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.

要研究G蛋白偶联受体（GPCR）在其天然细胞环境中的定位，需要通过荧光标记使其可视化。为了克服对受体进行基因修饰的要求或可解离的荧光配体的限制，在此我们描述了一种化合物的合理设计，该化合物在具有荧光部分的活细胞中共价和选择性地标记GPCR。我们设计了一种荧光拮抗剂，其中在药效团（ZM241385）和荧光团（磺基花青5）之间掺入的接头能够促进荧光团与腺苷A _2A的共价连接。受体。我们在药理和生化方面证明了不可逆的荧光标记，而不会阻碍对正构结合位点的访问，并证明了其在内源性表达系统中的使用。这提供了一种非侵入性和选择性的方法来研究天然GPCR的功能和定位。