Regulation of phospholipase C-beta1 by Gq and m1 muscarinic cholinergic receptor. Steady-state balance of receptor-mediated activation and GTPase-activating protein-promoted deactivation.
The phospholipase C-beta1 (PLC-beta1) signaling pathway was reconstituted by addition of purified PLC to phospholipid vesicles that contained purified recombinant m1 muscarinic 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 nM). PLC-beta1 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, approximately 10-fold faster than previously described. These and other kinetic data indicate that the receptor and PLC-beta1 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.
Biddlecome
G H
GH
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041, USA.
Berstein
G
G
Ross
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000502:physiology; D002474:Cell-Free System; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D051381:Rats; D011976:Receptors, Muscarinic / Q000502:physiology*; D011994:Recombinant Proteins; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*
D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010738:Type C Phospholipases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D002118:Calcium