©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Phospholipase C-1 by G and m1 Muscarinic Cholinergic Receptor
STEADY-STATE BALANCE OF RECEPTOR-MEDIATED ACTIVATION AND GTPase-ACTIVATING PROTEIN-PROMOTED DEACTIVATION (*)

(Received for publication, December 4, 1995; and in revised form, January 23, 1996)

Gloria H. Biddlecome Gabriel Berstein (§) Elliott M. Ross (¶)

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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, G(q), 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-beta1 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, 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, 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.


INTRODUCTION

Heterotrimeric G proteins transmit signals from cell-surface receptors to intracellular effectors (such as PLC-beta (^1)enzymes, adenylyl cyclase, or ion channels) by transiting a controlled cycle of GTP binding and hydrolysis. GTP binding to the alpha subunit activates G proteins and allows them, in turn, to activate effectors. They are deactivated when they hydrolyze bound GTP to GDP. Receptors increase the rate at which G proteins bind GTP. Agonist-bound receptor thus determines the rate of G protein activation, and hydrolysis of bound GTP determines the rate of deactivation. The relative rates of activation and deactivation determine the steady-state concentration of the activated G protein and, therefore, the amplitude of the signal transmitted through the receptor-G protein-effector pathway (see (1, 2, 3) for reviews).

Until recently, the rate of GTP hydrolysis by G protein alpha subunits was not known to be regulated directly, although there was evidence that GTP hydrolysis in cells may occur faster than had been described for purified G proteins(1) . A model for such regulation was the small monomeric GTP-binding proteins, such as p21. For these proteins, GTP hydrolysis is negligible in the absence of GTPase-activating proteins (GAPs), which accelerate hydrolysis dramatically. For example, the rate of hydrolysis of GTP bound to p21 is increased by Ras GAP 10^5-fold (from 1.2 times 10 s to 19 s )(4) . Numerous GAPs specific for one or more small GTP-binding proteins have now been identified. They regulate signal duration and/or amplitude, but apparently do not directly transmit signal(5) .

Only two GAPs for heterotrimeric G proteins have been described so far, one each for G(q) and G(t). The G(q) GAP is PLC-beta, the principal effector regulated by G(q)alpha(6) . When purified G(q) and m1AChR were co-reconstituted into phospholipid vesicles, addition of purified recombinant PLC-beta1 stimulated steady-state GTPase activity >20-fold when the muscarinic agonist carbachol was added to stimulate GTP binding(6) . Steady-state kinetic arguments and preliminary measurements of the rate of hydrolysis of bound GTP in this system suggested that PLC-beta1 stimulates hydrolysis of G(q)-bound GTP >50-fold, from 0.8 min to at least 40 min(6) .

The G(q) GAP activity described for PLC-beta1 is reciprocally specific for the PLC-beta family and the G(q) family. PLC-beta1, PLC-beta2, and PLC-beta3 are all G(q) GAPs, (^2)and preliminary data suggest that the PLC-beta encoded by the Drosophila NorpA gene is a GAP for Drosophila visual G(q)(7) . In contrast, neither PLC- (not G protein-regulated) nor adenylyl cyclase (regulated by G(s), but not by G(q)) has G(q) GAP activity.^2 In addition, while PLC-beta1 is a GAP for the G(q) family members G(q), G, and G(14), it is not a GAP for G(i), G(o), G(s), or G(z)(6, 7, 8) .^2

The G(t) GAP also involves the G(t) effector protein, cyclic GMP phosphodiesterase. In this case, however, both the subunit of the phosphodiesterase and a second, unidentified membrane protein are required to stimulate GTP hydrolysis by G(t)(9, 10, 11, 12, 13) . Phosphodiesterase and the membrane-bound factor together can increase the hydrolysis rate constant of G(t) from 0.05 s to >1 s, approaching the physiological deactivation rate of photoreceptor signaling(13) .

Not all G protein-regulated effectors are GAPs. We have been unable to detect G(s) GAP activity in several preparations of adenylyl cyclase(14) . However, the existence of other G protein GAPs is strongly predicted because the rates of deactivation of many G protein-mediated signaling processes are significantly faster than the rates of hydrolysis of bound GTP by isolated G proteins (reviewed in (1) ).

