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.

我们试图构建脊椎动物视杆感光细胞G蛋白介导的信号转导系统的单分子生物化学基础。利用单荧光分子成像技术，我们成功地观察了脊椎动物光传导的三个关键成分，即典型的GPCR视紫质、G蛋白转导蛋白及其靶酶cGMP-PDE的单分子行为。我们用近红外线荧光标记的抗体Fab‘片段或这些染料的直接偶联来标记这些蛋白质。用抗视紫红质C末端的单抗(1D4)的Cy7标记Fab‘片段证实了视紫红质。在视频率和空间分辨率为70 nm/像素的条件下，观察到视紫红质在盘膜上的单分子行为。原子力显微镜观察发现，视紫红质并不像最近报道的那样形成静态的准晶。视紫红质发生异常扩散，扩散速率频繁变化。FRET实验表明，视紫红质形成二聚体或一簇短寿命。利用免疫球蛋白进行人工二聚的视紫红质也表现出缓慢的扩散速度。低扩散速率团簇的形成明显不利于光传导中的快速扩增。因此，自相矛盾的是，团簇的形成对光传导具有重要意义。此外，我还成功地通过Cy7标记了转导蛋白，保持了它与视紫红质和PDE结合的活性。转导蛋白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进化。

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)