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

2013 Fiscal Year Final Research Report

• Project/Area Number: 24790064
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Biological pharmacy
• Research Institution: Tohoku 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
• Keywords: G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

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.

Research Products

• [Journal Article] Overexpression of autotaxin, a lysophosphatidic acid-producing enzyme, enhances cardia bifida induced by hypo-sphingosine-1-phosphate signaling in zebrafish embryo (2014)
  • 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: Journal of Biochemistry Volume: 155 Pages: 235-41
  • DOI: 10.1093/jb/mvt114
  • Peer Reviewed
• [Journal Article] Prestwich GD. Phosphorothioate analogs of sn-2 radyl lysophosphatidic acid (LPA) : metabolically stabilized LPA receptor agonists (2013)
  • Author(s): Jiang G, Inoue A, Aoki J
  • Journal Title: Bioorganic & Medicinal Chemistry Letters Volume: 23 Pages: 1865-9
  • DOI: 10.1016/j.bmcl.2013.01.002
  • 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-PLA1a<alpha> in autosomal recessive woolly hair/hypotrichosis (2013)
  • Author(s): Yoshizawa M, Nakamura M, Farooq M, Inoue A, Aoki J, Shimomura Y
  • Journal Title: Journal of Dermatological Science Volume: 72 Pages: 61-4
  • DOI: 10.1016/j.jdermsci.2013.05.001
  • Peer Reviewed
• [Journal Article] TGFαshedding assay : an accurate and versatile method for detecting GPCR activation (2012)
  • Author(s): Inoue A, Ishiguro J, Kitamura H, Arima N, Okutani M, Shuto A, Higashiyama S, Ohwada T, Arai H, Makide K, and Aoki J
  • Journal Title: Nature Methods Volume: 9 Pages: 1021-9
  • DOI: 10.1038/nmeth.2172
  • Peer Reviewed
• [Presentation] Dissecting G protein coupling of GPCRs using chimeric Gαsubunits and TGFαshedding assay (2013)
  • Author(s): Inoue A, Aoki J
  • Organizer: International Conference on Pharmacology and Drug Development (ICoPaDD)
  • Place of Presentation: University of Singapore, Singapore
  • Year and Date: 20131209-11
• [Presentation] Identification and analysis of GPCRs that recognize oxidized phospholipids (2013)
  • 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
  • Year and Date: 20130911-13
• [Presentation] A new tool to analyze G protein coupling of lipid mediator receptors (2013)
  • 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.
  • Year and Date: 20130804-09
• [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
  • Year and Date: 20130510-11
• [Presentation] A tool to comprehensively dissect G protein coupling of GPCRs : application of TGFαshedding assay and co-expression of chimeric Gαsubunits (2013)
  • Author(s): Inoue A, Aoki J
  • Organizer: Gordon Research Conferences, Molecular Pharmacology. Renaissance Tuscany Il Ciocco Resort
  • Place of Presentation: Lucca (Barga), Italy.
  • Year and Date: 20130428-0503
• [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
  • Year and Date: 20130328-30
• [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
  • Year and Date: 20121214-16
• [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
  • Year and Date: 20120607-08
• [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248 (2014)
  • Author(s): 井上飛鳥、青木淳賢
• [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 24 (2013)
  • Author(s): 井上飛鳥、青木淳賢
• [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
• [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31 (2013)
  • Author(s): 井上飛鳥、青木淳賢
• [Remarks]
  • URL: http://www.pharm.tohoku.ac.jp/~seika/H24/index.html
• [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
  • Industrial Property Number: PCT/JP2014/000992
  • Filing Date: 2014-02-25
  • Overseas