Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, NG7 2UH, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, The Midlands, UK; These authors contributed equally to this work.
Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, NG7 2UH, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, The Midlands, UK.
Trends Pharmacol Sci. 2018 Feb;39(2):136-147. doi: 10.1016/j.tips.2017.10.006. Epub 2017 Nov 10.
Recent advances in the development of fluorescent ligands for G-protein-coupled receptors (GPCRs) and receptor tyrosine kinase receptors (RTKs) have facilitated the study of these receptors in living cells. A limitation of these ligands is potential uptake into cells and increased nonspecific binding. However, this can largely be overcome by using proximity approaches, such as bioluminescence resonance energy transfer (BRET), which localise the signal (within 10nm) to the specific receptor target. The recent engineering of NanoLuc has resulted in a luciferase variant that is smaller and significantly brighter (up to tenfold) than existing variants. Here, we review the use of BRET from N-terminal NanoLuc-tagged GPCRs or a RTK to a receptor-bound fluorescent ligand to provide quantitative pharmacology of ligand-receptor interactions in living cells in real time.
近年来，用于 G 蛋白偶联受体 (GPCR) 和受体酪氨酸激酶受体 (RTK) 的荧光配体的发展取得了进展，这使得这些受体在活细胞中的研究变得更加便利。这些配体的一个局限性是潜在的细胞摄取和增加的非特异性结合。然而，通过使用接近方法（例如生物发光共振能量转移 (BRET)）可以在很大程度上克服这一问题，这种方法可以将信号（在 10nm 内）定位到特定的受体靶标。最近对 NanoLuc 的工程改造产生了一种荧光素酶变体，其体积更小，亮度显著提高（高达十倍）。在这里，我们回顾了从 N 端 NanoLuc 标记的 GPCR 或 RTK 到受体结合的荧光配体的 BRET 的使用，以实时提供活细胞中配体-受体相互作用的定量药理学。
