Photoaffinity labeling of the porcine brain α2-adrenergic receptor using a radioiodinated arylazide derivative of rauwolscine: Identification of the hormone-binding subunit

S. M. Lanier, R. M. Graham, H. J. Hess, A. Grodski, M. G. Repaske, J. M. Nunnari, L. E. Limbird, C. J. Homcy

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

A functionalized derivative of the α2-selective antagonist rauwolscine formed the basis for a photoaffinity adduct that has allowed identification of the hormone-binding subunit of the brain α2-adrenergic receptor protein. Rauwolscine carboxylate underwent reaction with 4-N-t-butyloxycarbonylaminoaniline, leading to the synthesis of rauwolscine 4-aminophenyl carboxamide (Rau-AmPC). Rau-AmPC was radioiodinated and converted to the arylazide derivative, 17α-hydroxy-20α-yohimban-16β-[N-(4-azido-3-[125I]iodo) phenyl] carboxamide (125I-Rau-AzPC), via a diazonium salt intermediate. The characterization of 125I-Rau-AzPC as a photolabile probe employed α2-adrenergic receptors, which were first solubilized from porcine brain membranes and partially purified by affinity chromatography utilizing a yohimbine-agarose affinity matrix. In the partially purified receptor preparation incubated with 125I-Rau-AzPC, photolysis resulted in covalent labeling of a major (M(r), 62,000) peptide as determined by NaDodSO4/PAGE and autogradiography. Labeling of this peptide was inhibited by the α2-selective antagonist, yohimbine, and the non-subtype-selective α-antagonist, phentolamine, but not by the α1-antagonist, prazosin, or the β-receptor antagonist, (-)-alprenolol. The α-adrenergic agonist epinephrine also inhibited labeling in a stereoselective manner. These data indicate that the photolabeled M(r) 62,000 peptide is the hormone-binding subunit of the α2-adrenergic receptor protein. The availability of this radioiodinated photoaffinity probe for the α2-adrenergic receptor should facilitate further structural and biophysical characterization of the receptor protein.

Original languageEnglish (US)
Pages (from-to)9358-9362
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume83
Issue number24
StatePublished - 1986
Externally publishedYes

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Yohimbine
Adrenergic Receptors
Swine
Hormones
Brain
Alprenolol
Adrenergic Agonists
Peptides
Proteins
Peptide Hormones
Prazosin
Phentolamine
Photolysis
Affinity Chromatography
Epinephrine
Salts
Membranes

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Photoaffinity labeling of the porcine brain α2-adrenergic receptor using a radioiodinated arylazide derivative of rauwolscine : Identification of the hormone-binding subunit. / Lanier, S. M.; Graham, R. M.; Hess, H. J.; Grodski, A.; Repaske, M. G.; Nunnari, J. M.; Limbird, L. E.; Homcy, C. J.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 83, No. 24, 1986, p. 9358-9362.

Research output: Contribution to journalArticle

Lanier, S. M. ; Graham, R. M. ; Hess, H. J. ; Grodski, A. ; Repaske, M. G. ; Nunnari, J. M. ; Limbird, L. E. ; Homcy, C. J. / Photoaffinity labeling of the porcine brain α2-adrenergic receptor using a radioiodinated arylazide derivative of rauwolscine : Identification of the hormone-binding subunit. In: Proceedings of the National Academy of Sciences of the United States of America. 1986 ; Vol. 83, No. 24. pp. 9358-9362.
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abstract = "A functionalized derivative of the α2-selective antagonist rauwolscine formed the basis for a photoaffinity adduct that has allowed identification of the hormone-binding subunit of the brain α2-adrenergic receptor protein. Rauwolscine carboxylate underwent reaction with 4-N-t-butyloxycarbonylaminoaniline, leading to the synthesis of rauwolscine 4-aminophenyl carboxamide (Rau-AmPC). Rau-AmPC was radioiodinated and converted to the arylazide derivative, 17α-hydroxy-20α-yohimban-16β-[N-(4-azido-3-[125I]iodo) phenyl] carboxamide (125I-Rau-AzPC), via a diazonium salt intermediate. The characterization of 125I-Rau-AzPC as a photolabile probe employed α2-adrenergic receptors, which were first solubilized from porcine brain membranes and partially purified by affinity chromatography utilizing a yohimbine-agarose affinity matrix. In the partially purified receptor preparation incubated with 125I-Rau-AzPC, photolysis resulted in covalent labeling of a major (M(r), 62,000) peptide as determined by NaDodSO4/PAGE and autogradiography. Labeling of this peptide was inhibited by the α2-selective antagonist, yohimbine, and the non-subtype-selective α-antagonist, phentolamine, but not by the α1-antagonist, prazosin, or the β-receptor antagonist, (-)-alprenolol. The α-adrenergic agonist epinephrine also inhibited labeling in a stereoselective manner. These data indicate that the photolabeled M(r) 62,000 peptide is the hormone-binding subunit of the α2-adrenergic receptor protein. The availability of this radioiodinated photoaffinity probe for the α2-adrenergic receptor should facilitate further structural and biophysical characterization of the receptor protein.",
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AU - Lanier, S. M.

