Abstract
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.
Highlights
Heterotrimeric G proteins transmit signals from cell-surface receptors to intracellular effectors by transiting a controlled cycle of GTP binding and hydrolysis
When purified Gq and m1AChR were co-reconstituted into phospholipid vesicles, addition of purified recombinant phospholipase C-1 (PLC-1) stimulated steadystate GTPase activity Ͼ20-fold when the muscarinic agonist carbachol was added to stimulate GTP binding [6]
To study the regulation of PLC-1 by muscarinic agonist and GTP in a system composed of known concentrations of pure proteins, purified recombinant Gq and m1AChR were co-reconstituted in a single population of phospholipid vesicles that contained 2– 4 mol % [3H]PIP2; purified PLC-1 was added subsequently
Summary
Materials—[␥-32P]GTP was synthesized as described by Johnson and Walseth [16] and purified by anion-exchange high pressure liquid chromatography using a 20 –350 potassium Pi gradient (pH 7.0). For vesicles that lacked [3H]PIP2, m1AChR was measured by [3H]QNB binding in the filtration assay described previously [15]. For vesicles that contained [3H]PIP2, m1AChR was measured by a modified centrifugal gel filtration [3H]QNB binding assay in which receptor-bound [3H]QNB was separated from [3H]PIP2 by thin-layer chromatography. M1AChR-Gq-[3H]PIP2 vesicles (30 l) were mixed with assay buffer (60 l) so that final concentrations were 70 mM NaHepes (pH 7.5), 100 mM NaCl, 3 mM EGTA, and 2.3–3.0 mM total MgCl2. The assay mixture (90 l) was warmed for 3 min at 30 °C, and 10 l of PLC-1 (1 nM final concentration in 20 mM NaHepes (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, 10% glycerol, and 0.1 mg/ml BSA) was added to initiate the assay. Proteins were detected as described in the ECL kit (Amersham Corp.)
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