CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes.

Autor:	White CW; 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. Electronic address: carl.white@perkins.uwa.edu.au., Caspar B; 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., Vanyai HK; Epithelial Biology Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK., Pfleger KDG; 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; Dimerix Limited, Nedlands, WA 6009, Australia., Hill SJ; 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. Electronic address: stephen.hill@nottingham.ac.uk.
Jazyk:	angličtina
Zdroj:	Cell chemical biology [Cell Chem Biol] 2020 May 21; Vol. 27 (5), pp. 499-510.e7. Date of Electronic Publication: 2020 Feb 12.
DOI:	10.1016/j.chembiol.2020.01.010
Abstrakt:	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. Competing Interests: Declaration of Interests K.D.G.P. receives funding from Promega, BMG Labtech, and Dimerix as Australian Research Council Linkage Grant participating organizations. These participating organizations played no role in any aspect of the conception or design of the research, collection, analysis, and interpretation of results, or writing and editing of the manuscript. K.D.G.P. is Chief Scientific Advisor of Dimerix, of which he maintains a shareholding. The authors declare no other competing interests. (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)
Databáze:	MEDLINE
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