Regulation of phospholipase C-β1 by Gq and m1 muscarinic cholinergic receptor: Steady-state balance of receptor-mediated activation and GTPase-activating protein-promoted deactivation

Gloria H. Biddlecome, Gabriel Berstein, Elliott M. Ross

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208 Scopus citations

Abstract

The phospholipase C-β1 (PLC-β1) signaling pathway was reconstituted by addition of purified PLC to phospholipid vesicles that contained purified recombinant m1 musearinic cholinergic receptor, Gq, and 2-4 mol % [3H]phosphatidylinositol 4,5-bisphosphate. In this system, the muscarinic agonist carbachol stimulated steady-state PLC activity up to 90-fold in the presence of GTP. Both GTP and agonist were required for PLC activation, which was observed at physiological levels of Ca2+ (10-100 mM). PLC-β1 is also a GTPase-activating protein for Gq. It accelerated steady-state GTPase activity up to 60-fold in the presence of carbachol, which alone stimulated activity 6-10-fold, and increased the rate of hydrolysis of Gq-bound GTP by at least 100-fold. Despite this rapid hydrolysis of Gq-bound GTP, the receptor maintained >10% of the total Gq in the active GTP-bound form by catalyzing GTP binding at a rate of at least 20-25 min-1, ∼10-fold faster than previously described. These and other kinetic data indicate that the receptor and PLC-β1 coordinately regulate the amplitude of the PLC signal and the rates of signal initiation and termination. They also suggest a mechanism in which the receptor, Gq, and PLC form a three-protein complex in the presence of agonist and GTP (stable over multiple GTPase cycles) that is responsible for PLC signaling.

Original languageEnglish (US)
Pages (from-to)7999-8007
Number of pages9
JournalJournal of Biological Chemistry
Volume271
Issue number14
DOIs
StatePublished - Apr 5 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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