Cloning of the protease that cleaves the extracellular domin of diphtheria toxin receptor

• Project/Area Number: 13670287
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Bacteriology (including Mycology)
• Research Institution: Kurume University
• Principal Investigator: UMATA Toshiyuki Kurume University, Institute of Life Science, Assistant Professor, 分子生命科学研究所, 講師 (30213482)
• Co-Investigator(Kenkyū-buntansha): MIURA Yoshiki Kurume University, Institute of Life Science, Research Associate, 分子生命科学研究所, 助手 (90279240)
• Project Period (FY): 2001 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥3,800,000 (Direct Cost: ¥3,800,000); Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000); Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
• Keywords: DTR / HB-EGF / LPA / membrane type metalloprotease / cleavage of extracellular domain / 細胞外領域切断 / リゾフォスファチジン酸 / 膜型メタロプロテアーゼ

Research Abstract

We demonstrated that lysophosphatidic acid (LPA) induces cleavage the extracellular domain of diphtheria toxin receptor/ membrane anchored heparin binding EOF like growth factor (DTR/HB-EGF). Dominant negative forms of several metalloprotease could not inhibit LPA-induced cleavage of DTR. As these results indicated that unknown protease implicated in LPA-induced cleavage of DTR, We tried cloning new protease cleaving DTR.
Three strategies were employed 1. Cloning from the subject associates with DTR induced by LPA. 2. Cloning from the subject condensed by metalloprotease inhibitor. 3. Cloning using monoclonal antibody that inhibits the cleavage of DTR Unfortunately, we could not clone the protease in the term of this project. However, We look forward to cloning the protease from the subject associates with DTR.
On the other hand, we demonstrated that the cleavage of DTR is induced by various stress-inducing stimuli, including UV light, osmotic pressure, hyperoxidation, and translation inhibitors. The pro-inflammatory cytokine interleukin-1β also stimulated the cleavage of DTR. An inhibitor of p38 MAPK (SB203580) or the expression of a dominant-negative (dn) form of p38 MAPK inhibited the stress-induced cleavage of DTR. However, stress-induced cleavage was not inhibited by the inhibitors of TPA- or LPA-induced cleavage or by dn forms of molecules involved in the TPA- or LPA-induced cleavage. These results indicate that stress-induced cleavage of DTR is mediated by p38 MAPK and that the signaling pathway induced by stress is distinct from the TPA- or LPA-induced cleavage of DTR.

Research Products

• [Publications] Takenobu T.: "The Stress-and Inflammatory Cytokine-induced Ectodomain Shedding of Heparin-binding Epidermal Growth Factor-like Growth Factor Is Mediated by p38 MAPK, Distinct from the 12-O-Tetradecanoylphorbol-13-acetate-and Lysophosohatidic Acid-induced Signaling Cascades"J. Biol. Chem.. (in press).
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Umata T.: "A Dual Signaling Cascade That Regulates the Ectodomain Shedding of Heparin-binding Epidermal Growth Factor-like Growth Factor"J. Biol. Chem.. 276・32. 30475-30482 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Hirata M.: "Identification of serum factor inducing ectodomain shedding of proHB-EGF and Studies of noncleavable mutants of proHB-EGF"Rinchem. Riophys. Res. Commun.. 283. 915-922 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Takenobu, H., Yamazaki, A., Hirata, M., Umata, T. & Mekada, E.: "The Stress- and Inflammatory Cytokine-induced Ectodomain Shedding of Heparin-binding Epidermal Growth Factor-like Growth Factor Is Mediated by p38 MARK, Distinct from the 12-O-Tetradecanoylphorbol-13-acetate- and Lysophosohatidic Acid-induced Signaling Cascades"J Biol Chem. in press.
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Umata, T., Hirata, M., Takahashi, T., Ryu, F., Shida, S., Takahashi, Y., Tsuneoka, M., Miura, Y., Masuda, M., Horiguchi, Y. & Mekada, E.: "A Dual Signaling Cascade That Regulates the Ectodomain Shedding of Heparin-binding Epidermal Growth Factor-like Growth Factor"J Biol Chem. 276. 304754-304782 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Hirata, M., Umata, T., Takahashi, T., Ohnuma, M., Miura Y., Iwamoto, R. & Mekada, E.: "Identification of Serum Factor Inducing Ectodomain Shedding of proHB-EGF and Studies of Noncleavable Mutants of proHB-EGF"Biochem Biophys Res Commun. 283. 915-922 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Takenobu T., Yamazaki A., Hirata M., Umata T., Mekada E.: "The Stress-and the Inflammatory Cytokine-induced Ectodomain Shedding of HB-EGF is mediated by p38 MAPK, distinct from TPA-induced signaling cascades"J. Biol. Chem.. (in press).
• [Publications] Michinari Hirata: "Identification of Serum Factor Inducing Ectodomain Shedding of proHB-EGF and Studies of Noncleavable Mutants of proHB-EGF"Biochemical and Biophysical Research Communications. 283・4. 915-922 (2001)
• [Publications] Toshiyuki Umata: "A Dual Signaling Cascade That Regulates the Ectodomain Shedding of Heparin-binding Epidermal Growth Factor-like Growth Factor"The Journal Biological Chemistry. 276・32. 30475-30482 (2001)