Skip to main content

Research Repository

Advanced Search

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

White, Carl W.; Caspar, Birgit; Vanyai, Hannah K.; Pfleger, Kevin D.G.; Hill, Stephen J.

Authors

Carl W. White

Birgit Caspar

Hannah K. Vanyai

Kevin D.G. Pfleger

STEPHEN HILL steve.hill@nottingham.ac.uk
Professor of Molecular Pharmacology



Abstract

© 2020 The Authors 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.

Citation

White, C. W., Caspar, B., Vanyai, H. K., Pfleger, K. D., & Hill, S. J. (2020). CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes. Cell Chemical Biology, 27(5), 499-510.e7. https://doi.org/10.1016/j.chembiol.2020.01.010

Journal Article Type Article
Acceptance Date Jan 24, 2020
Online Publication Date Feb 12, 2020
Publication Date May 21, 2020
Deposit Date Feb 4, 2020
Publicly Available Date Feb 13, 2021
Journal Cell Chemical Biology
Electronic ISSN 2451-9448
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 27
Issue 5
Pages 499-510.e7
DOI https://doi.org/10.1016/j.chembiol.2020.01.010
Keywords fluorescent ligands G protein-coupled receptor NanoLuc NanoBiT NanoBRET ligand binding β-arrestin2 CXCR4 ACKR3 endogenous
Public URL https://nottingham-repository.worktribe.com/output/3883039
Publisher URL https://www.sciencedirect.com/science/article/pii/S2451945620300350?via%3Dihub

Files





You might also like



Downloadable Citations