| Project/Area Number |
15390094
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General medical chemistry
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| Research Institution | HIROSHIMA UNIVERSITY |
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Principal Investigator |
KIKUCHI Akira Hiroshima University, Graduate School of Biomedical Sciences, Professor, 大学院・医歯薬学総合研究科, 教授 (10204827)
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| Co-Investigator(Kenkyū-buntansha) |
KISHIDA Shosei Hiroshima University, Graduate School of Biomedical Sciences, Associate Professor, 大学院・医歯薬学総合研究科, 助教授 (50274064)
YAMAMOTO Hideki Hiroshima University, Graduate School of Biomedical Sciences, Research Associate, 大学院・医歯薬学総合研究科, 助手 (20372691)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥15,400,000 (Direct Cost: ¥15,400,000)
Fiscal Year 2004: ¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 2003: ¥10,300,000 (Direct Cost: ¥10,300,000)
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| Keywords | 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 |
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| Research Abstract |
The following observations concerning Wnt signal and endocytosis were obtained in this research.
(1)Purification of Wnt proteins
Approximately 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 proteins
Wnt 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 Wnt
We 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.
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