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

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

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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
Volume259
Issue number12
StatePublished - 1984
Externally publishedYes

Fingerprint

Phenoxybenzamine
Adrenergic Receptors
Binding Sites
Epinephrine
Yohimbine
Prazosin
Phentolamine
Platelets
Labeling
Blood Platelets
Affinity chromatography
Digitonin
Peptides
Alkylating Agents
Molecular mass
Electrophoresis
Autoradiography
Affinity Chromatography
Adenylyl Cyclases
Sodium Dodecyl Sulfate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of the subunit-binding site of α2-adrenergic receptors using [3H]phenoxybenzamine. / Regan, John W; DeMarinis, R. M.; Caron, M. G.; Lefkowitz, R. J.

In: Journal of Biological Chemistry, Vol. 259, No. 12, 1984, p. 7864-7869.

Research output: Contribution to journalArticle

Regan, John W ; DeMarinis, R. M. ; Caron, M. G. ; Lefkowitz, R. J. / Identification of the subunit-binding site of α2-adrenergic receptors using [3H]phenoxybenzamine. In: Journal of Biological Chemistry. 1984 ; Vol. 259, No. 12. pp. 7864-7869.
@article{4fc9580a49514314a6a8b796f3d484c6,
title = "Identification of the subunit-binding site of α2-adrenergic receptors using [3H]phenoxybenzamine",
abstract = "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.",
author = "Regan, {John W} and DeMarinis, {R. M.} and Caron, {M. G.} and Lefkowitz, {R. J.}",
year = "1984",
language = "English (US)",
volume = "259",
pages = "7864--7869",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "12",

}

TY - JOUR

T1 - Identification of the subunit-binding site of α2-adrenergic receptors using [3H]phenoxybenzamine

AU - Regan, John W

AU - DeMarinis, R. M.

AU - Caron, M. G.

AU - Lefkowitz, R. J.

PY - 1984

Y1 - 1984

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0021257076&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021257076&partnerID=8YFLogxK

M3 - Article

C2 - 6330087

AN - SCOPUS:0021257076

VL - 259

SP - 7864

EP - 7869

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 12

ER -