1998 年度実績報告書

シグナル伝達系におけるレセプター・G蛋白質相互作用の原子レベルでの解析

研究課題

研究課題/領域番号	08308038
研究機関	京都大学
研究代表者	七田芳則京都大学, 大学院・理学研究科, 教授 (60127090)
研究分担者	藤吉好則京都大学, 大学院・理学研究科, 教授 (80142298) 徳永史生大阪大学, 大学院・理学研究科, 教授 (80025452) 津田基之姫路工業大学, 理学部, 教授 (60045458) 加茂直樹北海道大学, 薬学部, 教授 (10001976) 垣谷俊昭名古屋大学, 大学院・理学研究科, 教授 (90027350)
キーワード	受容体 / G蛋白質 / ロドプシン / 光 / バクテリオロドプシン / 情報 / 構造 / 機能
研究概要	本年度の研究実施計画に基づいて研究を行い、以下の成果を得た。 1, レセプターの機能を司るアミノ酸残基のうち、G蛋白質の活性化やG蛋白質サブタイプの選択性に関与するアミノ酸残基の同定を試みた。具体的には、ロドプシンとムスカリン受容体のキメラ蛋白質を作成することにより、G蛋白質の選択性には細胞内第3ループに存在する残基が、また、ロドプシンのG蛋白質活性化には細胞内第2ループのN端7残基が必須であることを明らかにした。また、ロドプシンとG蛋白質との相互作用部位の構造解析にフーリエ変換赤外スペクトル法を適用し、両蛋白質の相互作用構造を検討した。さらに、発色団のシス-トランス異性化反応の効率に影響するアミノ酸残基を、部位特異的変異と分光法を用いて同定した。また、G蛋白質と相互作用するタコロドプシンの中間状態を同定した。さらに、ウシロドプシンの3次元結晶を作製し、X線回折パターンを得た。 2. 分子動力学シミュレーションにより、バクテリオロドプシンにおける発色団の高効率光異性化反応を解析し、この反応に関与する発色団周りの原子配置について考察した。また、バクテリオロドプシンの光反応中間体におけるヘリックス構造の変化について理論計算を行った。さらに、ファラオニスフォボロドプシンを大腸菌中で発現させることに成功し、変異蛋白質の作製により、吸収極大や中間状態の寿命に関与するアミノ酸残基のいくつかを同定した。また、反応動力学的シミュレーションにより、レセプターからG蛋白質へ伝達効率と細胞応答との連関を考察した。 3. エンドセリン受容体のG蛋白質選択性を検討し、選択性を決定するアミノ酸残基を同定した。また、受容体の2次元結晶化条件を検討した。さらに、ニワトリ松果体に存在する光レセプターピノプシンが活性化するG蛋白質を同定した。

研究成果
(44件)

すべてその他

すべて文献書誌 (44件)

