Ligand-directed covalent labelling of a GPCR with a fluorescent tag in live cells

Stoddart, Leigh A.; Kindon, Nicholas D.; Otun, Omolade; Harwood, Clare R.; Patera, Foteini; Veprintsev, Dmitry B.; Woolard, Jeanette; Briddon, Stephen J.; Franks, Hester A.; Hill, Stephen J.; Kellam, Barrie

Abstract

© 2020, The Author(s). To study the localisation of G protein-coupled receptors (GPCR) in their native cellular environment requires their visualisation through fluorescent labelling. To overcome the requirement for genetic modification of the receptor or the limitations of dissociable fluorescent ligands, here we describe rational design of a compound that covalently and selectively labels a GPCR in living cells with a fluorescent moiety. We designed a fluorescent antagonist, in which the linker incorporated between pharmacophore (ZM241385) and fluorophore (sulfo-cyanine5) is able to facilitate covalent linking of the fluorophore to the adenosine A2A receptor. We pharmacologically and biochemically demonstrate irreversible fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.

Citation

Stoddart, L. A., Kindon, N. D., Otun, O., Harwood, C. R., Patera, F., Veprintsev, D. B., …Kellam, B. (2020). Ligand-directed covalent labelling of a GPCR with a fluorescent tag in live cells. Communications Biology, 3(1), Article 722. https://doi.org/10.1038/s42003-020-01451-w

• Journal Article Type: Article
• Acceptance Date: Nov 3, 2020
• Online Publication Date: Nov 27, 2020
• Publication Date: Dec 1, 2020
• Deposit Date: Nov 16, 2020
• Publicly Available Date: Nov 27, 2020
• Journal: Communications Biology
• Electronic ISSN: 2399-3642
• Publisher: Nature Publishing Group
• Peer Reviewed: Peer Reviewed
• Volume: 3
• Issue: 1
• Article Number: 722
• DOI: https://doi.org/10.1038/s42003-020-01451-w
• Public URL: https://nottingham-repository.worktribe.com/output/5048162
• Publisher URL: https://www.nature.com/articles/s42003-020-01451-w
• Additional Information: Received: 15 May 2020; Accepted: 3 November 2020; First Online: 27 November 2020; : The authors declare no competing interests.