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.

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. 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: Jan 22, 2021
• Publicly Available Date: Jan 22, 2021
• Journal: Cell Chemical Biology
• Electronic ISSN: 2451-9456
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 27
• Issue: 5
• Pages: 499-510
• DOI: https://doi.org/10.1016/j.chembiol.2020.01.010
• Keywords: Clinical Biochemistry; Molecular Medicine; Biochemistry; Molecular Biology; Pharmacology; Drug Discovery
• Public URL: https://nottingham-repository.worktribe.com/output/3965382
• Publisher URL: https://www.cell.com/cell-chemical-biology/fulltext/S2451-9456(20)30035-0?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2451945620300350%3Fshowall%3Dtrue