Investigation of GPCRs interacting with RAMPs an functional analysis of their complex.

• Project/Area Number: 22570175
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Molecular biology
• Research Institution: Osaka University (2012); The Institute of Physical and Chemical Research (2010-2011)
• Principal Investigator: HINO Nobumasa 大阪大学, 大学院・薬学研究科, 助教 (90469916)
• Project Period (FY): 2010 – 2012
• Project Status: Completed (Fiscal Year 2012)
• Budget Amount: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2012: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000); Fiscal Year 2011: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2010: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
• Keywords: タンパク質間相互作用 / 膜タンパク質 / 光クロスリンク / 非天然型アミノ酸 / 質量分析 / RAMP / GPCR / プロテオミクス / 非天然アミノ酸 / クロスリンク / 拡張遺伝暗号 / RAMP1 / SILAC法

Research Abstract

(I) I developed a system for the identification of proteins interacting with a given target protein, by site-specific incorporation of a photo-reactive amino acid into the target protein, in vivo photo-cross-linking to interacting proteins, and identification of thus captured proteins by comparative quantitative mass spectrometry analysis. A novel long chain photoreactive amino acid was alsosuccessfully incorporated into proteins, which enables wide-range photo-cross-linking of proteins.
(II) I showed how RAMP and calcitonin receptor-like receptor interact with each other on the membrane of living cells by X-ray crystal structure analysis and site-specific photo-cross-linking assay.

