1996 Fiscal Year Annual Research Report

神経伝達物質の放出におけるRab3A-Rabphilin-3A系の機能と作用機構

Research Project

Project/Area Number	08680706
Research Institution	Osaka University
Principal Investigator	白滝博通大阪大学, 医学部, 助手 (90249962)
Co-Investigator(Kenkyū-buntansha)	佐々木卓也大阪大学, 医学部, 助手 (40241278)
Keywords	神経伝達物質 / Rab3A / Rabphilin-3A / Rab GDT / Rab3 GAP
Research Abstract	私共の研究室では、神経伝達物質の放出反応における低分子量G蛋白質Rab3Aの活性化機構と作用機構について解析を進めており、Rab3Aの標的蛋白質としてC2様領域を2個有するRabphilin-3Aを見い出している。さらに、Rab3Aの活性制御蛋白質としてRab GDIを見い出している。本研究で、私共は、Rab3AのGTPase活性を促進する活性制御蛋白質Rab3 GAPをラット大脳より精製してcDNAをクローニングし、その一次構造を決定した。Rab3 GAPは、C末端に翻訳後脂質修飾を受けたRab3Aを含むRab3ファミリー全てに作用したが、その他のRabには作用しなかった。さらに、私共は、Rab3A-Rac GDI複合体に作用してRab3AをRab GDIから解離させる活性制御蛋白質Rab3A GDFをラットのシナプス小胞膜画分において同定し、種々のカラムクロマトグラムにて高度に精製した。Rab3A GDFは、熱処理およびトリプシン処理にて失活する蛋白分子であった。一方、私共は、Rabphilin-3AがRab3 GAPに抑制的に作用してRab3AをGTP結合型に保持することを明らかにしているが、本研究で、私共は、Rab3AのC末端の翻訳後脂質修飾部がシナプス小胞膜リン脂質に結合することにより、GTP-Rab3AからのGTPの解離が抑制されることを明らかにした。このことから、Rab3AはRabphilin-3Aと蛋白質-蛋白質結合すると共に、シナプス小胞膜リン脂質とlipid-lipid結合してシナプス小胞に局在し、GTP結合型に保持されている可能性が高くなった。このように、本研究は予想以上に進展し、当初の目的はほぼ完全に達成することができた。

Research Products

(17 results)

All Other

All Publications (17 results)

[Publications] Orita,S.: "Doc2 enhances Ca^<2+>-dependent exocytosis from PC12 cells." J.Biol.Chem.271. 7257-7260 (1996)
[Publications] Fujita,Y.: "Phosphorylation of Munc-18/n-Secl/rbSecl by protein kinase C-Its implication in regulating the interaction of Munc-18/n-Secl/rbSecl with Syntaxin." J.Biol.Chem.271. 7265-7268 (1996)
[Publications] Shimizu,K.: "SMAP,an Smg GDS-associating protein having Arm repeats and phosphorylated by Src tyrosine kinase." J.Biol.Chem.271. 27013-27017 (1996)
[Publications] Kato,M.: "Physical and functional interaction of Rabphilin-3A with α-Actinin." J.Biol.Chem.271. 31775-31778 (1996)
[Publications] Hirao,M.: "Regulation mechanism of ERM (Ezrin/Radixin/Moesin) protein/plasma membrane association-Possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway." J.Cell Biol.135. 37-51 (1996)
[Publications] Masumoto,N.: "Involvement of Rabphilin-3A in cortical granule exocytosis in mouse eggs." J.Cell Biol.135. 1741-1747 (1996)
[Publications] Matsuda,S.: "A membrane-associated GDP/GTP exchange protein specific for Rho small GTP-binding protein-Partial purification and characterization from rat brain." Oncogene. 12. 915-920 (1996)
[Publications] Taniguchi,T.: "Cholecystokinin-B/gastrin receptors mediate rapid formation of actin stress fibers." Oncogene. 12. 1357-1360 (1996)
[Publications] Komuro,R.: "Involvement of rabphilin-3A in Ca^<2+>-dependent exocytosis from PC12 cells." Biochem.Biophys.Res.Commun.219. 435-440 (1996)
[Publications] Senbonmatsu,T.: "Interaction of Rabphilin3 with synaptic vesicles through multiple regions." Biochem.Biophys.Res.Commun.228. 567-572 (1996)
[Publications] Oishi,H.: "Interaction of both the C2A and C2B domains of rabphilin3 with Ca^<2+> and pospholipid." Biochem.Biophys.Res.Commun.229. 498-503 (1996)
[Publications] Komuro,R.: "Involvement of Rho and Rac small G proteins and Rho GDI in Ca^<2+>-dependent exocytosis from PC12 cells." Genes to Cells. 1. 943-951 (1996)
[Publications] Takai,Y.: "Rab3A small GTP-binding protein in Ca^<2+>-dependent exocytosis." Genes to Cells. 1. 615-632 (1996)
[Publications] Fukui,K.: "Isolation and characterization of a GTPase activating protein specific for the Rab3 subfamily of small G proteins." J.Biol.Chem.272. 4655-4658 (1997)
[Publications] Kotani,H.: "Rho regulates association of both the ERM family and vinculin with the plasma membrane in MDCK cells." Oncogene. (in press). (1997)
[Publications] Naito,A.: "Molecular cloning of mouse Doc2α and distribution of its mRNA in adult mouse brain." Mol.Brain.Res.(in press). (1997)
[Publications] Jin-no,Y.: "A novel function of the C-terminal lipid moieties of Ran3A small G protein implicated in Ca^<2+>-dependent exocytosis-Inhibition of interaction with GTP and reduction of this inhibition by phospholipid." Genes to Cells. (in press). (1997)

