Department of Medicine, Research Institute of the McGill University Health Center (RI-MUHC), McGill University, Montréal, Québec, Canada H4A 3J1.
Department of Biochemistry and Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montréal, Québec, Canada H3C 1J4.
Nat Commun. 2016 Jul 11;7:12178. doi: 10.1038/ncomms12178.
Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes.
内吞作用和受体的细胞内转运对于维持生理功能和药物作用至关重要；然而，目前缺乏强有力的定量方法来研究活细胞中的这些过程。在这里，我们提出了新的生物发光共振能量转移（BRET）传感器，用于定量监测 G 蛋白偶联受体（GPCR）和β-arrestin 转运。这些传感器基于旁观者 BRET，并使用来自海肾萤光素酶（RLuc）和绿色荧光蛋白（rGFP）的天然相互作用的生色团。该方法的多功能性和稳健性通过将 rGFP 锚定在质膜或内体上来证明，以在配体促进的募集或隔离 RLuc 标记蛋白到或从特定细胞区室时产生高动态光谱 BRET 信号，以及用于蛋白质易位可视化的敏感亚细胞 BRET 成像。这些传感器可扩展到高通量格式，并允许实时、在活细胞中对 GPCR 转运进行定量药理学研究，揭示受体/β-arrestin 复合物的配体依赖性偏向转运。
