| Abstract
| - Structure−affinity relationships for ligand binding at the humanA2A adenosine receptor havebeen probed using site-directed mutagenesis in the transmembranehelical domains (TMs).The mutant receptors were expressed in COS-7 cells andcharacterized by binding of theradioligands [3H]CGS21680,[3H]NECA, and [3H]XAC. Threeresidues, at positions essentialfor ligand binding in other G protein-coupled receptors, wereindividually mutated. The residueV(3.32) in the A2A receptor that is homologous to theessential aspartate residue of TM3 in thebiogenic amine receptors, i.e., V84(3.32), may besubstituted with L (present in the A3receptor)but not with D (in biogenic amine receptors) or A.H250(6.52), homologous to the critical N507of rat m3 muscarinic acetylcholine receptors, may be substituted withother aromatic residuesor with N but not with A (Kim et al. J. Biol.Chem.1995, 270, 13987−13997).H278(7.43),homologous to the covalent ligand anchor site in rhodopsin, may not besubstituted with eitherA, K, or N. Both V84L(3.32) and H250N(6.52) mutantreceptors were highly variable in theireffect on ligand competition depending on the structural class of theligand. Adenosine-5‘-uronamide derivatives were more potent at the H250N(6.52) mutantreceptor than at wildtype receptors. Xanthines tended to be close in potency(H250N(6.52)) or less potent (V84L(3.32)) than at wild type receptors. The affinity of CGS21680increased as the pH was loweredto 5.5 in both the wild type and H250N(6.52) mutant receptors.Thus, protonation of H250(6.52) is not involved in this pH dependence. These data areconsistent with a molecular modelpredicting the proximity of bound agonist ligands to TM3, TM5, TM6, andTM7.
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