CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes. Issue 5 (21st May 2020)

• Record Type: Journal Article
• Authors: White, Carl W.; Caspar, Birgit; Vanyai, Hannah K.; Pfleger, Kevin D.G.; Hill, Stephen J.
• Is Part Of: Cell chemical biology. Volume 27:Issue 5(2020)
• Page Start: 499
• Page End: 510.e7
• Publication Date: 2020-05-21
• Subjects: fluorescent ligands -- G protein-coupled receptor -- NanoLuc -- NanoBiT -- NanoBRET -- ligand binding -- β-arrestin2 -- CXCR4 -- ACKR3 -- endogenous.; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2020.01.010
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: 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. Graphical Abstract: Highlights: Utilizing CRISPR/Cas9-mediated knockin to investigate GPCR function NanoBRET ligand binding at CRISPR/Cas9 genome-edited chemokine receptors Real time monitoring of live cell interactions between native CXCR4 and β-arrestin2 Development of an approach to examine extracellular CXCR4 conformational changes Abstract : White et al. demonstrate the utility of combining bioluminescence resonance energy transfer (BRET) and CRISPR/Cas9 genome editing to investigate the function of G protein-coupled receptors that are natively expressed. They also demonstrate that nanoluciferase complementation can be used to investigate receptor conformational changes.