Regulation of Phospholipase C-1 by G and m1 Muscarinic Cholinergic Receptor

The phospholipase C-β1 (PLC-β1) signaling pathway was reconstituted by addition of purified PLC to phospholipid vesicles that contained purified recombinant m1 muscarinic cholinergic receptor, G, 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 Ca (10-100 nM). PLC-β1 is also a GTPase-activating protein for G. 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-bound GTP by at least 100-fold. Despite this rapid hydrolysis of G-bound GTP, the receptor maintained >10% of the total G in the active GTP-bound form by catalyzing GTP binding at a rate of at least 20-25 min, 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, G, 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.