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Screening for ligands of G protein-coupled receptors using the newly developed TGFalpha shedding assay

Research Project

Project/Area Number 24790064
Research Category

Grant-in-Aid for Young Scientists (B)

Allocation TypeMulti-year Fund
Research Field Biological pharmacy
Research InstitutionTohoku University

Principal Investigator

INOUE Asuka  東北大学, 薬学研究科(研究院), 助手 (50525813)

Project Period (FY) 2012-04-01 – 2014-03-31
Project Status Completed (Fiscal Year 2013)
Budget Amount *help
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2013: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2012: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
KeywordsGタンパク質共役型受容体 / GPCR / TGFalpha / リゾリン脂質 / リゾホスファチジルセリン / CRISPR / HEK293 / エクトドメイン切断 / TRPチャネル / TGFα / オーファン / P2Y10 / GPR174
Research Abstract

In this KAKENHI project, by employing the newly developed transforming growth factor-alpha (TGFalpha) shedding assay, I screened for G protein-coupled receptors (GPCRs) that respond to bioactive molecules and developed an assay system that can quantify coupling efficiency of heterotrimeric G proteins. I identified three orphan GPCRs (P2Y10, GPR174, A630033H20) as lysophoshadylserine-specific receptors, one GPCR as a lysophoshadylglucoside receptor and two GPCRs as oxidized phospholipid receptors. I generated G protein-deficient HEK293 cells using the CRISPR-Cas9 system and successfully applied them to analyze G protein coupling of GPCRs.

Report

(3 results)
  • 2013 Annual Research Report   Final Research Report ( PDF )
  • 2012 Research-status Report
  • Research Products

    (34 results)

All 2014 2013 2012 Other

All Journal Article (7 results) (of which Peer Reviewed: 6 results,  Open Access: 2 results) Presentation (17 results) (of which Invited: 2 results) Book (4 results) Remarks (3 results) Patent(Industrial Property Rights) (3 results) (of which Overseas: 2 results)

  • [Journal Article] Overexpression of autotaxin, a lysophosphatidic acid-producing enzyme, enhances cardia bifida induced by hypo-sphingosine-1-phosphate signaling in zebrafish embryo2014

    • Author(s)
      Nakanaga K, Hama K, Kano K, Sato T, Yukiura H, Inoue A, Saigusa D, Tokuyama H, Tomioka Y, Nishina H, Kawahara A, Aoki J
    • Journal Title

      J. Biochem

      Volume: 155 Issue: 4 Pages: 235

    • DOI

      10.1093/jb/mvt114

    • NAID

      40020035029

    • Related Report
      2013 Annual Research Report 2013 Final Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Prestwich GD. Phosphorothioate analogs of sn-2 radyl lysophosphatidic acid (LPA) : metabolically stabilized LPA receptor agonists2013

    • Author(s)
      Jiang G, Inoue A, Aoki J
    • Journal Title

      Bioorganic & Medicinal Chemistry Letters

      Volume: 23 Issue: 6 Pages: 1865-9

    • DOI

      10.1016/j.bmcl.2013.01.002

    • Related Report
      2013 Final Research Report 2012 Research-status Report
    • Peer Reviewed
  • [Journal Article] A novel mutation, c.699C>G (p.C233W), in the LIPH gene leads to a loss of the hydrolytic activity and the LPA6 activation ability of PA-PLA1α in autosomal recessive wooly hair/hypotrichosis.2013

    • Author(s)
      Yoshizawa M, Nakamura M, Farooq M, Inoue A, Aoki J, Shimomura Y.
    • Journal Title

      J Dermatol Sci

      Volume: 72 Issue: 1 Pages: 61-4

    • DOI

      10.1016/j.jdermsci.2013.05.001

    • Related Report
      2013 Annual Research Report 2013 Final Research Report
    • Peer Reviewed
  • [Journal Article] Mitochondria-type GPAT is required for mitochondrial fusion2013

    • Author(s)
      Yohsuke Ohba, Takeshi Sakuragi, Eriko Kage-Nakadai, Naoko H Tomioka, Nozomu Kono, Rieko Imae, Asuka Inoue, Junken Aoki, Naotada Ishihara, Takao Inoue, Shohei Mitani and Hiroyuki Arai
    • Journal Title

      The EMBO Journal

      Volume: 32 Issue: 9 Pages: 1265

    • DOI

      10.1038/emboj.2013.77

    • Related Report
      2013 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] 新規GPCR活性化検出法を利用したオーファン受容体のリガンドの同定2013

    • Author(s)
      井上飛鳥
    • Journal Title

      生化学

      Volume: 85 Pages: 1029-33

    • NAID

      40019893957

    • Related Report
      2013 Annual Research Report
  • [Journal Article] TGFα shedding assay: An accurate and versatile method for detecting GPCR activation2012

