TY - JOUR
T1 - Regulation of phospholipase C-β1 by Gq and m1 muscarinic cholinergic receptor
T2 - Steady-state balance of receptor-mediated activation and GTPase-activating protein-promoted deactivation
AU - Biddlecome, Gloria H.
AU - Berstein, Gabriel
AU - Ross, Elliott M.
PY - 1996/4/5
Y1 - 1996/4/5
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.271.14.7999
DO - 10.1074/jbc.271.14.7999
M3 - Article
C2 - 8626481
AN - SCOPUS:0029961069
SN - 0021-9258
VL - 271
SP - 7999
EP - 8007
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 14
ER -