The finding that PLC-beta1 is a GAP for G(q) raises the question of how a signal can be generated when an effector that is a GAP terminates its own stimulus. Indeed, the discovery of PLC-beta1 as a G(q) GAP was sparked by our failure to detect GTP-supported stimulation of PLC activity by muscarinic agonists when m1AChR, G(q), and PLC-beta1 were co-reconstituted into phospholipid vesicles(15) . Although our measurements of GTP binding, GTP hydrolysis, and accumulation of activated G(q)-bound GTP suggested that added PLC-beta1 should be substantially activated, the G(q) GAP activity of PLC-beta1 accelerated GTP hydrolysis, and no phospholipase stimulation was observed. More recently, Nakamura et al.(8) succeeded in demonstrating stimulation of PLC activity by carbachol plus GTP in a similarly reconstituted system, suggesting that the three proteins are sufficient to generate signal in response to agonist in the presence of GTP. However, activation was modest (2.2-fold), leaving open the question of how receptor-promoted GTP binding can keep up with GAP-stimulated hydrolysis.

For this study, we have modified our previous reconstitution protocol (6, 15) to develop a system that generates a large IP(3) signal in response to GTP and carbachol (50-100-fold stimulation). This system provides an experimental model in which to study PLC-beta1 activity in terms of the regulation of the G(q) GTPase cycle. The data reported here reconcile the rates of receptor-stimulated GTP binding by G(q) and of GAP-stimulated GTP hydrolysis with the steady-state concentration of G(q)-bound GTP and activated PLC-beta1.


EXPERIMENTAL PROCEDURES

Materials

[-P]GTP was synthesized as described by Johnson and Walseth (16) and purified by anion-exchange high pressure liquid chromatography using a 20-350 potassium P(i) gradient (pH 7.0). Background in the GTPase assay was <0.3%. [alpha-P]GDP was prepared as described by Ferguson and Higashijima (17) and purified as described for [-P]GTP. NTA-Ni-agarose was from QIAGEN Inc. Lubrol 12A9 was a gift from Zeneca. Cholate and deoxycholate were from Sigma and were purified as described previously (18) . CHAPS was from Sigma. 3-(2`-Aminobenzhydryloxy)tropane-agarose (19) was the gift of T. Haga and K. Haga (University of Tokyo). Sources of other materials are listed elsewhere(15) .

Recombinant Proteins

All proteins used in this study were expressed in Sf9 cells using baculovirus vectors. The rat PLC-beta1 cDNA (20) was modified to replace the first seven codons with Met-Gly-His(6) and ligated into pVL1393(21) . Mouse G(q)alpha cDNA (22) that extended to a site 170 base pairs beyond the termination codon was cut with EcoNI just after the initiation codon and ligated with a 5`-BamHI/EcoNI duplex linker into pVL1393(21) . This construct does not contain a His(6) tag, and the coding sequence begins at the first of two in-frame methionine residues (MTLESIMACC . . . ). All cDNA constructs were prepared using standard procedures(23) , and recombinant viruses were prepared as described previously(24, 25) . Baculoviruses that encode Gbeta subunits, G subunits, and Galpha (26, 27) were gifts from J. Iñiguez-Lluhí, T. Kozasa, and A. G. Gilman (Department of Pharmacology, University of Texas Southwestern Medical Center). Methods for the growth of Sf9 cells and expression of recombinant proteins have been described previously(25) .

Membrane Preparation and Protein Extraction

All protein purification steps were conducted at 4 °C except as indicated. Sf9 cells were harvested, and membranes were prepared 48-60 h after infection as described by Parker et al.(25) , except that 5 mM MgCl(2) and 10 µg/ml DNase I were included in the final step to disrupt DNA aggregates. Thawed membranes (5 mg/ml) were extracted by stirring for 1 h in extraction buffer, and the mixture was centrifuged at 100,000 times g for 1 h. m1AChR was purified as described previously(19, 25) .

