Vol. 59, Issue 5, 1187-1195, May 2001

Determination of Amino Acid Residues that are Accessible from the Ligand Binding Crevice in the Seventh Transmembrane-Spanning Region of the Human A₁ Adenosine Receptor

Eric S. Dawson and Jack N. Wells

Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee

The substituted-cysteine accessibility method (SCAM) was applied to transmembrane span seven of the human A₁ adenosine receptor (hA₁AR) to reveal a subset of amino acids that are exposed to the ligand-binding crevice. The SCAM approach involved a systematic probe of receptor structure by individual substitutions of residues K265 (7.30) to R296 (7.61) with cysteine. In most cases, hA₁AR substituted-cysteine mutant membranes displayed antagonist dissociation binding constants that did not differ significantly from wild-type (WT). Radioligand binding assays were used to compare cell membranes that were treated with hydrophilic, sulfhydryl-specific methanethiosulfonate derivatives with control cell membranes. Position H278 was previously reported to be required for A₁AR ligand binding; however, that report did not establish that H278 represents a contact point for ligands. Cysteine-substitution at H278 yields membrane preparations with greatly decreased receptor density compared with WT membranes from cells in the same transfection experiment. However, H278C membranes retain a measurable fraction of antagonist binding. This observation allows for the investigation of binding-crevice accessibility at position 278 and suggests that H278 may not be required for binding of antagonist ligands. Our data reveal the binding-crevice accessibility of residues T270 (7.35), A273 (7.38), I274 (7.39), T277 (7.42), H278 (7.43), N284 (7.49), and Y288 (7.53) in the hA₁AR. These data are consistent with the high-resolution structure of bovine rhodopsin that features three -helical turns in this region that are interrupted by an elongated, nonhelical structure from positions 7.43 to 7.48 in the primary amino acid sequence.