Determination of amino acid residues that are accessible from the ligand binding crevice in the seventh transmembrane-spanning region of the human A(1) adenosine receptor.
The substituted-cysteine accessibility method (SCAM) was applied to transmembrane span seven of the human A(1) adenosine receptor (hA(1)AR) 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, hA(1)AR 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 A(1)AR 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 hA(1)AR. These data are consistent with the high-resolution structure of bovine rhodopsin that features three alpha-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.
Dawson
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ES
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Wells
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JN
D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008698:Mesylates / Q000494:pharmacology; D058915:Purinergic P1 Receptor Antagonists; D011869:Radioligand Assay; D018047:Receptors, Purinergic P1 / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection; D014316:Tritium; D014867:Water / Q000737:chemistry; D014970:Xanthines / Q000494:pharmacology
D007202:Indicators and Reagents; D008565:Membrane Proteins; D008698:Mesylates; D058915:Purinergic P1 Receptor Antagonists; D018047:Receptors, Purinergic P1; D014970:Xanthines; C093171:methanethiosulfonate ethylammonium; D014867:Water; D014316:Tritium; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D005020:Ethyl Methanesulfonate; C051360:1,3-dipropyl-8-cyclopentylxanthine