Purification of G(q) Subunits

Gbeta(1)(2) was purified exactly as described by Kozasa and Gilman(26) . Purification of G(q)alpha was based on the same approach. Sf9 cells were infected with baculoviruses encoding G(q)alpha, Gbeta(2)His(6), and G(2)His(6). Membrane proteins were extracted with 1% cholate in 20 mM Tris-Cl (pH 8.0), 100 mM NaCl, 5 mM 2-mercaptoethanol, 10 µM GDP, 0.1 mM PMSF, 10 µg/ml leupeptin, and 1 µg/ml aprotinin. All chromatography buffers contained 20 mM NaHepes (pH 8.0), 100 mM NaCl, 1 mM MgCl(2), 5 mM 2-mercaptoethanol, 0.1 mM PMSF, 10 µg/ml leupeptin, and 1 µg/ml aprotinin. Buffer G1 also contained 0.5% Lubrol 12A9 and 10 µM GDP. Buffer G2 contained the components of buffer G1 plus 0.2% cholate and 3 µM GTPS. Buffer G3 contained 0.3% cholate. Buffer G4 contained 1% cholate, 20 µM AlCl(3), 10 mM NaF, and 10 mM total MgCl(2). Buffer G5 contained 0.4% cholate and 1 mM dithiothreitol (but no 2-mercaptoethanol).

The extraction supernatant (100 ml) was diluted 5-fold into buffer G1 and mixed with 12 ml of NTA-Ni-agarose. The mixture was stirred for 1 h, and the NTA-Ni-agarose was collected in a chromatography column. The resin was washed with buffer G1, buffer G1 plus 0.5 M NaCl, buffer G1 plus 0.5 M NaCl and 10 mM imidazole, buffer G1 plus 1 M NaCl, and buffer G1. Each wash step was deemed complete when the absorbance base line (280 nm) remained flat for at least 24 ml. The column was then moved to room temperature and washed with buffer G2 (100 ml) to remove the endogenous Sf9 alpha(i)-like protein and with buffer G3 (80 ml) to remove residual GTPS and Lubrol. G(q)alpha was separated from the Gbeta(2)His(6)bulletG(2)His(6) dimer at room temperature in buffer G4 (100 ml), and the eluate was collected at 0 °C. The Al/F concentration in the eluate was reduced at least 1000-fold by repeated concentration (Amicon PM-10 membrane) and dilution in buffer G5. G(q) was prepared by mixing G(q)alpha and Gbeta(1)(2) in a 1:2 molar ratio. The concentration of active G(q)alpha was determined according to bound GDP(28) , and the Gbeta(1)(2) concentration was based on total protein(29) .

The estimated purity of G(q)alpha was >95%, and that of Gbeta(1)(2) was >85% (Fig. 1). No Sf9 cell G(i)alpha was detectable in the G(q)alpha preparation by silver staining or by immunoblotting (P-960 antibody) (30) . We were also unable to detect contamination with Sf9 cell G(q)alpha by comparing the bands detected on a single immunoblot that was sequentially probed with an antibody that recognizes Sf9 cell G(q)alpha (Z811) (31) and with a second antibody that recognizes only recombinant G(q)alpha. The yield of G(q)alpha was 130-150 µg/liter of Sf9 cells; the yield of Gbeta(1)(2) was 340 µg/liter of Sf9 cells.


Figure 1: SDS-polyacrylamide gel electrophoresis of purified proteins. Each protein, purified as described under ``Experimental Procedures,'' was electrophoresed and silver-stained. Two samples of each protein (nanograms applied) are shown, one of which is 10-fold overloaded to highlight impurities. Arrowheads indicate the major protein bands. The lower arrowhead for the Gbeta gel indicates G, which stains poorly but is visible on the original gel.



