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

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

Research output: Contribution to journalArticle

52 Scopus citations

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 - Dec 1 1986
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    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.