Mammalian α1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit

J. W. Lomasney, L. M F Leeb-Lundberg, S. Cotecchia, John W Regan, J. F. DeBernardis, M. G. Caron, R. J. Lefkowitz

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Abstract

α1-Adrenergic receptors from the cultured smooth muscle cell line (DDT1 MF-2) have been solubilized with digitonin and purified to apparent homogeneity by sequential chromatography on a biospecific affinity support (Sepharose-A55453 (4-amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-phenyl)pentanoyl]- 1-piperazinyl]-quinazoline), an α1 receptor-selective antagonist), a wheat germ agglutinin-agarose gel, and a high performance steric exclusion liquid chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveals a peptide with an apparent M(r) = 80,000 that co-migrates with the peptide labeled by the specific α1-adrenergic receptor photoaffinity probe 4-amino-6,7-dimethoxy-2-[4-[5[(4-azido-3-[125I]iodophenyl )pentanoyl]-1-piperazinly] quinazoline. The specific activity (~13,600 pmol of ligand binding/mg of protein) of purified receptor preparations is consistent with that expected for a pure peptide of M(r) = 80,000 containing a single ligand binding site. Overall yields approximate 14% of initial crude particulate binding. The purified receptor preparations bind agonist and antagonist ligands with appropriate α1-adrenergic specificity, stereoselectivity, and affinity. Peptide maps of the pure α1-adrenergic receptor and the pure human platelet α2-adrenergic receptor (Regan, J.W., Nakata H., DeMarinis, R.M., Caron, M.G., and Lefkowitz, R.J. (1986) J. Biol. Chem. 261, 3894-3900) using several different proteases suggest that these two receptors show little if any structural homology.

Original languageEnglish (US)
Pages (from-to)7710-7716
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number17
StatePublished - 1986
Externally publishedYes

Fingerprint

Adrenergic Receptors
Purification
Quinazolines
Ligands
A 55453
Sepharose
Peptides
Stereoselectivity
Digitonin
Wheat Germ Agglutinins
Liquid chromatography
Platelets
Chromatography
Electrophoresis
Autoradiography
Liquid Chromatography
Sodium Dodecyl Sulfate
Adrenergic Agents
Smooth Muscle Myocytes
Gel Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Lomasney, J. W., Leeb-Lundberg, L. M. F., Cotecchia, S., Regan, J. W., DeBernardis, J. F., Caron, M. G., & Lefkowitz, R. J. (1986). Mammalian α1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit. Journal of Biological Chemistry, 261(17), 7710-7716.

Mammalian α1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit. / Lomasney, J. W.; Leeb-Lundberg, L. M F; Cotecchia, S.; Regan, John W; DeBernardis, J. F.; Caron, M. G.; Lefkowitz, R. J.

In: Journal of Biological Chemistry, Vol. 261, No. 17, 1986, p. 7710-7716.

Research output: Contribution to journalArticle

Lomasney, JW, Leeb-Lundberg, LMF, Cotecchia, S, Regan, JW, DeBernardis, JF, Caron, MG & Lefkowitz, RJ 1986, 'Mammalian α1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit', Journal of Biological Chemistry, vol. 261, no. 17, pp. 7710-7716.
Lomasney JW, Leeb-Lundberg LMF, Cotecchia S, Regan JW, DeBernardis JF, Caron MG et al. Mammalian α1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit. Journal of Biological Chemistry. 1986;261(17):7710-7716.
Lomasney, J. W. ; Leeb-Lundberg, L. M F ; Cotecchia, S. ; Regan, John W ; DeBernardis, J. F. ; Caron, M. G. ; Lefkowitz, R. J. / Mammalian α1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit. In: Journal of Biological Chemistry. 1986 ; Vol. 261, No. 17. pp. 7710-7716.
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AU - Lomasney, J. W.

AU - Leeb-Lundberg, L. M F

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AU - Regan, John W

AU - DeBernardis, J. F.

AU - Caron, M. G.

AU - Lefkowitz, R. J.

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