Molecular mechanism of trafficking regulation of G protein-coupled receptor

• Project/Area Number: 25460326
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: General pharmacology
• Research Institution: Shiga University of Medical Science (2014-2015); Hokkaido University (2013)
• Principal Investigator: Terada Koji 滋賀医科大学, 医学部, 准教授 (70342722)
• Co-Investigator(Renkei-kenkyūsha): MIWA Soichi 北海道大学, 大学院医学研究科, 教授 (40157706)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2015: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2014: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2013: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: エンドセリン / ユビキチン / エンドサイトーシス / リサイクリング / エンドセリン受容体 / ユビキチン化 / Gタンパク質共役受容体

Outline of Final Research Achievements

Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, resemble each other, but upon ET-1 stimulation, they follow different intracellular trafficking pathways. In this study, we showed that ETBR but not ETAR was ubiquitinated upon ET-1 stimulation, and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated “5KR mutant”) in which 5 lysine residues in C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was with Rab7, a marker of late endosome/lysosome. These results demonstrate that agonist-induced ubiquitination in C-tail of ETBR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome.

Research Products

• [Journal Article] Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells (2016)
  • Author(s): Horinouchi T, Hoshi A, Harada T, Higa T, Karki S, Terada T, Higashi T, Mai Y, Nepal P, Mazaki Y, Miwa S
  • Journal Title: Br J Pharmacol Volume: 173 Issue: 6 Pages: 1018-1032
  • DOI: 10.1111/bph.13406
  • NAID: 120005981358
  • Peer Reviewed
• [Journal Article] Agonist-promoted ubiquitination differentially regulates receptor trafficking of endothelin type A and type B receptors. (2014)
  • Author(s): Terada K., Horinouchi T., Fujioka Y., Higashi T., Nepal P., Horiguchi M., Sarita K., Hatate C., Hoshi A., Harada T., Mai Y., Ohba Y. and Miwa S.
  • Journal Title: J. Biol. Chem. Volume: 289 Issue: 51 Pages: 35283-35295
  • DOI: 10.1074/jbc.m113.544171
  • NAID: 120005549588
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] A simple and rapid method for standard preparation of gas phase extract of cigarette smoke. (2014)
  • Author(s): Higashi T., Mai Y., Noya Y., Horinouchi T., Terada K., Hoshi A., Nepal P., Harada T., Horiguchi M., Hatate C., Kuge Y. and Miwa S.
  • Journal Title: PLos ONE Volume: 9 Issue: 9 Pages: e107856-e107856
  • DOI: 10.1371/journal.pone.0107856
  • NAID: 120005496512
  • Peer Reviewed / Open Access
• [Journal Article] Endothelin-1 activates extracellular signal-regulated kinases 1/2 via transactivation of platelet-derived growth factor receptor in rat L6 myoblasts. (2014)
  • Author(s): Harada T, Horinouchi T, Higa T, Hoshi A, Higashi T, Terada K, Mai Y, Nepal P, Horiguchi M, Hatate C, Miwa S.
  • Journal Title: Life Sci. Volume: 104 Issue: 1-2 Pages: 24-31
  • DOI: 10.1016/j.lfs.2014.04.002
  • Peer Reviewed
• [Journal Article] Filamin-interacting proteins, Cfm1 and Cfm2, are essential for the formation of cartilaginous skeletal elements. (2014)
  • Author(s): Mizuhashi K, Kanamoto T, Moriishi T, Muranishi Y, Miyazaki T, Terada K, Omori Y, Ito M, Komori T, Furukawa T.
  • Journal Title: Human molecular genetics Volume: - Issue: 11 Pages: 2953-2967
  • DOI: 10.1093/hmg/ddu007
  • Peer Reviewed / Open Access
• [Journal Article] Endothelin receptor signaling: new insight into its regulatory mechanisms (2013)
  • Author(s): Harada T., Horinouchi T., Higa T., Hoshi A., Higashi T., Terada K., Mai Y., Nepal P., Horiguchi M., Hatate C. and Miwa S.
  • Journal Title: J Pharmacol Sci. Volume: 123 Pages: 85-101
  • NAID: 130003382599
  • Peer Reviewed
• [Journal Article] Identification of stable cytotoxic factors in the gas phase extract of cigarette smoke and pharmacological characterization of their cytotoxicity. (2013)
  • Author(s): Noya Y, Seki K, Asano H, Mai Y, Horinouchi T, Higashi T, Terada K, Hatate C, Hoshi A, Nepal P, Horiguchi M, Kuge Y, Miwa S.
  • Journal Title: Toxicology Volume: 314 Issue: 1 Pages: 1-10
  • DOI: 10.1016/j.tox.2013.08.015
  • Peer Reviewed
• [Presentation] エンドセリンB型受容体の細胞内動態におけるユビキチン化の役割 (2014)
  • Author(s): 寺田晃士、堀之内孝広、東恒仁、堀口美香、Prabha Nepal、Sarita Karki、旗手千鶴、原田拓弥、眞井洋輔、星暁壮、三輪聡一
  • Organizer: 第65回日本薬理学会北部会
  • Place of Presentation: コラッセふくしま（福島県）
  • Year and Date: 2014-09-26 – 2014-09-27
• [Presentation] エンドセリンB型受容体のトラフィッキングにおける受容体ユビキチン化の役割 (2014)
  • Author(s): 寺田晃士、堀之内孝広、東恒仁、Nepal Prabha、堀口美香、旗手千鶴、星暁壮、眞井洋輔、原田拓弥、三輪聡一
  • Organizer: 第87回日本薬理学会年会
  • Place of Presentation: 仙台（仙台国際センター）
• [Presentation] エンドセリンB型受容体のET-1刺激誘発的な細胞内移行とシグナルの制御におけるユビキチン修飾の役割
  • Author(s): 寺田晃士、堀之内孝広、東恒仁、Nepal Prabha、堀口美香、旗手千鶴、星暁壮、三輪聡一
  • Organizer: 第64回日本薬理学会北部会
  • Place of Presentation: 旭川（大雪クリスタルホール）