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

1998 Fiscal Year Annual Research Report

• Project/Area Number: 08308038
• Research Institution: KYOTO UNIVERSITY
• Principal Investigator: 七田 芳則 京都大学, 大学院・理学研究科, 教授 (60127090)
• Co-Investigator(Kenkyū-buntansha): 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)
• Keywords: 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

Research Abstract

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。

Research Products

• [Publications] T.Kakitani: "Deuterium substitution effect on the excited state dynamics of rhodopsin." J.Phys.Chem.107. 1334-1339 (1998)
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• [Publications] 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)
• [Publications] H.Kandori: "Protein structural changes in bacteriorhodopsin upon photoisomerization as revealed by polarized FTIR spectroscopy." J.Phys.Chem.102. 7899-7905 (1998)
• [Publications] H.Chosrowjan: "Rhodopsin emission in real time: A new aspect of the primary event in vision." J.Am.Chem.Soc.120. 9706-9707 (1998)
• [Publications] 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)
• [Publications] A.Terakita: "Selective activation of G-protein subtypes by vertebrate and invertebrate rhodopsins." FEBS Lett.439. 110-114 (1998)
• [Publications] 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)
• [Publications] Y.Shichida: "Visual Pigment: G-protein-coupled receptor for light signal." Cell Mol Life Sci.54. 1299-1315 (1998)
• [Publications] 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)
• [Publications] T.Doi: "Interactions of Endothelin Receptor Subtypes A and B with Gi,Go,and Gq in Reconstituted Phospholipid vesicles." Biochemistry. (in press.).
• [Publications] Y.Hiroaki: "Influence of Various Nucleotides on the in situ Crystallization of Ca^<2+>-ATPase." Biochemica et Biophysica Acta.(in press.).
• [Publications] I.Yamaguchi: "A novel method of estimating real 〔Ca2+〕i dynamics from fluorescence signals." Neurosci.Res.30. 91-98 (1998)
• [Publications] 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)
• [Publications] Y.Hirako: "Cleavage of BP 180,a 180kDa bullous pemphigoid antigens, yields a 120kDa collagenous extracellular polypeptide." J.Biol.Chem.273. 9711-9717 (1998)
• [Publications] 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)
• [Publications] 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)
• [Publications] K.Shimono: "V108 mutant of pharaonis phoborhodopsin: Substitution caused no absorption change but affected its M-state." J.Biochem.124. 404-409 (1998)
• [Publications] K.Takeda: "A novel three-dimensional crystal of bacteriorhodopsin obtained by successive fusion of the vesicular assemblies." J.Mol.Biol.283. 463-474 (1998)
• [Publications] T.Iwasa: "NMR and UV-VIS absorption spectroscopic studies on fluorinated octopus rhodopsin and its photoproducts." J.Phys.Chem.B. 102. 5602-5610 (1998)
• [Publications] M.Nakagawa: "A novel photointermediate of octopus rhodopsin activates its G-protein." FEBS Lett.436. 259-262 (1998)
• [Publications] 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)
• [Publications] 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)
• [Publications] 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.).
• [Publications] O.Hisatomi: "A novel type of G-protein-coupled receptor kinase, GRK7, in teleost cone photoreceptors." FEBS Lett.424. 159-164 (1998)
• [Publications] T.Yoshikawa: "A deep brain photoreceptive molecule in the toad hypothalamus." FEBS Lett.424. 69-72 (1998)
• [Publications] 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)
• [Publications] S.Matsuda: "Role of carboxyl-terminalcharges on S-modulin membrane affinity and inhibition of rhodopsin phosphorylation." Biochemistry. 38. 1310-1315 (1999)
• [Publications] A.K.Satoh: "Rhodopsin transport and Rab expression in the carotenoid-deprived Drosophila Melanogaster" Zool.Sci.15. 651-659 (1998)
• [Publications] Y.Wada: "Identification of rhodopsin in the pigeon deep brain." FEBS Lett.424. 53-56 (1998)
• [Publications] 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)
• [Publications] S.Nishimura: "Interaction between Photoactivated Rhodopsin and the C-Terminal Peptide of Transducin a Subunit Studied by FTIR Spectroscopy." Biochemistry. 37. 15816-15824 (1998)
• [Publications] K.Mitsuoka: "Charge Distribution in the Structure of Bacteriorhodopsin Based on Electron Crystallography at 3A Resolution." J.Mol.Biol.(in press). (1999)
• [Publications] K.Koyama: "Photoelectric response of halorhodopsin from Natronobacterium pharaonis." Bioelectrochem.Bioenerg.46. 289-292 (1998)
• [Publications] K.Takao: "Azide accelerates the decay of M-intermediate of pharaonis phoborhodopsin." Biophys.Chem.73. 145-153 (1998)
• [Publications] 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)
• [Publications] 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)
• [Publications] Yoshinori Shichida: "Heterogeniety of Rhodopsin Intermediate State Interacting with Transducin. (Methods in Enzynology 分担執筆)(印刷中)" Academic Press, (1999)
• [Publications] Y.Shichida: "Visual pigment: Photochemistry and molecular evolution. in The Retinal Basis of Vision.(ed.J.Toyoda)(in press)" Elsevier Science, The Netherlands.,
• [Publications] H.Okamoto: "Amplification of switching characteristics of biochemical-reaction networks involving Ca^<2+>/calmodulin-dependent protein kinase." Plenum Press.New York, 12 (1998)
• [Publications] 深田吉孝: "視覚異常症" 中外医学社, 6 (1998)
• [Publications] 垣谷俊昭: "光・物質・生命と反応" 丸善, 447 (1998)
• [Publications] 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)
• [Publications] 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)