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Regulation of the Wnt signal by receptor-mediated endocytosis

受体介导的内吞作用对 Wnt 信号的调节

基本信息

批准号：
15390094
负责人：
KIKUCHI Akira
金额：
$ 9.86万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15390094/
关键词：
Wnt endocytosis Rho-kinase conditioned medium lipidation gycosylation β-catenin Frizzled LRP ダイナミン受容体 Rhoキナーゼ PC12細胞

项目摘要

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.

(1)Wnt蛋白的纯化用柱层析法从1 L条件培养液中纯化出约40~60μg的Wnt-3a和Wnt-5a。纯化的WNT-3a在不同的细胞系中积累β-连环蛋白并激活Rho-激酶。纯化的WNT-5a通过激活NLK抑制依赖Wnt-3a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蛋白被认为是翻译后修饰；然而，使用纯化的蛋白质进行分析的工作很少。质谱学结果表明，WNT-5a分子筛被棕榈酸SCID修饰。当WNT-3a和WNT-5a被糖苷酶-F处理时，其在十二烷基硫酸钠凝胶上的迁移率发生了移动，表明它们是糖基化的。它的作用需要WNT-5a的棕榈酰化。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反应的方式观察Frizzled5和LRP6的内吞作用的方法。在没有Wnt刺激的情况下，两种受体均可在质膜上检测到。WNT-3a在30-60min内诱导Frizzled5和LRP6内化，并与早期的内吞标志物EEA1共定位形成一个小泡。刺激后2~3h，Frizzled5和LRP6重新出现在细胞膜上。一种抑制受体内化的显性负性动力蛋白，抑制了WNT-3a依赖的β-连环蛋白的积累。针对小窝蛋白或笼状蛋白的小干扰RNA也抑制了依赖WNT-3a的β-连环蛋白的积累。这些结果表明，Wnt的受体内吞作用是激活Wnt信号所必需的。