[文献書誌] T.Kakitani: "Deuterium substitution effect on the excited state dynamics of rhodopsin." J.Phys.Chem.107. 1334-1339 (1998)
[文献書誌] H.Kandori: "Cysteine S-H as a hydrogen-bonding probe in proteins." J.Am.Chem.Soc.120. 5828-5829 (1998)
[文献書誌] S.Tachibanaki: "Identification of a new intermediate state that binds but not activates transducin in the bleaching process of bovine rhodopsin." FEBS Lett.425. 126-130 (1998)
[文献書誌] H.Kandori: "Protein structural changes in bacteriorhodopsin upon photoisomerization as revealed by polarized FTIR spectroscopy." J.Phys.Chem.102. 7899-7905 (1998)
[文献書誌] H.Chosrowjan: "Rhodopsin emission in real time: A new aspect of the primary event in vision." J.Am.Chem.Soc.120. 9706-9707 (1998)
[文献書誌] T.Nagata: "The hydrogen bonding network of water molecules and the peptide backbone in the region connecting Asp83, Gly120 and Glu113 in bovine rhodopsin." Biochemistry. 37. 17216-17222 (1998)
[文献書誌] A.Terakita: "Selective activation of G-protein subtypes by vertebrate and invertebrate rhodopsins." FEBS Lett.439. 110-114 (1998)
[文献書誌] A.Wada: "Stereoselective synthesis of 11Z-9-demethyl-9-benzyl- and 9-phenyl-retinals and their interaction with bovine opsin." Bioorg.Med.Chem.Lett.8. 423-426 (1998)
[文献書誌] Y.Shichida: "Visual Pigment: G-protein-coupled receptor for light signal." Cell Mol Life Sci.54. 1299-1315 (1998)
[文献書誌] 寺北明久: "視物質と視細胞光情報伝達系の多様性-機能解析と分子系統解析の接点" 蛋白質・核酸・酵素. 44. 217-225 (1999)
[文献書誌] T.Doi: "Interactions of Endothelin Receptor Subtypes A and B with Gi,Go,and Gq in Reconstituted Phospholipid vesicles." Biochemistry. (in press.).
[文献書誌] Y.Hiroaki: "Influence of Various Nucleotides on the in situ Crystallization of Ca^<2+>-ATPase." Biochemica et Biophysica Acta.(in press.).
[文献書誌] I.Yamaguchi: "A novel method of estimating real 〔Ca2+〕i dynamics from fluorescence signals." Neurosci.Res.30. 91-98 (1998)
[文献書誌] T.Matsuhashi: "Complete suppression of ethanol-induced formation of megamitochondria by 4-hydroxy-2,2,6,6-tetra-methyl-piperidine-1-oxil (4-oh-TEMPO)." Free Radic.Biol.Med.24. 139-147 (1998)
[文献書誌] Y.Hirako: "Cleavage of BP 180,a 180kDa bullous pemphigoid antigens, yields a 120kDa collagenous extracellular polypeptide." J.Biol.Chem.273. 9711-9717 (1998)
[文献書誌] A.Yamazaki: "A possible role of RGS 9 in phototransduction: A bridge between the cGMP-phosphodiesterase system and the guanylyl cyclase system." J.Biol.Chem.273. 22169-22172 (1998)
[文献書誌] T.Okada: "Highly selective separation of rhodopsin from bovine rod outer segment membranes using combination of divalent cation and alkyl(thio)glucoside." Photochem.Photobiol.67. 495-499 (1998)
[文献書誌] K.Shimono: "V108 mutant of pharaonis phoborhodopsin: Substitution caused no absorption change but affected its M-state." J.Biochem.124. 404-409 (1998)
[文献書誌] K.Takeda: "A novel three-dimensional crystal of bacteriorhodopsin obtained by successive fusion of the vesicular assemblies." J.Mol.Biol.283. 463-474 (1998)
[文献書誌] T.Iwasa: "NMR and UV-VIS absorption spectroscopic studies on fluorinated octopus rhodopsin and its photoproducts." J.Phys.Chem.B. 102. 5602-5610 (1998)
[文献書誌] M.Nakagawa: "A novel photointermediate of octopus rhodopsin activates its G-protein." FEBS Lett.436. 259-262 (1998)
