Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-β

Gabriel Berstein, Jonathan L. Blank, Alan V. Smrcka, Tsutomu Higashijima, Paul C. Sternweis, John H. Exton, Elliott M. Ross

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Abstract

We describe the reconstitution using purified proteins of the m1 muscarinic cholinergic pathway that activates phosphatidylinositol 4,5-bisphosphate-specific phospholipase C via the G protein Gq/11. Recombinant m1 muscarinic receptor was co-reconstituted in lipid vesicles with either hepatic Gq/11 or with cerebral αq/11 and βγ subunits. The rate of [35S]GTPγS binding to the reconstituted vesicles was stimulated 20-50-fold by agonist. Maximal receptor-catalyzed binding was 7 mol of GTPγS bound per mol of receptor. The m2 muscarinic receptor was a poor activator of Gq/11. The binding of [α-32P]GTP and [γ-32P]GTP to m1/Gq/11 vesicles indicated that the receptor could maintain up to 40% of the total coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 mm-1, is consistent with the rate predicted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeric G proteins. Agonist-stimulated photoaffinity labeling with γ-(4-azidoanilido)-[α-32P]GTP indicated that the receptor catalyzed binding to both αq and α11 with about equal efficiency. Receptor-catalyzed activation of Gq/11 by GTPγS, measured as the ability to activate purified phospholipase C-β1, paralleled receptor-catalyzed [35S]GTPγS binding. Co-reconstitution of receptor, Gq/11, and phospholipase C-β1 restored GTPγS-dependent carbachol-stimulated hydrolysis of phosphatidylinositol 4,5-bis-phosphate. The m1 receptor, Gq/11, and phospholipase C-β1 are thus sufficient to initiate the hormonal inositol trisphosphate/diacylglycerol signaling pathway without additional proteins.

Original languageEnglish (US)
Pages (from-to)8081-8088
Number of pages8
JournalJournal of Biological Chemistry
Volume267
Issue number12
Publication statusPublished - Apr 25 1992

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ASJC Scopus subject areas

  • Biochemistry

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