    • Author(s)
      Asuka Inoue
    • Journal Title

      Nat Methods

      Volume: 9 Issue: 10 Pages: 1021

    • DOI

      10.1038/nmeth.2172

    • Related Report
      2013 Final Research Report 2012 Research-status Report
    • Peer Reviewed
  • [Journal Article] Synthesis and biological evaluation of optically active Ki164252012

    • Author(s)
      Sato, T. Sugimoto, K. Inoue, A. Okudaira, S. Aoki, J. Tokuyama, H.
    • Journal Title

      Bioorg Med Chem Lett

      Volume: 22 Issue: 13 Pages: 4323-4326

    • DOI

      10.1016/j.bmcl.2012.05.012

    • Related Report
      2012 Research-status Report
    • Peer Reviewed
  • [Presentation] Development of a versatile method to quantify coupling of all the four heterotrimeric G proteins2014

    • Author(s)
      Asuka Inoue and Junken Aoki
    • Organizer
      Keystone Symposia Z1: G Protein-Coupled Receptors: Structural Dynamics and Functional Implications (2014), USA
    • Place of Presentation
      Snowbird Resort, Snowbird, Utah, USA
    • Related Report
      2013 Annual Research Report
  • [Presentation] Dissecting G protein coupling of GPCRs using chimeric Gαsubunits and TGFαshedding assay2013

    • Author(s)
      Inoue A, Aoki J
    • Organizer
      International Conference on Pharmacology and Drug Development (ICoPaDD)
    • Place of Presentation
      University of Singapore, Singapore
    • Related Report
      2013 Final Research Report
  • [Presentation] Identification and analysis of GPCRs that recognize oxidized phospholipids2013

    • Author(s)
      Inoue A, Ishiguro J, Aoki J
    • Organizer
      第86回日本生化学会大会
    • Place of Presentation
      横浜、神奈川
    • Related Report
      2013 Annual Research Report 2013 Final Research Report
  • [Presentation] A new tool to analyze G protein coupling of lipid mediator receptors2013

    • Author(s)
      Inoue A, Aoki J
    • Organizer
      FASEB Summer Research Conferences, Lysophospholipid and other Related Mediators - From Bench to Clinic
    • Place of Presentation
      Niseko, Hokkaido, Japan.
    • Related Report
      2013 Final Research Report
  • [Presentation] TGFαのエクトドメイン切断を利用した新規GPCRリガンド探索のアッセイ系2013

    • Author(s)
      井上飛鳥、青木淳賢
    • Organizer
      第10回GPCR研究会
    • Place of Presentation
      お台場、東京
    • Related Report
      2013 Annual Research Report 2013 Final Research Report
  • [Presentation] A tool to comprehensively dissect G protein coupling of GPCRs : application of TGFαshedding assay and co-expression of chimeric Gαsubunits2013

    • Author(s)
      Inoue A, Aoki J
    • Organizer
      Gordon Research Conferences, Molecular Pharmacology. Renaissance Tuscany Il Ciocco Resort
    • Place of Presentation
      Lucca (Barga), Italy.
    • Related Report
      2013 Final Research Report
  • [Presentation] TGFα切断を用いたGPCRと三量体Gタンパク質の共役活性の評価系の確立2013

    • Author(s)
      井上飛鳥、青木淳賢
    • Organizer
      日本薬学会第133年会
    • Place of Presentation
      横浜、パシフィコ横浜
    • Related Report
      2013 Final Research Report
  • [Presentation] A tool to comprehensively dissect G protein coupling of GPCRs: application of TGFα shedding assay and co-expression of chimeric Gα subunits2013

    • Author(s)
      Asuka Inoue and Junken Aoki
    • Organizer
      Gordon Research Conferences, Molecular Pharmacology
    • Place of Presentation
      Renaissance Tuscany Il Ciocco Resort, Lucca (Barga), Italy
    • Related Report
      2013 Annual Research Report
  • [Presentation] 酸化リン脂質を認識するGタンパク質共役型受容体2013

    • Author(s)
      井上飛鳥、石黒純、青木淳賢
    • Organizer
      第55回日本脂質生化学会
    • Place of Presentation
      松島、宮城
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] A new tool to analyze G protein coupling of lipid mediator receptors2013

    • Author(s)
      Asuka Inoue and Junken Aoki
    • Organizer
      FASEB Summer Research Conferences, Lysophospholipid and other Related Mediators - From Bench to Clinic
    • Place of Presentation
      ニセコ、北海道
    • Related Report
      2013 Annual Research Report
  • [Presentation] Dissecting G protein coupling of GPCRs using chimeric Gα subunits and TGFα shedding assay2013

    • Author(s)
      Asuka Inoue and Junken Aoki
    • Organizer
      International Conference on Pharmacology and Drug Development (ICoPaDD) Singapore
    • Place of Presentation
      University of Singapore, Singapore
    • Related Report
      2013 Annual Research Report
    • Invited
  • [Presentation] TGFα切断を用いたGPCRと三量体Gタンパク質の共役活性の評価系の確立2013

