Reconstitution of resolved muscarinic cholinergic receptors with purified GTP-binding proteins

V. A. Florio, P. C. Sternweis

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Abstract

The association of agonists with muscarinic receptors in membranes from bovine brain was affected only slightly by guanine nucleotides. However, solubilization of these membranes with deoxycholate and subsequent removal of detergent resulted in a preparation of receptors with increased affinity for agonists and a large increase in response to guanine nucleotides. Chromatography of deoxycholate extracts of membranes on DEAE-Sephacel resulted in the separation of receptors from 95% of the guanine nucleotide-binding activity. Guanine nucleotides had no effect on the binding of agonists to these resolved receptors. The effect of guanine nucleotides was restored after the addition of either of two purified guanine nucleotide-binding proteins from bovine brain. One of these proteins, presumably brain G(I), is composed of subunits with the same molecular weights (α, 41,000; β, 35,000; γ, 11,000) and functions as the inhibitory guanine nucleotide-binding protein isolated from liver. The other protein, termed G(O), is a novel guanine nucleotide-binding protein that possesses a similar subunit composition (α, 39,000; β, 35,000; γ, 11,000) but whose function is not yet known. Addition of either protein to the resolved receptor preparation increased agonist affinity by at least 10-20-fold, and low concentrations of guanine nucleotides specifically reversed this effect. Reconstitution of receptors with the resolved subunits of G(O) demonstrates that the β-subunit alone had no effect on agonist binding, but that this subunit does appear to enhance the effects observed with the α subunit alone.

Original languageEnglish (US)
Pages (from-to)3477-3483
Number of pages7
JournalJournal of Biological Chemistry
Volume260
Issue number6
StatePublished - Jan 1 1985

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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