Abstract
alpha 2-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 indentification of the subunit-binding site of alpha 2-adrenergic receptors. alpha 2-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 alpha 2-adrenergic characteristics were retained by the partially purified receptor, i.e. the following potency series (based on Ki values) was obtained: phentolamine approximately equal to yohimbine much greater than prazosin and (-)epinephrine greater than (+)epinephrine. Phenoxybenzamine was found to have a Ki for the partially purified alpha 2-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 alpha 2-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 Mr = 61,000 represents the major subunit binding site of the human platelet alpha 2-adrenergic receptor.
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