[文献書誌] S.Kikkawa: "A novel rhodopsin kinase in octopus photoreceptor possesses a pleckstrin homology domain and is activated by G protein bg subunits." J.Biol.Chem.273. 7441-7447 (1998)
[文献書誌] Y.Yamamoto: "Ab initio MO study on the potential energy surface of low-lying excited states of protonated Schiff base retinal." THEOCHEM. 451. 151-162 (1998)
[文献書誌] S.Yamamoto: "Ab initio MO study on the potential energy surface for twisting around C11=C12 bond of protonated Schiff base of retinal." THEOCHEM. (in press.).
[文献書誌] O.Hisatomi: "A novel type of G-protein-coupled receptor kinase, GRK7, in teleost cone photoreceptors." FEBS Lett.424. 159-164 (1998)
[文献書誌] T.Yoshikawa: "A deep brain photoreceptive molecule in the toad hypothalamus." FEBS Lett.424. 69-72 (1998)
[文献書誌] T.Matsuda: "Specific isoprenyl group linked to transducin gamma-subunit is a determinant of unique signaling properties among G proteins." Biochemistry. 37. 9843-9850 (1998)
[文献書誌] S.Matsuda: "Role of carboxyl-terminalcharges on S-modulin membrane affinity and inhibition of rhodopsin phosphorylation." Biochemistry. 38. 1310-1315 (1999)
[文献書誌] A.K.Satoh: "Rhodopsin transport and Rab expression in the carotenoid-deprived Drosophila Melanogaster" Zool.Sci.15. 651-659 (1998)
[文献書誌] Y.Wada: "Identification of rhodopsin in the pigeon deep brain." FEBS Lett.424. 53-56 (1998)
[文献書誌] Y.Yamazaki: "Interaction of the Protonated Schiff Base with the Peptide Backbone of Valine 49 and the Intervening Water Molecule in the N Photointermediate of Bacteriorhodopsin." Biochemistry. (1998)
[文献書誌] S.Nishimura: "Interaction between Photoactivated Rhodopsin and the C-Terminal Peptide of Transducin a Subunit Studied by FTIR Spectroscopy." Biochemistry. 37. 15816-15824 (1998)
[文献書誌] K.Mitsuoka: "Charge Distribution in the Structure of Bacteriorhodopsin Based on Electron Crystallography at 3A Resolution." J.Mol.Biol.(in press). (1999)
[文献書誌] K.Koyama: "Photoelectric response of halorhodopsin from Natronobacterium pharaonis." Bioelectrochem.Bioenerg.46. 289-292 (1998)
[文献書誌] K.Takao: "Azide accelerates the decay of M-intermediate of pharaonis phoborhodopsin." Biophys.Chem.73. 145-153 (1998)
[文献書誌] 小島大輔: "視物質の進化から見た色覚" 医学のあゆみ. 186. 175-179 (1998)
[文献書誌] Hiroo Imai: "Analysis of Amino Acid Residuesin Rhodopsin and Cone Visual Pigments that Determine their Molecular Properties. (Methods in Enzynology 分担執筆)(印刷中)" Academic Press, (1999)
[文献書誌] Yoshinori Shichida: "Heterogeniety of Rhodopsin Intermediate State Interacting with Transducin. (Methods in Enzynology 分担執筆)(印刷中)" Academic Press, (1999)
[文献書誌] Y.Shichida: "Visual pigment: Photochemistry and molecular evolution. in The Retinal Basis of Vision.(ed.J.Toyoda)(in press)" Elsevier Science, The Netherlands.,
[文献書誌] H.Okamoto: "Amplification of switching characteristics of biochemical-reaction networks involving Ca^<2+>/calmodulin-dependent protein kinase." Plenum Press.New York, 12 (1998)
[文献書誌] 深田吉孝: "視覚異常症" 中外医学社, 6 (1998)
[文献書誌] 垣谷俊昭: "光・物質・生命と反応" 丸善, 447 (1998)
[文献書誌] 津田基之: "視細胞における光電変換-その分子メカニズムを探る" ぶんしん出版, 185 (1998)
[文献書誌] 徳永史生: "動物の色覚色彩科学ハンドブック(日本色彩学会編)" 東大出版会, 4 (1998)

