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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization
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Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization

机译：荧光标记的G蛋白偶联受体的单分子分析揭示了具有不同动力学和组织的复合物

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Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany,Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine,University of Wiirzburg, 97078 Wurzburg, Germany;Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany,Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine,University of Wiirzburg, 97078 Wurzburg, Germany;Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany,Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine,University of Wiirzburg, 97078 Wurzburg, Germany;Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany,Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine,University of Wiirzburg, 97078 Wurzburg, Germany,Department of Physiology, Faculty of Science, Mahidol University, 10400 Bangkok, Thailand;Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany,Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine,University of Wiirzburg, 97078 Wurzburg, Germany;Institute of Mathematics, University of Wurzburg, 97074 Wurzburg, Germany;lmmune Disease Institute, Program in Cellular and Molecular Medicine at Boston Children's Hospital and Department of Cell Biology, Harvard Medical School, Boston, MA 02115;Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany;Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany,Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine,University of Wiirzburg, 97078 Wurzburg, Germany;%G-protein-coupled receptors (GPCRs) constitute the largest family of receptors and major pharmacological targets. Whereas many GPCRs have been shown to form di-/oligomers, the size and stability of such complexes under physiological conditions are largely unknown. Here, we used direct receptor labeling with SNAP-tags and total internal reflection fluorescence microscopy to dynamically monitor single receptors on intact cells and thus compare the spatial arrangement, mobility, and supramolecular organization of three prototypical GPCRs: the β_1-adrenergic receptor (β_1AR), the β_2-adrenergic receptor (β_2AR), and the y-aminobutyric acid (GABA_B) receptor. These GPCRs showed very different degrees of di-/oligo-merization, lowest for β_1ARs (monomers/dimers) and highest for GABA_B receptors (prevalently dimers/tetramers of heterodimers). The size of receptor complexes increased with receptor density as a result of transient receptor-receptor interactions. Whereas β_1-/ β_2ARs were apparently freely diffusing on the cell surface, GABA_B receptors were prevalently organized into ordered arrays, via interaction with the actin cytoskeleton. Agonist stimulation did not alter receptor di-/oligomerization, but increased the mobility of GABA_B receptor complexes. These data provide a spatiotemporal characterization of β_1/β_2ARs and GABA_B receptors at single-molecule resolution. The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.
机译：维尔茨堡大学药理毒理研究所，德国维尔茨堡97078，德国Forschungsgemeinschaft-德国鲁德尔夫·维尔奇中心-实验生物医学研究中心，德国维尔茨堡97078维尔茨堡，德国；维尔茨堡药理毒理研究所，维尔茨堡大学，970德国，Deutsche Forschungsgemeinschaft-Rudolf Virchow中心-维尔茨堡大学实验生物医学研究中心，德国维尔茨堡97078; Wiirzburg大学药理与毒理学研究所，97078维尔茨堡，德国，Rudolf Virchows中心，Deutsche Forsch实验维尔茨堡大学，生物医学，97078德国维尔茨堡；维尔茨堡大学，药理毒理学研究所，97078维尔茨堡，德国，德国Deutsche Forschungsgemeinschaft-Rudolf Virchow中心，德国实验生物医学研究中心，维尔茨堡大学，97078理学部生理学，马哈迪尔大学，泰国曼谷10400；维尔茨堡大学药理毒理研究所，德国维尔茨堡97078，德国Forschungsgemeinschaft德国鲁道夫·维尔奇霍夫实验生物医学研究中心，德国维尔茨堡大学实验生物医学研究中心，德国维尔茨堡97078维尔茨堡大学，德国97074维尔茨堡；波士顿儿童医院细胞和分子医学程序免疫学研究所，哈佛医学院细胞生物学系，波士顿，马萨诸塞州02115；维尔茨堡大学药理毒理学研究所，97078维尔茨堡；德国；维尔茨堡大学药理毒理研究所，德国维尔茨堡97078，德国Forschungsgemeinschaft-德国实验生物医学研究中心Rudolf Virchow中心，维尔茨堡大学，97078维尔茨堡，德国；％G蛋白偶联受体（GPCR）构成最大的受体家族和主要的药理靶标。尽管许多GPCR已显示可形成二聚/寡聚体，但在生理条件下此类复合物的大小和稳定性尚不清楚。在这里，我们使用了带有SNAP标签的直接受体标记和全内反射荧光显微镜技术来动态监测完整细胞上的单个受体，从而比较三种原型GPCR的空间排列，活动性和超分子组织：β_1-肾上腺素能受体（β_1AR） ，β_2-肾上腺素能受体（β_2AR）和γ-氨基丁酸（GABA_B）受体。这些GPCR显示出不同程度的二聚/寡聚，β_1ARs最低（单体/二聚体），GABA_B受体最高（通常是异二聚体的二聚体/四聚体）。受体复合物的大小随着受体密度的增加而增加，这是瞬时受体与受体相互作用的结果。 β_1-/β_2ARs明显在细胞表面自由扩散，而GABA_B受体通过与肌动蛋白细胞骨架的相互作用而普遍组织成有序阵列。激动剂刺激不改变受体二-/低聚，但增加了GABA_B受体复合物的流动性。这些数据以单分子分辨率提供了β_1/β_2ARs和GABA_B受体的时空特征。结果表明，GPCR以动态平衡的形式存在于细胞表面，具有不断形成和解离的新受体复合物，这些受体复合物可以以配体调节的方式靶向不同的细胞表面微结构域。

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  • 作者

    Davide Calebiro; Finn Rieken; Julia Wagner; Titiwat Sungkaworn; Ulrike Zabel; Alfio Borzi; Emanuele Cocucci; Alexander Zuern; Martin J. Lohse;

  • 作者单位

    Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany,Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine,University of Wiirzburg, 97078 Wurzburg, Germany;

    Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany,Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine,University of Wiirzburg, 97078 Wurzburg, Germany;

    Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany,Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine,University of Wiirzburg, 97078 Wurzburg, Germany;

    Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany,Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine,University of Wiirzburg, 97078 Wurzburg, Germany,Department of Physiology, Faculty of Science, Mahidol University, 10400 Bangkok, Thailand;

    Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany,Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine,University of Wiirzburg, 97078 Wurzburg, Germany;

    Institute of Mathematics, University of Wurzburg, 97074 Wurzburg, Germany;

    lmmune Disease Institute, Program in Cellular and Molecular Medicine at Boston Children's Hospital and Department of Cell Biology, Harvard Medical School, Boston, MA 02115;

    Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany;

    Institute of Pharmacology and Toxicology,University of Wiirzburg, 97078 Wurzburg, Germany,Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine,University of Wiirzburg, 97078 Wurzburg, Germany;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    live cell imaging; protein-protein interactions;

    机译：活细胞成像;蛋白质相互作用;

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