COVALENT MODIFICATION OF TRANSMEMBRANE SPAN-III OF THE A(1) ADENOSINERECEPTOR WITH AN ANTAGONIST PHOTOAFFINITY PROBE

Structure-based design of subtype-selective ligands for the A(1) adeno sine receptor will require a reliable model of the ligand-binding pock et. It should be possible to develop a reliable model based on the res ults of affinity labeling experiments that provide atomic coordinates for the ligand in relation to predicted receptor helices. A high affin ity, A(1)-selective xanthine antagonist photoaffinity probe, (4-azidop henethyl)-1-propyl-8-cyclopentylxanthine, was used to covalently modif y the A(1) receptor. Chemical or enzymatic fragmentation experiments w ere performed to localize the region or regions of incorporation withi n the receptor. The fragmentation profiles for radiolabeled A(1) recep tor obtained with endoproteinase Glu-C, endoproteinase Lys-C, cyanogen bromide, and hydroxylamine were consistent with the interpretation th at the covalent linkage was within the first four predicted transmembr ane regions. This interpretation was confirmed by the demonstration th at the radioactive endoproteinase Glu-C fragment derived from an A(1) receptor that contains an amino-terminal FLAG epitope was recognized b y an anti-FLAG monoclonal antibody. Sequential digestion with endoprot einase Glu-C/endoproteinase Lys-C limited the possible labeling to the first three predicted transmembrane spans, and endoproteinase Glu-C/t rypsin digestion refined this prediction to include only transmembrane spans III and IV. Taken together, our findings suggest that the adeno sine antagonist (4-azidophenethyl)-1-propyl-8-cyclopentyl-xanthine cov alently modifies transmembrane III of the A(1) receptor because this w as the only receptor region common to all radiolabeled fragments.