Research Products

• [Journal Article] Tetrameric interaction of the ectoenzyme CD38 on the cell surface enables its catalytic and raft-association activities (2012)
  • Author(s): Hara-Yokoyama M., Kukimoto-Niino M., Terasawa K., Harumiya S., Podyma-Inoue K. A., Hino N., Sakamoto K., Itoh S., Hashii N., Hiruta Y., Kawasaki N., Mishima-Tsumagari C., Kaitsu Y., Matsumoto T., Wakiyama M., Shirouzu M., Kasama T., Ta
  • Journal Title: Structure Volume: 20 Issue: 9 Pages: 1585-1595
  • DOI: 10.1016/j.str.2012.06.017
  • Peer Reviewed
• [Journal Article] Structural basis of interleukin-5 dimer recognition by its alpha receptor (2012)
  • Author(s): Kusano S., Kukimoto-Niino M., Hino N., Ohsawa N., Ikutani M., Takaki S., Sakamoto K., Hara-Yokoyama M., Shirouzu M., Takatsu K., Yokoyama S
  • Journal Title: Protein Sci Volume: 21 Issue: 6 Pages: 850-864
  • DOI: 10.1002/pro.2072
  • Peer Reviewed
• [Journal Article] Wade-range protein photo-crosslinking achieved by a genetically encoded N^ε-(benzyloxycarbonyl) lysine derivative with a diazirinyl moiety (2012)
  • Author(s): T. Yanagisawa, N. Hino, F. Iraha, T. Mukai, K. Sakamoto, S. Yokoyama
  • Journal Title: Mol. Bio Syst. Volume: 8 Issue: 4 Pages: 1131-1135
  • DOI: 10.1039/c2mb05321g
  • Peer Reviewed
• [Journal Article] Structural basis for extracellular interactions between calcitonin receptor-like receptor and receptor activity-modifying protein 2 for adrenomedullin-specific binding (2012)
  • Author(s): Kusano S., Kukimoto-Niino M., Hino N., Ohsawa N., Okuda K., Sakamoto K., Shirouzu M., Shindo T., Yokoyama S
  • Journal Title: Protein Sci Volume: 21 Issue: 2 Pages: 199-210
  • DOI: 10.1002/pro.2003
  • Peer Reviewed
• [Journal Article] Site-specific incorporation of unnatural amino acids into proteins in mammalian cells (2012)
  • Author(s): Hino N., Sakamoto K., Yokoyama S
  • Journal Title: Methods Mol. Biol Volume: 794 Pages: 215-228
  • DOI: 10.1007/978-1-61779-331-8_13
  • ISBN: 9781617793301, 9781617793318
• [Journal Article] Genetic incorporation of a photo-crosslinkable amino acid reveals novel protein complexes with GRB2 in mammalian cells (2011)
  • Author(s): Hino N., Oyama M., Sato A., Mukai T., Iraha F., Hayashi A., Kozuka-Hata H., Yamamoyo T., Yokoyama S., Sakamoto K
  • Journal Title: J. Mol. Biol. Volume: 406 Issue: 2 Pages: 343-353
  • DOI: 10.1016/j.jmb.2010.12.022
  • Peer Reviewed
• [Journal Article] Dissecting Cell Signaling Pathways with Genetically Encoded 3-Iodo-L-tyrosine (2011)
  • Author(s): Hayashi A., Hino N., Kobayashi T., Arai R., Shirouzu M., Yokoyama S., Sakamoto K
  • Journal Title: ChemBioChem Volume: 12 Issue: 3 Pages: 387-389
  • DOI: 10.1002/cbic.201000665
  • Peer Reviewed
• [Journal Article] Crystallographic study of a site-specifically crosslinked protein complex with a genetically incorporated photo-reactive amino acid (2011)
  • Author(s): Sato S., Mimasu S., Sato A., Hino N., Sakamoto K., Umehara T., Yokoyama S.
  • Journal Title: Biochemistry Volume: 50 Pages: 250-257
  • Peer Reviewed
• [Journal Article] Genetic Incorporation of a Photo-Crosslinkable Amino Acid Reveals Novel Protein Complexes with GRB2 in Mammalian Cells. (2011)
  • Author(s): 樋野展正、尾山大明、佐藤文、向井崇人、伊良波史枝、林明子、秦裕子、山本雅、横山茂之、坂本健作
  • Journal Title: Journal of molecular biology Volume: 406 Pages: 343-353
  • Peer Reviewed
• [Journal Article] Crystallographic Study of a Site-Specifically Cross-Linked Protein Complex with a Genetically Incorporated Photoreactive Amino Acid. (2011)
  • Author(s): 佐藤心、三桝信哉、佐藤文、樋野展正、坂本健作、梅原崇史、横山茂之
  • Journal Title: Biochemistry Volume: 50 Pages: 250-257
  • Peer Reviewed
• [Journal Article] Dissecting Cell Signaling Pathways with Genetically Encoded 3-Iodo-L-tyrosine. (2011)
  • Author(s): 林明子、樋野展正、小林隆嗣、新井亮一、白水美香子、横山茂之、坂本健作
  • Journal Title: Chembiochem Volume: 12 Pages: 387-389
  • Peer Reviewed
• [Journal Article] Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein (2010)
  • Author(s): Tagami S., Sekine S., Kumarevel T., Hino N., Murayama Y., Kamegamori S., Yamamoto M., Sakamoto K., Yokoyama S.
  • Journal Title: Nature Volume: 468 Issue: 7326 Pages: 978-982
  • DOI: 10.1038/nature09573
  • Peer Reviewed
• [Journal Article] Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein. (2010)
  • Author(s): 田上俊輔、関根俊一、Thirumananseri Kumarevel、樋野展正、村山祐子、亀ヶ盛俊介、山本雅貴、坂本健作、横山茂之
  • Journal Title: Nature Volume: 468 Pages: 978-982
  • Peer Reviewed
• [Presentation] インターロイキン-5受容体α鎖による2量体インターロイキン-5認識の構造的基盤 (2012)
  • Author(s): 草野清輔,新野睦子,樋野展正,大沢登,生谷尚士,高木智,坂本健作,横山三紀,白水美香子,高津聖志,横山茂之
  • Organizer: 第12回日本蛋白質科学会年会
  • Place of Presentation: 名古屋
• [Presentation] 新たなクロスリンカー含有非天然型アミノ酸のタンパク質への部位特異的導入と,その広域性光クロスリンク能 (2012)
  • Author(s): 柳沢達男, 樋野展正, 伊良波史枝, 向井崇人, 坂本健作, 横山茂之
  • Organizer: 第14回日本RNA学会年会
  • Place of Presentation: 仙台
• [Presentation] ジアジリン含有ベンジルオキシカルボニルリジン誘導体のタンパク質への部位特異的導入と広域性光クロスリンク能 (2012)
  • Author(s): 柳沢達男,樋野展正, 伊良波史枝, 向井崇人, 坂本健作, 横山茂之
  • Organizer: 日本ケミカルバイオロジー学会第七回年会
  • Place of Presentation: 京都
• [Presentation] RNAポリメラーゼの新規コンフォメーション状態 (2010)
  • Author(s): 田上俊輔, 関根俊一, Thirumananseri Kumarevel, 樋野展正, 村上裕子, 亀ヶ盛俊介, 山本雅貴, 坂本健作, 横山茂之
  • Organizer: 第12回日本RNA学会年会
• [Remarks]
  • URL: http://www.phs.osaka-u.ac.jp/homepage/b018/
• [Remarks]
  • URL: http://protein.gsc.riken.jp/sakamoto/
• [Remarks]
  • URL: http://protein.gsc.rikeni.jp/sakamoto/
• [Patent(Industrial Property Rights)] 新規な非天然タンパク質及びその利用(N-(p-trifluoromethyldiazirinylbenzyloxycarbonyl)-lysine) (2011)
  • Inventor(s): 坂本健作,柳沢達男,樋野展正,伊良波史枝,横山茂之
  • Industrial Property Rights Holder: 理化学研究所
  • Filing Date: 2011-11-18
• [Patent(Industrial Property Rights)] 新規な非天然タンパク質、及びその利用(N-(p-trifluoromethyldiazirinylbenzyloxycarbonyl)-lysine) (2011)
  • Inventor(s): 坂本健作、柳沢達男、樋野展正、伊良波史枝、横山茂之
  • Industrial Property Rights Holder: 理化学研究所
  • Filing Date: 2011-11-18