Affinity labeling of the DDT1 MF-2 cell alpha 1-adrenergic receptor with [3H]phenoxybenzamine

In this study, we used phenoxybenzamine to label the alpha 1-adrenergic receptor of a smooth muscle cell line. Our results demonstrate a dose-dependent occupancy of alpha 1-adrenergic receptors by phenoxybenzamine determined by competition for the [3H]prazosin binding site. Following incorporation of [3H]phenoxybenzamine, partially purified membranes were solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Despite numerous Coomassie blue-stained bands, only three bands, Mr = 80,000 +/- 500, Mr = 33,000 +/- 2,000, and Mr = 21,000 +/- 400 (N = 4), were labeled with [3H]phenoxybenzamine as determined by autofluorography. Incorporation of [3H]phenoxybenzamine into the Mr = 80,000 band, but not the Mr = 33,000 and Mr = 21,000 bands, was affected by adrenergic agonists and antagonists in a manner consistent with an alpha 1-adrenergic interaction. Labeling of the Mr = 33,000 and Mr = 21,000 bands was partially blocked by phenoxybenzamine. We conclude that [3H]phenoxybenzamine can be used as an affinity probe for the alpha 1-adrenergic receptor and that the ligand binding site of the alpha 1-adrenergic receptor resides in a Mr = 80,000 protein.