AU - Graham, R. M.

AU - Hess, H. J.

AU - Grodski, A.

AU - Repaske, M. G.

AU - Nunnari, J. M.

AU - Limbird, L. E.

AU - Homcy, C. J.

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N2 - A functionalized derivative of the α2-selective antagonist rauwolscine formed the basis for a photoaffinity adduct that has allowed identification of the hormone-binding subunit of the brain α2-adrenergic receptor protein. Rauwolscine carboxylate underwent reaction with 4-N-t-butyloxycarbonylaminoaniline, leading to the synthesis of rauwolscine 4-aminophenyl carboxamide (Rau-AmPC). Rau-AmPC was radioiodinated and converted to the arylazide derivative, 17α-hydroxy-20α-yohimban-16β-[N-(4-azido-3-[125I]iodo) phenyl] carboxamide (125I-Rau-AzPC), via a diazonium salt intermediate. The characterization of 125I-Rau-AzPC as a photolabile probe employed α2-adrenergic receptors, which were first solubilized from porcine brain membranes and partially purified by affinity chromatography utilizing a yohimbine-agarose affinity matrix. In the partially purified receptor preparation incubated with 125I-Rau-AzPC, photolysis resulted in covalent labeling of a major (M(r), 62,000) peptide as determined by NaDodSO4/PAGE and autogradiography. Labeling of this peptide was inhibited by the α2-selective antagonist, yohimbine, and the non-subtype-selective α-antagonist, phentolamine, but not by the α1-antagonist, prazosin, or the β-receptor antagonist, (-)-alprenolol. The α-adrenergic agonist epinephrine also inhibited labeling in a stereoselective manner. These data indicate that the photolabeled M(r) 62,000 peptide is the hormone-binding subunit of the α2-adrenergic receptor protein. The availability of this radioiodinated photoaffinity probe for the α2-adrenergic receptor should facilitate further structural and biophysical characterization of the receptor protein.

AB - A functionalized derivative of the α2-selective antagonist rauwolscine formed the basis for a photoaffinity adduct that has allowed identification of the hormone-binding subunit of the brain α2-adrenergic receptor protein. Rauwolscine carboxylate underwent reaction with 4-N-t-butyloxycarbonylaminoaniline, leading to the synthesis of rauwolscine 4-aminophenyl carboxamide (Rau-AmPC). Rau-AmPC was radioiodinated and converted to the arylazide derivative, 17α-hydroxy-20α-yohimban-16β-[N-(4-azido-3-[125I]iodo) phenyl] carboxamide (125I-Rau-AzPC), via a diazonium salt intermediate. The characterization of 125I-Rau-AzPC as a photolabile probe employed α2-adrenergic receptors, which were first solubilized from porcine brain membranes and partially purified by affinity chromatography utilizing a yohimbine-agarose affinity matrix. In the partially purified receptor preparation incubated with 125I-Rau-AzPC, photolysis resulted in covalent labeling of a major (M(r), 62,000) peptide as determined by NaDodSO4/PAGE and autogradiography. Labeling of this peptide was inhibited by the α2-selective antagonist, yohimbine, and the non-subtype-selective α-antagonist, phentolamine, but not by the α1-antagonist, prazosin, or the β-receptor antagonist, (-)-alprenolol. The α-adrenergic agonist epinephrine also inhibited labeling in a stereoselective manner. These data indicate that the photolabeled M(r) 62,000 peptide is the hormone-binding subunit of the α2-adrenergic receptor protein. The availability of this radioiodinated photoaffinity probe for the α2-adrenergic receptor should facilitate further structural and biophysical characterization of the receptor protein.

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