Structural insights into the agonist selectivity of the adenosine A₃ receptor

Adenosine receptors play pivotal roles in physiological processes. Adenosine A₃ receptor (A₃R), the most recently identified adenosine receptor, is expressed in various tissues, exhibiting important roles in neuron, heart, and immune cells, and is often overexpressed in tumors, highlighting the therapeutic potential of A₃R-selective agents. Recently, we identified RNA-derived N⁶-methyladenosine (m⁶A) as an endogenous agonist for A₃R, suggesting the relationship between RNA-derived modified adenosine and A₃R. Despite extensive studies on the other adenosine receptors, the selectivity mechanism of A₃R, especially for A₃R-selective agonists such as m⁶A and namodenoson, remained elusive. Here, we identify tRNA-derived N⁶-isopentenyl adenosine (i⁶A) as an A₃R-selective ligand via screening of modified nucleosides against the adenosine receptors. Like m⁶A, i⁶A is found in the human body and may be an endogenous A₃R ligand. Our cryo-EM analyses elucidate the A₃R-G_i complexes bound to adenosine, 5'-N-ethylcarboxamidoadenosine (NECA), m⁶A, i⁶A, and namodenoson at overall resolutions of 3.27 Å (adenosine), 2.86 Å (NECA), 3.19 Å (m⁶A), 3.28 Å (i⁶A), and 3.20 Å (namodenoson), suggesting the selectivity and activation mechanism of A₃R. We further conduct structure-guided engineering of m⁶A-insensitive A₃R, which may aid future research targeting m⁶A and A₃R, providing a molecular basis for future drug discovery.