Single Molecular Biochemistry of G protein signaling system in photoreceptor cell

感光细胞G蛋白信号系统的单分子生物化学

• 批准号: 15201027
• 负责人: HAYASHI Fumio
• 金额: $ 28.7万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15201027/
• 关键词: single molecule observation; rhodopsin; G protein; GPCR; photoreceptor; dimerization; transducin; signaling system; 光受容; 全反射顕微鏡; ホスホジエステラーゼ; ホスホジエステラーゼ6; エバネッセンス

We have tried to construct a foundation of single molecular biochemistry of G protein mediated signal transduction system in vertebrate rod photoreceptor cells. By using a single fluorescent molecule imaging technique, we have succeeded to observe single molecular behavior of three key components of vertebrate phototransduction, i.e. a typical GPCR rhodopsin, G protein transducin, and its target enzyme cGMP-PDE. We have employed near far-red fluorescent-labeled antibody Fab' fragment or direct conjugation of these dyes for labeling these proteins. Rhodopsin was proved with Cy7-labeled Fab' fragment of monoclonal antibody (1D4) against the C-terminus of rhodopsin. The single molecular behavior of rhodopsin on the disc membrane was observed at video rate and 70 nm/pixel in spatial resolution. It was found that the rhodopsin does not form static paracrystalline as reported recently on the basis of AFM observation. Rhodopsin undergoes anomalous diffusion changing diffusion rate frequently. FRET experiments revealed that rhodopsins form dimer or a cluster of short lifetime. Artificially dimerized rhodopsin by the use of IgG also shows slow diffusion rate. The formation of cluster of low diffusion rate is apparently disadvantageous for rapid amplification in the phototransduction. Thus, it was suggested paradoxically that the cluster formation has an important meaning for photo-transduction. In addition, I succeeded in labeling transducin by Cy7 maintaining its activity to bind rhodopsin and PDE. Single molecular behavior of transducin was totally different from that of rhodopsin. Single molecular biochemistry of phototransduction has started to evolve.

本研究试图建立G蛋白介导的视杆细胞信号转导系统的单分子生物学基础。利用单荧光分子成像技术，我们成功地观察了脊椎动物光转导的三个关键组分，即典型的GPCR视紫红质、G蛋白转导素及其靶酶cGMP-PDE的单分子行为。我们采用近远红荧光标记抗体Fab'片段或直接偶联这些染料来标记这些蛋白。用Cy 7标记的抗视紫红质C端的单克隆抗体（1D 4）的Fab'片段证明了视紫红质。在视频速率和70 nm/像素的空间分辨率下观察了视紫红质在盘膜上的单分子行为。通过AFM观察发现，视紫红质不像最近报道的那样形成静态的次晶。视紫红质经历异常扩散，经常改变扩散速率。FRET实验显示视紫红质形成二聚体或短寿命的簇。通过使用IgG的牺牲性二聚视紫红质也显示出缓慢的扩散速率。低扩散速率团簇的形成明显不利于光转导过程中的快速放大。因此，这是矛盾的建议，集团的形成有一个重要的意义，光转导。此外，我成功地标记转导蛋白的Cy 7保持其活性，结合视紫红质和PDE。转导素的单分子行为与视紫红质完全不同。光传导的单分子生物化学已经开始发展。

项目成果

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Ahamed，A.：“通过亲和层析分离铬皂苷 I 特异性抗体”Biochem。

Liu, H.: "Active Transducin a Subunit Carries PDE6 to Detergent-Resistant Membranes in Rod Photoreceptor Outer Segments"Biochem.Biophys.Res.Commun.. 303. 19-23 (2003)

Liu, H.：“活性转导蛋白亚基将 PDE6 携带到视杆光感受器外节中的去垢剂抗性膜”Biochem.Biophys.Res.Commun.. 303. 19-23 (2003)