Synthesis and characterization of a radioiodinated photoaffinity probe for the alpha 1-adrenergic receptor

A radioiodinated aryl azide analog, 2-[4-(4-azido-3- iodobenzoyl ) piperazin -1-yl]-4-amino-6, 7- dimethoxyquinazoline [(125I] CP65 ,526), of the highly selective alpha 1-adrenergic antagonist prazosin was synthesized and characterized using rat hepatic plasma membranes. Prior to photolysis, this ligand bound with high affinity (Kd 0.3 nM), stereoselectively and in a saturable manner to sites with an alpha 1-adrenergic specificity. When membranes pretreated with [125I] CP65 ,526 were irradiated with ultraviolet light, the ligand incorporated irreversibly into the receptor-binding sites, also with typical alpha 1-adrenergic specificity. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of such labeled membranes followed by radioautography revealed major bands at Mr = 77,000, 68,000, and 59,000 daltons. Labeling of each of these bands was inhibitable by a variety of adrenergic ligands, stereoselectively and with a specificity typical of the alpha 1-adrenergic receptor. Smaller peptides with molecular weights of 42,000 and 31,000 daltons also displayed prazosin-inhibitable [125] CP65 ,526-binding. However, as the labeling of these protein species was not inhibitable by other adrenergic agonists or antagonists, they are unlikely to represent subunits of the receptor. Further evidence that [125I] CP65 ,526 incorporates covalently upon photolysis was the ability to specifically label immunoglobulin heavy and light chains of an antiserum that recognized both this ligand and the parent compound, prazosin. This new, radioiodinated, high-affinity probe should thus be uniquely valuable for the molecular characterization of the alpha 1-adrenergic receptor.