Purification of PLC-beta1

Sf9 cells were infected with baculovirus encoding PLC-beta1-His(6). Peripheral membrane proteins were extracted with 20 mM Tris-Cl (pH 7.5), 1 M NaCl, 5 mM 2-mercaptoethanol, 0.1 mM PMSF, 20 µg/ml leupeptin, and 1 µg/ml aprotinin, and the extract (220 ml) was stirred with 8 ml of NTA-Ni-agarose for at least 1 h. All wash buffers contained the components of buffer P (20 mM Tris (pH 7.5), 5 mM 2-mercaptoethanol, 20% glycerol, 0.1 mM PMSF, 20 µg/ml leupeptin, and 1 µg/ml aprotinin) plus NaCl or imidazole as indicated. The resin was washed as follows: 1) with 1 M NaCl until A returned to zero; 2) with a gradient of 1 M NaCl to 100 mM NaCl plus 10 mM imidazole (12 ml); 3) with 100 mM NaCl plus 10 mM imidazole (30 ml); 4) with a gradient of 100 mM NaCl plus 10 mM imidazole to 1 M NaCl plus 10 mM imidazole (12 ml); and 5) with 1 M NaCl plus 10 mM imidazole (30 ml). PLC-beta1 was eluted with 100 mM NaCl plus 50 mM EDTA in buffer P (30 ml). The NTA-Ni-agarose eluate was loaded onto a Pharmacia Biotech HR5/5 MonoQ column equilibrated with 10 mM NaCl in buffer Q (20 mM Tris-Cl (pH 7.5), 1 mM dithiothreitol, 20% glycerol, 0.1 mM PMSF, 20 µg/ml leupeptin, and 1 µg/ml aprotinin). Buffer Q, with the NaCl concentrations indicated below, was used for all steps of the MonoQ chromatography. The column was washed with 10 mM NaCl (5 ml) and then with 100 mM NaCl until A returned to zero. PLC-beta1 was eluted with a 15-ml gradient of 100-600 mM NaCl. PLC-beta1 eluted between 200 and 300 mM NaCl in four 1-ml fractions. The pooled MonoQ fractions were diluted 10-fold into buffer S (20 mM MES (pH 6.0), 1 mM dithiothreitol, 1 mM EDTA, 20% glycerol, 0.1 mM PMSF, 20 µg/ml leupeptin, and 1 µg/ml aprotinin) and loaded onto a Pharmacia Biotech HR5/5 MonoS column equilibrated with 10 mM NaCl in buffer S. Buffer S, with NaCl added as indicated below, was used for all steps of the MonoS chromatography. The column was washed with 5 mM NaCl (5 ml) and then with 200 mM NaCl until A returned to zero. PLC-beta1 was eluted with a 20-ml gradient of 200-700 mM NaCl. PLC-beta1 eluted between 350 and 450 mM NaCl in four 1-ml fractions, which were pooled, concentrated to at least 1 mg/ml (Amicon Centricon-30), and stored at -80 °C. PLC-beta1 was >98% pure (Fig. 1). Bands at 100 and 50 kDa are proteolytic products. The His(6) tag did not alter the behavior of the protein (compare with Refs. 6 and 15).

Protein-Phospholipid Vesicles

m1AChR and G(q) were co-reconstituted into lipid vesicles by gel filtration. A mixture (75 µl) of phosphatidylethanolamine (1650 µM), phosphatidylserine (980 µM), cholesteryl hemisuccinate (180 µM), and [^3H]PIP(2) (100 µM, 88-165 cpm/pmol) was dried under argon; rehydrated in 20 mM NaHepes (pH 7.5), 100 mM NaCl, 1 mM EGTA (pH 8.0), and 0.3% deoxycholate (pH 8.0); and sonicated under argon at room temperature for 20 min. The cloudy suspension was centrifuged for 10 min at 1300 times g at 4 °C, and the clear supernatant was removed to a separate tube. Recovery of [^3H]PIP(2) in this supernatant was at least 90% and usually >97%. Phospholipid-detergent micelles (75 µl, 205 nmol of phospholipid) were mixed with purified G(q) (35 pmol of G(q)alpha) and held on ice for 15 min before addition of purified m1AChR (10-13 pmol). Column buffer (20 mM NaHepes (pH 7.5), 2 mM MgCl(2), 1 mM EGTA (pH 8.0), and 100 mM NaCl) was added to bring the total volume to 150 µl, and the mixture was applied to a 6.6 times 250-mm column of Ultrogel AcA34. Protein-phospholipid vesicles (750 µl) were recovered in the void volume, 0.1 mg/ml protease-free BSA was added, and the vesicles were stored at -80 °C. Recovery in the vesicles of applied m1AChR was 5-13%, that of G(q) coupled to m1AChR was 11-28%, and that of [^3H]PIP(2) was 30-50%.

