White Carl W, Caspar Birgit, Vanyai Hannah K, Pfleger Kevin D G, Hill Stephen J
Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology & Neuroscience, School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, The Midlands, UK; Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia, QEII Medical Centre, Nedlands, WA 6009, Australia; Australian Research Council Centre for Personalised Therapeutics Technologies, Australia.
Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology & Neuroscience, School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, The Midlands, UK.
Cell Chem Biol. 2020 May 21;27(5):499-510.e7. doi: 10.1016/j.chembiol.2020.01.010. Epub 2020 Feb 12.
G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of β-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels.
G蛋白偶联受体是一类主要的膜受体，介导生理和病理生理细胞信号传导。受体激活和信号传导的许多方面可以使用基因编码的发光融合蛋白进行研究。然而，在活细胞系统中使用这些生物传感器需要外源表达感兴趣的标记蛋白。为了在此维持正常的细胞环境，我们使用CRISPR/Cas9介导的同源定向修复将发光标签插入内源性基因组。利用纳米荧光素酶和生物发光共振能量转移，我们证明了在基因组编辑的趋化因子受体上荧光配体的结合。我们还证明，分裂纳米荧光素酶互补可用于研究CXCR4的构象变化和内化，并且当两种蛋白均为天然表达时，可以监测β-抑制蛋白2向CXCR4的募集。这些结果表明，基因编码的发光生物传感器可用于在天然表达水平上研究受体功能的许多方面。
