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膜蛋白質の核輸送を規定する因子の同定

Research Project

Project/Area Number 16659073
Research Category

Grant-in-Aid for Exploratory Research

Allocation TypeSingle-year Grants
Research Field General medical chemistry
Research InstitutionEhime University

Principal Investigator

東山 繁樹  愛媛大学, 医学部, 教授 (60202272)

Co-Investigator(Kenkyū-buntansha) 松原 守  カルナバイオサイエンス株式会社, プロテオミクス室, 室長(研究職) (90288481)
Project Period (FY) 2004 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2005: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2004: ¥2,000,000 (Direct Cost: ¥2,000,000)
Keywords膜型増殖因子 / EGFファミリー / HB-EGF / amphiregulin / recycling endosome / 小胞輸送 / Rab / CTF signal / neuclear import / membrane protein / endocytosis / vesicle transport / shedding
Research Abstract

膜型増殖因子EGFファミリーメンバーは細胞膜表面でsheddingを受け、カルボキシ末端断片(C-terminal fragment ; CTF)を産生する。EGFファミリーメンバーのHB-EGFとamphiregulinのCTFについて解析をした結果、HB-EGF-CTF, amphiregulin-CTFともにER/核膜に移行し、集積した。本研究ではShedding後に細胞膜に残る膜ペプチドCTFがいかにしてER/核膜に移行するのか、またそれを規定する因子は何かを解析した。
HB-EGF-CTF核移行の分子機構解析 sheddingによって初めて露出するHB-EGF-CTFアミノ末端を特異的に認識する抗体(抗HB-EGF-CTF-N抗体)を開発した。Western blot解析からこの抗HB-EGF-CTF-N抗体は、HB-EGF全長は認識せず、sheddingによって産生されるHB-EGF-CTFを特異的に認識することを確認した。次に抗HB-EGF-CTF-N抗体を用いてHB-EGF発現HT1080細胞を免疫染色した。Shedding誘導剤非存在化では抗HB-EGF-CTF-N抗体陽性は認められなかったが、Shedding誘導剤であるTPAで処理1時間後の免疫染色で、ER/核膜の染色陽性像を得た。更にTPAで処理時間を短縮し、同様に染色を行い細胞内局在を解析したところ、recycling eondosomeと思われる領域を染色した。そこでrecycling eondosomeのマーカーとなるトランスフェリン受容体を標識トランスフェリンで細胞内に取り込ませ、recycling eondosomeに局在させるのを平行して行い抗HB-EGF-CTF-N抗で共局在を解析した。その結果、抗HB-EGF-CTF-N抗体はトランスフェリン受容体と共局在した。このことから、HB-EGF-CTFはshedding後にrecycling eondosomeを通過することが予測された。そこで、recycling eondosomeへの小胞輸送反応において重要な反応を担う低分子量G-蛋白質Rab5に焦点を絞り、Rab5のdominant negative formの発現による機能抑制、Rab5特異的siRNAによる遺伝子ノックダウンにより、HB-EGF-CTFのER/核膜に移行について解析を進めている。

Report

(2 results)
  • 2005 Annual Research Report
  • 2004 Annual Research Report
  • Research Products

    (12 results)

All 2006 2005 2004 Other

All Journal Article (12 results)

  • [Journal Article] PLZF regulates Pbx1 transcription and Pbx1-HoxC8 complex leads to androgen-independent prostate cancer proliferation.2006

    • Author(s)
      Kikugawa, T. et al.
    • Journal Title

      The Prostate (In Press)

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Ectodomain shedding of membrane-anchored heparin-binding EGF like growthfactor and subcellular localization of the C-terminal fragment in a cell cycle2005

    • Author(s)
      Toki, F. et al.
    • Journal Title

      J. Cell. Physiol 202

      Pages: 839-848

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Heparin-binding. EGF-like growth factor is essential for keratinocyte migration in skin wound heaking2005

    • Author(s)
      Shirakata, Y. et al.
    • Journal Title

      J. Cell Science 118

      Pages: 2363-2370

    • Related Report
      2005 Annual Research Report
  • [Journal Article] G protein coupling and second messenger generation are indispensable for metalloprotease-dependent, heparin-binding epidermal growth factor shedding through angiotensin II type-1 receptor.2005

    • Author(s)
      Mifune, M.et al.
    • Journal Title

      J. Biol. Chem. 280

      Pages: 26592-26599

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Induction of Keratinocyte Migration via Transactivation of the Epidermal Growth Factor Receptor by the Antimicrobial Peptide LL-37.2005

    • Author(s)
      Tokumaru, S. et al.
    • Journal Title

      J. Immunol. 175

      Pages: 4662-4668

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Alternative recycling of E-cadeherin depends on metalloprotease activity of ADAM92005

    • Author(s)
      Hirao, F. et al.
    • Journal Title

      Exp. Cell Res. 12

      Pages: 331-339

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Ectodomain shedding of membrane-anchored heparin-binding EGF like growth factor and subcellular localization of the C-terminal fragment in a cell cycle2005

    • Author(s)
      Toki, F.et al.
    • Journal Title

      J.Cell.Physiol. 202

      Pages: 839-848

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Distinct roles for ADAM10 and 17 in ectodomain shedding of six EGFR-ligands2004

    • Author(s)
      Sahin, U.et al.
    • Journal Title

      J.Cell Biol 164

      Pages: 769-779

    • Related Report
      2004 Annual Research Report
  • [Journal Article] The mechanism of cleavage of EGFR ligands induced by inflammatory cytokines in gastric epithelial cells ; ADAM10 mediates IL-8-induced EGFR transactivation.2004

    • Author(s)
      Tanida, S.
    • Journal Title

      Gastroenterology. 127

      Pages: 559-569

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Charged amino acid residues in the cytoplamic domain of proHB-EGF regulate its distribution and interaction with PLZF.2004

    • Author(s)
      Nanba, D.
    • Journal Title

      Biochem.Biophys.Res.Commun. 320

      Pages: 376-382

    • Related Report
      2004 Annual Research Report
  • [Journal Article] ADAM-binding protein Eve-1 is required for ectodomain shedding of EGF receptor ligands2004

    • Author(s)
      Tanaka, M.et al.
    • Journal Title

      J.Biol.Chem. 279

      Pages: 41950-41959

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Heparin-binding EGF-like growth factor is essential for keratinocyte migration in skin wound heaking

    • Author(s)
      Shirakata, Y.et al.
    • Journal Title

      J.Cell Science (in press)

    • Related Report
      2004 Annual Research Report

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Published: 2004-04-01   Modified: 2016-04-21  

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