1998 年度 実績報告書

シグナル伝達系におけるレセプター・G蛋白質相互作用の原子レベルでの解析

研究代表者

七田 芳則 京都大学, 大学院・理学研究科, 教授 (60127090)

研究成果

[文献書誌] T.Kakitani: "Deuterium substitution effect on the excited state dynamics of rhodopsin." J.Phys.Chem.107. 1334-1339 (1998)

[文献書誌] H.Kandori: "Cysteine S-H as a hydrogen-bonding probe in proteins." J.Am.Chem.Soc.120. 5828-5829 (1998)

[文献書誌] S.Tachibanaki: "Identification of a new intermediate state that binds but not activates transducin in the bleaching process of bovine rhodopsin." FEBS Lett.425. 126-130 (1998)

[文献書誌] H.Kandori: "Protein structural changes in bacteriorhodopsin upon photoisomerization as revealed by polarized FTIR spectroscopy." J.Phys.Chem.102. 7899-7905 (1998)

[文献書誌] H.Chosrowjan: "Rhodopsin emission in real time: A new aspect of the primary event in vision." J.Am.Chem.Soc.120. 9706-9707 (1998)

[文献書誌] T.Nagata: "The hydrogen bonding network of water molecules and the peptide backbone in the region connecting Asp83, Gly120 and Glu113 in bovine rhodopsin." Biochemistry. 37. 17216-17222 (1998)

[文献書誌] A.Terakita: "Selective activation of G-protein subtypes by vertebrate and invertebrate rhodopsins." FEBS Lett.439. 110-114 (1998)

[文献書誌] A.Wada: "Stereoselective synthesis of 11Z-9-demethyl-9-benzyl- and 9-phenyl-retinals and their interaction with bovine opsin." Bioorg.Med.Chem.Lett.8. 423-426 (1998)

[文献書誌] Y.Shichida: "Visual Pigment: G-protein-coupled receptor for light signal." Cell Mol Life Sci.54. 1299-1315 (1998)

[文献書誌] 寺北明久: "視物質と視細胞光情報伝達系の多様性-機能解析と分子系統解析の接点" 蛋白質・核酸・酵素. 44. 217-225 (1999)

[文献書誌] T.Doi: "Interactions of Endothelin Receptor Subtypes A and B with Gi,Go,and Gq in Reconstituted Phospholipid vesicles." Biochemistry. (in press.).

[文献書誌] Y.Hiroaki: "Influence of Various Nucleotides on the in situ Crystallization of Ca^<2+>-ATPase." Biochemica et Biophysica Acta.(in press.).

[文献書誌] I.Yamaguchi: "A novel method of estimating real 〔Ca2+〕i dynamics from fluorescence signals." Neurosci.Res.30. 91-98 (1998)

[文献書誌] T.Matsuhashi: "Complete suppression of ethanol-induced formation of megamitochondria by 4-hydroxy-2,2,6,6-tetra-methyl-piperidine-1-oxil (4-oh-TEMPO)." Free Radic.Biol.Med.24. 139-147 (1998)

[文献書誌] Y.Hirako: "Cleavage of BP 180,a 180kDa bullous pemphigoid antigens, yields a 120kDa collagenous extracellular polypeptide." J.Biol.Chem.273. 9711-9717 (1998)

[文献書誌] A.Yamazaki: "A possible role of RGS 9 in phototransduction: A bridge between the cGMP-phosphodiesterase system and the guanylyl cyclase system." J.Biol.Chem.273. 22169-22172 (1998)

[文献書誌] T.Okada: "Highly selective separation of rhodopsin from bovine rod outer segment membranes using combination of divalent cation and alkyl(thio)glucoside." Photochem.Photobiol.67. 495-499 (1998)

[文献書誌] K.Shimono: "V108 mutant of pharaonis phoborhodopsin: Substitution caused no absorption change but affected its M-state." J.Biochem.124. 404-409 (1998)

[文献書誌] K.Takeda: "A novel three-dimensional crystal of bacteriorhodopsin obtained by successive fusion of the vesicular assemblies." J.Mol.Biol.283. 463-474 (1998)

[文献書誌] T.Iwasa: "NMR and UV-VIS absorption spectroscopic studies on fluorinated octopus rhodopsin and its photoproducts." J.Phys.Chem.B. 102. 5602-5610 (1998)

[文献書誌] M.Nakagawa: "A novel photointermediate of octopus rhodopsin activates its G-protein." FEBS Lett.436. 259-262 (1998)

[文献書誌] S.Kikkawa: "A novel rhodopsin kinase in octopus photoreceptor possesses a pleckstrin homology domain and is activated by G protein bg subunits." J.Biol.Chem.273. 7441-7447 (1998)

[文献書誌] Y.Yamamoto: "Ab initio MO study on the potential energy surface of low-lying excited states of protonated Schiff base retinal." THEOCHEM. 451. 151-162 (1998)

[文献書誌] S.Yamamoto: "Ab initio MO study on the potential energy surface for twisting around C11=C12 bond of protonated Schiff base of retinal." THEOCHEM. (in press.).

