Photoaffinity labeling of the .alpha.1-adrenergic receptor using an 125I-labeled aryl azide analog of prazosin

Abstract

.alpha.1-Adrenergic receptor probes, which can be radioiodinated to yield high specific activity radioligands, were synthesized and characterized. 2-[4-(4-Aminobenzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline (CP63,155), an arylamine analog of the selective .alpha.1-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 (Kd = 1 and 0.6 nM, respectively) to rat hepatic membrane .alpha.1-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 (Kd = 0.3 nM). Binding of [125I]CP63,789 and [125I]CP65,526 (prior to photolysis) is rapid and saturable. Both ligands identify similar .alpha.1-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 .alpha.1-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 MW of 77K [kilodaltons], 68K and 59K. Each protein binds adrenergic ligands with stereoselectivity and with a specificity typical of the .alpha.1-adrenergic receptor. Inclusion of multiple protease inhibitors during membrane preparation prior to SDS-PAGE does not alter the labeling of these peptides. Smaller peptides with MW 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. These findings confirm and extend the observations on the subunit composition of the receptor determined with the purified protein and indicate the utility of these novel high-affinity radioiodinated probes as tools for more detailed elucidation and comparison of the molecular properties of the receptor in a variety of tissues.