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 language | English (US) |
|---|---|
| Pages (from-to) | 9358-9362 |
| Number of pages | 5 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 83 |
| Issue number | 24 |
| State | Published - 1986 |
| Externally published | Yes |
Fingerprint
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 journal › Article
}
TY - JOUR
T1 - Photoaffinity labeling of the porcine brain α2-adrenergic receptor using a radioiodinated arylazide derivative of rauwolscine
T2 - Identification of the hormone-binding subunit
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.
PY - 1986
Y1 - 1986
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.
UR - http://www.scopus.com/inward/record.url?scp=0022901665&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0022901665&partnerID=8YFLogxK
M3 - Article
C2 - 3025837
AN - SCOPUS:0022901665
VL - 83
SP - 9358
EP - 9362
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 24
ER -