Photoaffinity labeling of the α1-adrenergic receptor using an 125I-labeled aryl azide analogue of prazosin

α1-Adrenergic receptor probes, which can be radioiodinated to yield high specific activity radioligands, have been synthesized and characterized. 2-[4-(4-Amino-benzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline (CP63,155), an arylamine analogue of the selective α1-adrenergic antagonist prazosin, and its iodinated derivative, 2-[4-(4-amino-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline ([125I]CP63,789), bind reversibly and with high affinity (K/sub D/ = 1 nM and 0.6 nM, respectively) to rat hepatic membrane α1-adrenergic receptors. Conversion of [125I]CP63,789 to the aryl azide yields a photolabile derivative, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline ([125I]CP65,526), which prior to photolysis binds competitively and with high affinity (K/sub D/ = 0.3 nM). Binding of [125I]CP63,789 and [125I]CP65,526 (prior to photolysis) is rapid and saturable. Both ligands identify similar α1-adrenergic receptor binding site concentrations as the parent probe, [3H]prazosin. Specific binding by these iodinated ligands is stereoselective and inhibited by a variety of adrenergic agents with a specificity typical of the α1-adrenergic receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [125I]CP65,526-labeled rat hepatic membranes reveal major protein species with molecular weights of 77K, 68K and 59K. Each protein binds adrenergic ligands with stereoselectivity and with a specificity typical of the α1-adrenergic receptor. Smaller peptides with molecular weights of 42K and 31K display prazosin-inhibitable [125I]CP65,526 binding. Labeling of these protein species with [125I]CP65,526 is not inhibitable by other adrenergic agonists or antagonists. They are thus unlikely to represent subunits of the receptor