1996 Fiscal Year Annual Research Report

神経伝達物質の放出におけるRab3A-Rabphilin-3A系の機能と作用機構

Principal Investigator

白滝 博通 大阪大学, 医学部, 助手 (90249962)

Research Products

[Publications] Orita,S.: "Doc2 enhances Ca^<2+>-dependent exocytosis from PC12 cells." J.Biol.Chem.271. 7257-7260 (1996)

[Publications] Fujita,Y.: "Phosphorylation of Munc-18/n-Secl/rbSecl by protein kinase C-Its implication in regulating the interaction of Munc-18/n-Secl/rbSecl with Syntaxin." J.Biol.Chem.271. 7265-7268 (1996)

[Publications] Shimizu,K.: "SMAP,an Smg GDS-associating protein having Arm repeats and phosphorylated by Src tyrosine kinase." J.Biol.Chem.271. 27013-27017 (1996)

[Publications] Kato,M.: "Physical and functional interaction of Rabphilin-3A with α-Actinin." J.Biol.Chem.271. 31775-31778 (1996)

[Publications] Hirao,M.: "Regulation mechanism of ERM (Ezrin/Radixin/Moesin) protein/plasma membrane association-Possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway." J.Cell Biol.135. 37-51 (1996)

[Publications] Masumoto,N.: "Involvement of Rabphilin-3A in cortical granule exocytosis in mouse eggs." J.Cell Biol.135. 1741-1747 (1996)

[Publications] Matsuda,S.: "A membrane-associated GDP/GTP exchange protein specific for Rho small GTP-binding protein-Partial purification and characterization from rat brain." Oncogene. 12. 915-920 (1996)

[Publications] Taniguchi,T.: "Cholecystokinin-B/gastrin receptors mediate rapid formation of actin stress fibers." Oncogene. 12. 1357-1360 (1996)

[Publications] Komuro,R.: "Involvement of rabphilin-3A in Ca^<2+>-dependent exocytosis from PC12 cells." Biochem.Biophys.Res.Commun.219. 435-440 (1996)

[Publications] Senbonmatsu,T.: "Interaction of Rabphilin3 with synaptic vesicles through multiple regions." Biochem.Biophys.Res.Commun.228. 567-572 (1996)

[Publications] Oishi,H.: "Interaction of both the C2A and C2B domains of rabphilin3 with Ca^<2+> and pospholipid." Biochem.Biophys.Res.Commun.229. 498-503 (1996)

[Publications] Komuro,R.: "Involvement of Rho and Rac small G proteins and Rho GDI in Ca^<2+>-dependent exocytosis from PC12 cells." Genes to Cells. 1. 943-951 (1996)

[Publications] Takai,Y.: "Rab3A small GTP-binding protein in Ca^<2+>-dependent exocytosis." Genes to Cells. 1. 615-632 (1996)

[Publications] Fukui,K.: "Isolation and characterization of a GTPase activating protein specific for the Rab3 subfamily of small G proteins." J.Biol.Chem.272. 4655-4658 (1997)

[Publications] Kotani,H.: "Rho regulates association of both the ERM family and vinculin with the plasma membrane in MDCK cells." Oncogene. (in press). (1997)

[Publications] Naito,A.: "Molecular cloning of mouse Doc2α and distribution of its mRNA in adult mouse brain." Mol.Brain.Res.(in press). (1997)

[Publications] Jin-no,Y.: "A novel function of the C-terminal lipid moieties of Ran3A small G protein implicated in Ca^<2+>-dependent exocytosis-Inhibition of interaction with GTP and reduction of this inhibition by phospholipid." Genes to Cells. (in press). (1997)

白滝博通大阪大学, 医学部, 助手 (90249962)