    • Author(s)
      井上飛鳥、青木淳賢
    • Organizer
      日本薬学会第133年会
    • Place of Presentation
      横浜
    • Related Report
      2012 Research-status Report
  • [Presentation] 毛髪形成メカニズムから派生したGタンパク質共役型受容体(GPCR)の新規検出系の開発とその応用2012

    • Author(s)
      井上飛鳥、石黒純、巻出久美子、青木淳賢
    • Organizer
      第85回日本生化学会大会
    • Place of Presentation
      福岡、福岡国際会議場
    • Related Report
      2013 Final Research Report
  • [Presentation] TGFα切断を用いた脂質メディエーター受容体の検出法の開発2012

    • Author(s)
      井上飛鳥、石黒純、巻出久美子、青木淳賢
    • Organizer
      第54回日本脂質生化学
    • Place of Presentation
      福岡、九州大学
    • Related Report
      2013 Final Research Report
  • [Presentation] TGFα切断を用いた脂質メディエーター受容体の検出法の開発2012

    • Author(s)
      井上飛鳥、石黒純、巻出久美子、青木淳賢
    • Organizer
      第54回日本脂質生化学
    • Place of Presentation
      福岡
    • Related Report
      2012 Research-status Report
  • [Presentation] 毛髪形成メカニズムから派生したGタンパク質共役型受容体(GPCR)の新規検出系の開発とその応用2012

    • Author(s)
      井上飛鳥、石黒純、巻出久美子、青木淳賢
    • Organizer
      第85回日本生化学会大会
    • Place of Presentation
      福岡
    • Related Report
      2012 Research-status Report
  • [Presentation] Development of a versatile GPCR assay and its application for ligand identification of orphan GPCRs2012

    • Author(s)
      Asuka Inoue, Jun Ishiguro, Kumiko Makide and Junken Aoki
    • Organizer
      LIPID MAPS Annual Meeting 2012
    • Place of Presentation
      San Diego, USA
    • Related Report
      2012 Research-status Report
  • [Book] リゾホスファチジン酸(LPA)の生理的機能」(総説)、医学のあゆみ,生命を支える脂質−最新の研究と臨床2482014

    • Author(s)
      井上飛鳥、青木淳賢
    • Related Report
      2013 Final Research Report
  • [Book] 「【最新生理活性脂質研究-実験手法、基礎的知識とその応用-】(第1章)技術編TGFα切断を用いたGPCR活性化の新しい検出法」(解説)、遺伝子医学MOOK 242013

    • Author(s)
      井上飛鳥、青木淳賢
    • Related Report
      2013 Final Research Report
  • [Book] 【最新生理活性脂質研究-実験手法、基礎的知識とその応用-】(第1章)技術編TGFα切断を用いたGPCR活性化の新しい検出法(解説)2013

    • Author(s)
      井上飛鳥
    • Related Report
      2013 Final Research Report
  • [Book] TGFα切断を用いたGPCRの高感度・高精度な活性化検出法」(解説)、実験医学312013

    • Author(s)
      井上飛鳥、青木淳賢
    • Related Report
      2013 Final Research Report
  • [Remarks]

    • URL

      http://www.pharm.tohoku.ac.jp/~seika/H24/index.html

    • Related Report
      2013 Final Research Report
  • [Remarks] 東北大学大学院薬学研究科分子細胞生化学分野

    • URL

      http://www.pharm.tohoku.ac.jp/~seika/H24/index.html

    • Related Report
      2013 Annual Research Report
  • [Remarks] 東北大学大学院薬学研究科分子細胞生化学分野

    • URL

      http://www.pharm.tohoku.ac.jp/~seika/H24/index.html

    • Related Report
      2012 Research-status Report
  • [Patent(Industrial Property Rights)] Gタンパク質共役型受容体のシグナル伝達の検出方法2014

    • Inventor(s)
      青木淳賢、井上 飛鳥
    • Industrial Property Rights Holder
      青木淳賢、井上 飛鳥
    • Industrial Property Rights Type
      特許
    • Filing Date
      2014-02-25
    • Related Report
      2013 Final Research Report
    • Overseas
  • [Patent(Industrial Property Rights)] Gタンパク質共役型受容体のシグナル伝達の検出方法2014

    • Inventor(s)
      青木淳賢、井上飛鳥
    • Industrial Property Rights Holder
      青木淳賢、井上飛鳥
    • Industrial Property Rights Type
      特許
    • Filing Date
      2014-02-25
    • Related Report
      2013 Annual Research Report
    • Overseas
  • [Patent(Industrial Property Rights)] 自己免疫疾患治療薬2012

    • Inventor(s)
      青木淳賢、巻出久美子、井上飛鳥、大和田智彦他
    • Industrial Property Rights Holder
      青木淳賢、巻出久美子、井上飛鳥、大和田智彦他
    • Industrial Property Rights Type
      特許
    • Filing Date
      2012-05-18
    • Related Report
      2012 Research-status Report

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Published: 2013-05-31   Modified: 2019-07-29  

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