Identification of the subunit-binding site of α₂-adrenergic receptors using [³H]phenoxybenzamine

α₂-Adrenergic receptors are members of an important class of membrane-bound receptors which appear to mediate physiologic responses by decreasing the activity of the regulatory enzyme adenylate cyclase. This report describes the first direct identification of the subunit-binding site of α₂-adrenergic receptors. α₂-Adrenergic receptors from human platelets were solubilized with 1% digitonin and were purified approximately 600-fold by repetitive affinity chromatography. In saturation and competition binding studies using [³H]yohimbine the original α₂-adrenergic characteristics were retained by the partially purified receptor, i.e. the following potency series (based on K(i) values) was obtained: phentolamine ≃ yohimbine >> prazosin and (-)epinephrine > (+)epinephrine. Phenoxybenzamine was found to have a K(i) for the partially purified α₂-adrenergic receptor of 108 nM. As judged by the loss of specific [³H]yohimbine binding, phenoxybenzamine (a known alkylating agent) was found to bind irreversibly to the partially purified α₂-adrenergic receptor. Using [³H]phenoxybenzamine, covalent labeling of proteins in the partially purified receptor preparation was obtained. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, a specifically labeled peptide with a relative molecular mass of 61,000 was visualized. Irreversible labeling of this peptide by [³H]phenoxybenzamine could be prevented with either phentolamine or (-)epinephrine, but not with prazosin or (+)epinephrine, suggesting that this peptide of M(r) = 61,000 represents the major subunit binding site of the human platelet α₂-adrenergic receptor.