Determination of amino acid residues that contribute to an aqueous binding crevice in the human A(1) adenosine receptor

We attempted to map the accessibility of human A1 adenosine receptor (A1AR) amino acid residues from an aqueous ligand-binding crevice. Individual substituted-cysteine mutants H251C and H264C through R296C of the human A1AR were generated, expressed in mammalian cells, and characterized by [3H]-DPCPX saturation binding. Comparisons of binding dissociation constants for mutant proteins versus the wildtype protein addressed potential structural perturbations from the mutations. Cell membranes containing the substituted-cysteine mutant receptor proteins were exposed to ionic MTS-reagents of varying charges and sizes (MTS-X) to identify accessible residues by causing an inhibition of ligand binding in MTS-X-treated membranes containing accessible substituted-cysteines compared with control membranes. Accessibility of a substituted-cysteine was confirmed by reductions in the extent of MTS-X inhibition of binding by co-incubation with adenosine receptor agonists and antagonists as compared with controls that were exposed to MTS-X alone.; Our studies established the accessibility of a subset of amino acid residues from human A1AR TMVI and TMVII to an aqueous ligand-binding crevice. We presume that these residues are accessible in the G-protein-uncoupled state of the receptor because a non-hydrolyzable GTP-analog, GppNHp, was included in all assays involving agonists and [3H]-DPCPX, an A1-selective antagonist with reported inverse agonist activity, was used for detection of ligand binding. Conserved histidines H251 (TMVI) and H278 (TMVII) are accessible from the ligand-binding crevice and, therefore, available for direct interactions with ligands. Pharmacological characterizations of binding in H251C and H278C mutants with an N6-substituted agonist (R-PIA), a 5'-ribose-substituted agonist (NECA), and a non-selective xanthine antagonist (theophylline) were performed by competition of each ligand for [3H]-DPCPX binding at each mutant. Combined data from biochemical and pharmacological approaches provided evidence for distinct roles of these two conserved histidines in ligand binding. The accessible residues identified in this study are consistent with the high-resolution structure of bovine rhodopsin, a G-protein-coupled receptor, and may be included as constraints in molecular models of the G-protein-uncoupled state of the human A1AR.