Molecular basis of ligand binding and receptor activation at the human A3 adenosine receptor

Adenosine receptors (ARs: A₁AR, A_2AAR, A_2BAR, 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 M174^5.35. 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.