Regulation of phospholipase C-beta 1 by G(q) and m1 muscarinic cholinergic receptor - Steady-state balance of receptor-mediated activation and GTPase-activating protein-promoted deactivation

The phospholipase C-beta 1 (PLC-beta 1) signaling pathway was reconstituted by addition of purified PLC to phospholipid vesicles that contained purified recombinant mi muscarinic cholinergic receptor, G(q), and 2-4 mol % [H-3]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 nM). PLC-beta 1 is also a GTPase-activating protein for G(q). 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 G(q)-bound GTP by at least 100-fold. Despite this rapid hydrolysis of G(q)-bound GTP, the receptor maintained >10% of the total G(q) in the active GTP-bound form by catalyzing GTP binding at a rate of at least 20-25 min(-1), similar to 10-fold faster than previously described. These and other kinetic data indicate that the receptor and PLC-beta 1 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, G(q), 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.