Identification and characterization of amino acid residues essential for human A2A adenosine receptor : ZM241385 binding and subtype selectivity.

J Biol Chem 285(17):13032-13044 (2010) PMID 20147292

The crystal structure of the human A2A adenosine receptor bound to the A2A receptor-specific antagonist, ZM241385 was recently determined at 2.6 A resolution. Surprisingly, the antagonist binds in an extended conformation, perpendicular to the plane of the membrane and indicates a number of interactions unidentified before in ZM241385 recognition. To further understand ZM241385s selectivity for the human A2A adenosine receptor, we examined the effect of mutating amino acid residues within the binding cavity likely to have key interactions and that have not been previously examined. Mutation of Phe168 to Ala abolishes both agonist and antagonist binding as well as receptor activity, whereas mutation of this residue to Trp or Tyr had only moderate effects. The Met177Ala mutation impeded antagonist but not agonist binding. Finally, the Leu249Ala mutant showed neither agonist nor antagonist binding affinity. From our results and previously published mutagenesis data, we conclude that conserved residues Phe168(5.29), Glu169(5.30), Asn253(6.55) and Leu249(6.51) play a central role in coordinating the bicyclic core present in both agonists and antagonists. By combining the analysis of the mutagenesis data with a comparison of the sequences of different adenosine receptor subtypes from different species, we predict that the interactions that determine subtype selectivity reside in the more divergent upper region of the binding cavity while the lower part of the binding cavity is conserved across adenosine receptor subtypes.