Research Program on Biomedical Informatics (GRIB), IMIM/Pompeu Fabra University, Barcelona, Spain.
Curr Med Chem. 2011;18(30):4606-34. doi: 10.2174/092986711797379285.
Dimerization and oligomerization of G protein-coupled receptors (GPCRs), proposed almost 30 years ago, have crucial relevance for drug design. Indeed, formation of GPCR oligomers may affect the diversity and performance by which extracellular signals are transferred to G proteins in the process of receptor transduction. Thus, the control of oligomer assembly/disassembly and signaling will be a powerful pharmacological tool. This, however, requires (i) the determination that oligomerization takes place between particular receptors, (ii) the confirmation that the oligomer has pharmacological importance and (iii) the availability of the oligomer 3D structure. This review aims at presenting experimental methods which unveil the complexity of GPCR dimerization/oligomerization focusing on biochemical and biophysical approaches. In total, we review 22 methods, including biochemical methods (radiation inactivation technique, receptor co-expression and trans-complementation studies, cross-linking experiments, co-immunoprecipitation and immunoblotting studies and analysis of receptor mutants and chimeras) and biophysical methods (Fluorescence Resonance Energy Transfer, (FRET), including photobleaching FRET (pb-FRET) and Time-Resolved FRET (TR-FRET), Luminescence Resonance Energy Transfer (LRET), Bioluminescence Resonance Energy Transfer (BRET), Bimolecular Fluorescence Complementation (BiFC), Luminescence Fluorescence Complementation (BiLC), Fluorescence Recovery after Photobleaching (FRAP), Confocal Microscopy, Immunofluorescence Microscopy, Single Fluorescent-Molecule Imaging, Transmission Electron Microscopy, Immunoelectron Microscopy, Atomic Force Microscopy, Total Internal Reflectance Fluorescence Microscopy (TIRFM) and X-ray Crystallography). For each method the scientific basis of the approach is shortly described followed by the extensive description of its application for studying GPCR oligomers presented according to their classes and families. Based on the wealth of experimental evidence, there is no doubt about the existence of GPCR dimers, oligomers and receptor mosaics which constitute a new and highly promising group of novel drug targets for more selective and safer drugs.
G 蛋白偶联受体（GPCRs）的二聚化和寡聚化，这一概念在大约 30 年前被提出，对药物设计具有至关重要的意义。事实上，GPCR 寡聚体的形成可能会影响到受体转导过程中细胞外信号向 G 蛋白传递的多样性和效率。因此，控制寡聚体的组装/解组装和信号转导将成为一种强大的药理学工具。然而，这需要（i）确定特定受体之间存在寡聚化，（ii）确认寡聚体具有药理学重要性，以及（iii）获得寡聚体的 3D 结构。本综述旨在介绍揭示 GPCR 二聚化/寡聚化复杂性的实验方法，重点介绍生化和生物物理方法。总共，我们回顾了 22 种方法，包括生化方法（辐射失活技术、受体共表达和互补研究、交联实验、共免疫沉淀和免疫印迹研究以及受体突变体和嵌合体分析）和生物物理方法（荧光共振能量转移（FRET），包括光漂白 FRET（pb-FRET）和时间分辨 FRET（TR-FRET）、发光共振能量转移（LRET）、生物发光共振能量转移（BRET）、双分子荧光互补（BiFC）、发光荧光互补（BiLC）、荧光漂白后恢复（FRAP）、共聚焦显微镜、免疫荧光显微镜、单荧光分子成像、透射电子显微镜、免疫电子显微镜、原子力显微镜、全内反射荧光显微镜（TIRFM）和 X 射线晶体学）。对于每种方法，我们简要描述了方法的科学基础，然后根据其分类和家族，详细描述了其用于研究 GPCR 寡聚体的应用。基于丰富的实验证据，GPCR 二聚体、寡聚体和受体镶嵌体的存在毋庸置疑，它们构成了一组新的、极具前景的新型药物靶点，可用于开发更具选择性和更安全的药物。
