An Affinity-Based Probe for the Human Adenosine A_2A Receptor

Using activity-based protein profiling (ABPP), functional proteins can be interrogated in their native environment. Despite their pharmaceutical relevance, G protein-coupled receptors (GPCRs) have been difficult to address through ABPP. In the current study, we took the prototypical human adenosine A_2A receptor (hA_2AR) as the starting point for the construction of a chemical toolbox allowing two-step affinity-based labeling of GPCRs. First, we equipped an irreversibly binding hA_2AR ligand with a terminal alkyne to serve as probe. We showed that our probe irreversibly and concentration-dependently labeled purified hA_2AR. Click-ligation with a sulfonated cyanine-3 fluorophore allowed us to visualize the receptor on SDS-PAGE. We further demonstrated that labeling of the purified hA_2AR by our probe could be inhibited by selective antagonists. Lastly, we showed successful labeling of the receptor in cell membranes overexpressing hA_2AR, making our probe a promising affinity-based tool compound that sets the stage for the further development of probes for GPCRs.