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

J. W. Regan, R. M. DeMarinis, M. G. Caron, R. J. Lefkowitz

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22 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)7864-7869
Number of pages6
JournalJournal of Biological Chemistry
Issue number12
StatePublished - 1984

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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