Abstract

Structure-based design of subtype-selective ligands for the A1 adenosine receptor will require a reliable model of the ligand-binding pocket. It should be possible to develop a reliable model based on the results of affinity labeling experiments that provide atomic coordinates for the ligand in relation to predicted receptor helices. A high affinity, A1-selective xanthine antagonist photoaffinity probe, 125l-3-(4-azidophenethyl)-1-propyl-B-cyclopentylxanthine, was used to covalently modify the A1 receptor. Chemical or enzymatic fragmentation experiments were performed to localize the region or regions of incorporation within the receptor. The fragmentation profiles for radiolabeled A1 receptor obtained with endoproteinase Glu-C, endoproteinase Lys-C, cyanogen bromide, and hydroxylamine were consistent with the interpretation that the covalent linkage was within the first four predicted transmembrane regions. This interpretation was confirmed by the demonstration that the radioactive endoproteinase Glu-C fragment derived from an A1 receptor that contains an amino-terminal FLAG epitope was recognized by an anti-FLAG monoclonal antibody. Sequential digestion with endoproteinase Glu-C/endoproteinase Lys-C limited the possible labeling to the first three predicted transmembrane spans, and endoproteinase Glu-C/trypsin digestion refined this prediction to include only transmembrane spans III and IV. Taken together, our findings suggest that the adenosine antagonist 125l-3-(4-azidophenethyl)-1-propyl-8-cyclopentyl-xanthine covalently modifies transmembrane III of the A1 receptor because this was the only receptor region common to all radiolabeled fragments.