Quantitation of Vesicle Proteins

Total receptor-coupled G(q) was measured by carbachol-stimulated [S]GTPS binding as described previously(15) . For vesicles that lacked [^3H]PIP(2), m1AChR was measured by [^3H]QNB binding in the filtration assay described previously(15) . For vesicles that contained [^3H]PIP(2), m1AChR was measured by a modified centrifugal gel filtration [^3H]QNB binding assay in which receptor-bound [^3H]QNB was separated from [^3H]PIP(2) by thin-layer chromatography. Vesicles were incubated for 1 h at 30 °C with 49 nM [^3H]QNB, with or without 23 µM atropine, and then solubilized in 1% digitonin at 0 °C. Solubilized vesicles were diluted 10-fold into column buffer that contained 0.1% digitonin and 2.5 nM [^3H]QNB, with or without 10 µM atropine, so that the final concentrations were 6.3 nM [^3H]QNB and 11 µM atropine. Receptor-bound [^3H]QNB, collected by centrifugal gel filtration, was separated from [^3H]PIP(2) by chromatographing the sample (lyophilized and resuspended in 10 µl of 70% ethanol) on silica gel in ethanol/acetic acid/water (60:30:10). [^3H]PIP(2) was retained near the origin (R(F) = 0-0.2), and [^3H]QNB migrated with an R(F) of 0.4-0.7. [^3H]QNB was quantitated by liquid scintillation counting. Each vesicle preparation was tested in quadruplicate for total binding (QNB) and in duplicate for nonspecific binding (QNB + atropine). Control experiments with vesicles that lacked [^3H]PIP(2) indicated that this method detects 80-90% of the m1AChR detected by the filtration assay. Receptor concentrations reported here were not corrected for this small loss of receptor.

GTPase Assays

Measurement of GTP hydrolysis has been described previously(6, 32) . The assay medium contained 20 mM NaHepes (pH 8.0), 100 mM NaCl, 1 mM EGTA, 2 mM MgCl(2), 0.1 mg/ml BSA, and 1 µM [-P]GTP (80-200 cpm/fmol) or 10 µM[-P]GTP (4-8 cpm/fmol for GAP-stimulated assays). Unless otherwise indicated, reactions were initiated by addition of vesicles and proceeded for 10 min at 30 °C.

Nucleotide Binding Assays

Binding of [alpha-P]GTP, [S]GTPS, or [alpha-P]GDP was measured by adsorption of G proteins to BA85 nitrocellulose membranes as described previously(33) . Final concentrations in the assay were 20 mM NaHepes (pH 7.5), 2 mM MgCl(2), 1 mM EGTA, 100 mM NaCl, and 0.1 mg/ml BSA. Assays were conducted at 30 °C. The concentration of nucleotide and the order of addition of assay components are indicated in the figure legends.

PLC Activity Assays

PLC activity was measured as described by Blank et al.(34) . m1AChR-G(q)-[^3H]PIP(2) vesicles (30 µl) were mixed with assay buffer (60 µl) so that final concentrations were 70 mM NaHepes (pH 7.5), 100 mM NaCl, 3 mM EGTA, and 2.3-3.0 mM total MgCl(2). GTP (10 µM), CaCl(2) (to give 10 nM free Ca), and either carbachol (1 mM) or atropine (10 µM) were included except as indicated in the figure legends. The concentrations of free Ca were calculated using constants from Martell and Calvin (35) adjusted for temperature, pH, and ionic strength. The assay mixture (90 µl) was warmed for 3 min at 30 °C, and 10 µl of PLC-beta1 (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. The zero time point was taken before addition of PLC-beta1. PLC-accessible [^3H]PIP(2) was measured as [^3H]IP(3) formed during a 40-min incubation with 1 mM carbachol, 100 nM GTPS, and 1 µM free Ca. No additional product was formed after 30 min, even if fresh PLC was added (data not shown). PLC-accessible PIP(2) is reported in the figure legends and ranged from 30 to 70% of the total PIP(2).

