Ward Richard J, Pediani John D, Godin Antoine G, Milligan Graeme
From the Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
the University of Bordeaux, LP2N, UMR 5298, F-33405 Talence, France, and the Institut d'Optique Graduate School and CNRS, LP2N, UMR 5298, F-33405 Talence, France.
J Biol Chem. 2015 May 15;290(20):12844-57. doi: 10.1074/jbc.M115.644724. Epub 2015 Mar 30.
The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT2C receptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor.
G蛋白偶联受体是以单体、二聚体和/或寡聚体形式存在，以及这些形式是否以配体依赖的方式相互转化，这些问题是当前生物学中最具争议性的问题之一。当对稳定表达单体增强型绿色荧光蛋白（eGFP）的质膜相关形式或该荧光团的串联形式的细胞进行激光扫描共聚焦显微镜图像的空间强度分布分析时，eGFP串联体被鉴定为二聚体。对稳定表达表皮生长因子受体的eGFP标记形式的细胞进行的类似研究表明，尽管该受体在基础状态下主要是单体，但在添加其配体表皮生长因子后，它迅速主要变成二聚体。在诱导表达5-羟色胺2C（5-HT2C）受体的eGFP标记形式的细胞中，对构建体量子亮度的整体分析与该受体的主要形式为二聚体一致。然而，详细的空间强度分布分析表明存在从单体到高阶寡聚体的多种形式。此外，用化学性质不同的5-HT2C受体拮抗剂处理导致四级结构随时间发生变化，变为受体单体占优势的结构。这种拮抗剂介导的效应是可逆的，因为洗脱配体导致受体的许多寡聚体形式再生。
