Zhang, L.; Mobbs, J. I.; Bennetts, F. M.; Venugopal, H.; Nguyen, A. T. N.; Christopoulos, A.; van der Es, D.; Heitman, L. H.; May, L. T.; Glukhova, A.; Thal, D. M.

Adenosine receptors (ARs: A1AR, A2AAR, A2BAR, and A3AR) are crucial therapeutic targets, yet developing selective, efficacious drugs remains challenging. Here, we present high-resolution cryo-electron microscopy (cryo-EM) structures of the human A3AR in three distinct functional states: bound to the endogenous agonist adenosine, the clinically relevant agonist Piclidenoson, and the covalent antagonist LUF7602. These structures, complemented by mutagenesis and pharmacological studies, reveal a unique A3AR activation mechanism involving an extensive hydrogen bond network from the extracellular surface down to the orthosteric binding site. In addition, we identify a cryptic pocket that accommodates the N6-iodobenzyl group of Piclidenoson through a ligand-dependent conformational change of M1745.35. Our comprehensive structural and functional characterization of A3AR advances understanding of adenosine receptor pharmacology and establishes a foundation for developing more selective therapeutics for various disorders including inflammatory diseases, cancer, and glaucoma.