β-Adrenergic receptors display intramolecular disulfide bridges in situ: Analysis by immunoblotting and functional reconstitution

C. P. Moxham, E. M. Ross, S. T. George, C. C. Malbon

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

The molecular nature of mammalian β-adrenergic receptors in situ was probed using immunoblotting and functional reconstitution techniques. Membrane proteins of cells replete with β-adrenergic receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferrd to nitrocellulose and then probed with anti-receptor antibodies. When cell membranes were first treated with agents that cleave disulfides of proteins, immunoblots of these membranes revealed intense immunoreactive bands with electrophoretic mobility similar to that of protein standards of M(r) 65,000-67,000, comigrating with purified, reduced, and alkylated β-adrenergic receptors. However, when cell membranes were prepared under anaerobic conditions, solubilized in the presence of agents that alkylate thiols, and denatured in the absence of added thiols, immunoblotting revealed receptor with M(r) 55,000, rather than 65,000. This faster electrophoretic mobility is associated with the presence of intramolecular disulfides in the purified receptor and demonstrates that β-adrenergic receptors possess intramolecular disulfide bridges in situ. Purified receptors that demonstrate this faster mobility (M(r) 55,000 under non-reducing conditions) were co-reconstituted into phospholipid vesicles with the stimulatory GTP-binding protein G(s) and their ability to catalyze the binding of [35S]guanosine-5'-O-(3-thio)triphosphate to G(s) was measured. Agonist (isoproterenol) as well as thiol increased the receptor-promoted activation of G(s). Taken together, these data demonstrate that native β-adrenergic receptors possess one or more intramolecular disulfide bridges in situ, reduction of which causes functional activation of the receptor.

Original languageEnglish (US)
Pages (from-to)486-492
Number of pages7
JournalMolecular Pharmacology
Volume33
Issue number5
StatePublished - Jan 1 1988

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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