[文献書誌] O.Hisatomi: "A novel type of G-protein-coupled receptor kinase, GRK7, in teleost cone photoreceptors." FEBS Lett.424. 159-164 (1998)

[文献書誌] T.Yoshikawa: "A deep brain photoreceptive molecule in the toad hypothalamus." FEBS Lett.424. 69-72 (1998)

[文献書誌] T.Matsuda: "Specific isoprenyl group linked to transducin gamma-subunit is a determinant of unique signaling properties among G proteins." Biochemistry. 37. 9843-9850 (1998)

[文献書誌] S.Matsuda: "Role of carboxyl-terminalcharges on S-modulin membrane affinity and inhibition of rhodopsin phosphorylation." Biochemistry. 38. 1310-1315 (1999)

[文献書誌] A.K.Satoh: "Rhodopsin transport and Rab expression in the carotenoid-deprived Drosophila Melanogaster" Zool.Sci.15. 651-659 (1998)

[文献書誌] Y.Wada: "Identification of rhodopsin in the pigeon deep brain." FEBS Lett.424. 53-56 (1998)

[文献書誌] Y.Yamazaki: "Interaction of the Protonated Schiff Base with the Peptide Backbone of Valine 49 and the Intervening Water Molecule in the N Photointermediate of Bacteriorhodopsin." Biochemistry. (1998)

[文献書誌] S.Nishimura: "Interaction between Photoactivated Rhodopsin and the C-Terminal Peptide of Transducin a Subunit Studied by FTIR Spectroscopy." Biochemistry. 37. 15816-15824 (1998)

[文献書誌] K.Mitsuoka: "Charge Distribution in the Structure of Bacteriorhodopsin Based on Electron Crystallography at 3A Resolution." J.Mol.Biol.(in press). (1999)

[文献書誌] K.Koyama: "Photoelectric response of halorhodopsin from Natronobacterium pharaonis." Bioelectrochem.Bioenerg.46. 289-292 (1998)

[文献書誌] K.Takao: "Azide accelerates the decay of M-intermediate of pharaonis phoborhodopsin." Biophys.Chem.73. 145-153 (1998)

[文献書誌] 小島大輔: "視物質の進化から見た色覚" 医学のあゆみ. 186. 175-179 (1998)

[文献書誌] Hiroo Imai: "Analysis of Amino Acid Residuesin Rhodopsin and Cone Visual Pigments that Determine their Molecular Properties. (Methods in Enzynology 分担執筆)(印刷中)" Academic Press, (1999)

[文献書誌] Yoshinori Shichida: "Heterogeniety of Rhodopsin Intermediate State Interacting with Transducin. (Methods in Enzynology 分担執筆)(印刷中)" Academic Press, (1999)

[文献書誌] Y.Shichida: "Visual pigment: Photochemistry and molecular evolution. in The Retinal Basis of Vision.(ed.J.Toyoda)(in press)" Elsevier Science, The Netherlands.,

[文献書誌] H.Okamoto: "Amplification of switching characteristics of biochemical-reaction networks involving Ca^<2+>/calmodulin-dependent protein kinase." Plenum Press.New York, 12 (1998)

[文献書誌] 深田吉孝: "視覚異常症" 中外医学社, 6 (1998)

[文献書誌] 垣谷俊昭: "光・物質・生命と反応" 丸善, 447 (1998)

[文献書誌] 津田基之: "視細胞における光電変換-その分子メカニズムを探る" ぶんしん出版, 185 (1998)

[文献書誌] 徳永史生: "動物の色覚 色彩科学ハンドブック(日本色彩学会編)" 東大出版会, 4 (1998)

1998 年度実績報告書

七田芳則京都大学, 大学院・理学研究科, 教授 (60127090)

[文献書誌] 徳永史生: "動物の色覚色彩科学ハンドブック(日本色彩学会編)" 東大出版会, 4 (1998)