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 A2A receptor (hA2AR) 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 hA2AR ligand with a terminal alkyne to serve as
probe. We showed that our probe irreversibly and concentration-dependently
labeled purified hA2AR. 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 hA2AR by our probe could be inhibited by selective antagonists. Lastly,
we showed successful labeling of the receptor in cell membranes overexpressing
hA2AR, making our probe a promising affinity-based tool
compound that sets the stage for the further development of probes
for GPCRs.