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

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 A(2A) 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. Stoddart et al. propose rational design of a covalent and selective ligand probe to fluorescently label GPCR in transiently transfected and endogenous systems. Using the adenosine A(2A) receptor as a model system, they show fluorescent labelling in an endogenous system, without impeding access to the orthosteric binding site. This study is useful to selectively and non-invasively study localisation and functions of GPCRs.