3）受体介导的Wnt内吞我们建立了一种以Wnt响应方式观察Frizzled 5和LRP 6内吞的测定法。在没有Wnt刺激的质膜上检测到这两种受体。Wnt-3a在30-60 min内诱导Frizzled 5和LRP 6的内吞，并形成与早期内吞标记物EEA 1共定位的小泡。刺激后2 ~ 3 h，Frizzled 5和LRP 6再次出现在质膜上。抑制受体内化的发动蛋白的显性负性形式抑制β-连环蛋白的Wnt-3a依赖性积累。针对小窝蛋白或网格蛋白的siRNA还抑制Wnt-3a依赖性的β-连环蛋白积累。这些结果表明，Wnt受体的内吞作用是Wnt信号激活所必需的。

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.