Ligand-Directed Labeling of the Adenosine A₁ Receptor in Living Cells

The study of protein function and dynamics in their native cellular environment is essential for progressing fundamental science. To overcome the requirement of genetic modification of the protein or the limitations of dissociable fluorescent ligands, ligand-directed (LD) chemistry has most recently emerged as a complementary, bioorthogonal approach for labeling native proteins. Here, we describe the rational design, development, and application of the first ligand-directed chemistry approach for labeling the A₁AR in living cells. We pharmacologically demonstrate covalent labeling of A₁AR expressed in living cells while the orthosteric binding site remains available. The probes were imaged using confocal microscopy and fluorescence correlation spectroscopy to study A₁AR localization and dynamics in living cells. Additionally, the probes allowed visualization of the specific localization of A₁ARs endogenously expressed in dorsal root ganglion (DRG) neurons. LD probes developed here hold promise for illuminating ligand-binding, receptor signaling, and trafficking of the A₁AR in more physiologically relevant environments.