Miscellaneous

For G(q)alpha, Gbeta, m1AChR, and PLC-beta1, total protein was estimated by Amido Black staining (29) using BSA as a standard. Active G(q)alpha was estimated by bound GDP (15, 28) and was 40-60% of the amount estimated by Amido Black staining(29) . SDS-polyacrylamide gel electrophoresis was performed according to Laemmli(36) . For enhanced resolution of closely migrating G(q)alpha species, a 42:1 acrylamide/bisacrylamide ratio was used, and electrophoresis was continued until the 30-kDa marker left the gel. For Western blots, proteins were transferred to nitrocellulose overnight in cold transfer buffer (37) containing 20% methanol. Proteins were detected as described in the ECL kit (Amersham Corp.).


RESULTS

Steady-state Regulation of PLC-beta1 Activity by G(q) and m1AChR

Successful reconstitution of the regulation of PLC-beta1 by receptor is complicated both by the G(q) GAP activity of PLC-beta1 and by the membrane-bound nature of its PIP(2) substrate. To study the regulation of PLC-beta1 by muscarinic agonist and GTP in a system composed of known concentrations of pure proteins, purified recombinant G(q) and m1AChR were co-reconstituted in a single population of phospholipid vesicles that contained 2-4 mol % [^3H]PIP(2); purified PLC-beta1 was added subsequently. In this system, addition of GTP plus carbachol stimulated steady-state PIP(2) hydrolysis substantially, 60-fold in the example shown in Fig. 2. Stimulation by carbachol plus GTP was blocked by atropine, a muscarinic antagonist. PIP(2) hydrolysis was increased only slightly above a low background by either carbachol or GTP alone, and ``-fold stimulation'' in this system depends largely on the basal activity, which may represent only 10-50 cpm. These responses compare well with those observed in natural membranes and many intact cells. The nonhydrolyzable GTP analog GTPS by itself increased PLC-beta1 activity as much or more than did GTP plus carbachol, and the combination of GTPS and carbachol increased activity 100-200-fold. Stimulation by GTPS alone evidently reflects nucleotide exchange promoted by unliganded m1AChR because GTPS-stimulated activity was inhibited 50% by atropine in the absence of any agonist (data not shown).


Figure 2: Reconstitution of G(q)-mediated activation of PLC-beta1 by m1AChR. m1AChR and G(q) were co-reconstituted with [^3H]PIP(2) as described under ``Experimental Procedures.'' The activity of added PLC-beta1 was measured in the presence of guanine nucleotide and/or muscarinic ligands. A, GTP-dependent PLC activity; B, GTPS-stimulated PLC activity. Conditions were as follows: no addition(-), 10 µM GTP (GTP), 1 mM carbachol (Cch), carbachol plus 10 µM atropine (C+A), and 100 nM GTPS (S). All samples contained 10 nM free Ca, 2.4 nM G(q), 0.33 nM m1AChR, 1 nM PLC-beta1, and 0.56 µM accessible PIP(2). A and B show data (mean ± S.D.) from the same experiment on different scales.



Regulation of PLC-beta1 by reconstituted m1AChR and G(q) displayed the dependence on the concentrations of carbachol, GTP, Ca, PIP(2), and PLC-beta1 that is typical of intact biological systems (Fig. 3Fig. 4Fig. 5). Carbachol plus GTP markedly stimulated the activity of PIP(2) even at very low Ca concentrations (Fig. 3). Stimulation was 100-fold at 10 nM free Ca, and this concentration was used in all other experiments shown here. Stimulation in the physiological range of 10-100 nM Ca was appreciable. Ca also stimulated PLC activity directly, and stimulation by Ca and that by G(q) were strongly synergistic. This overall pattern of regulation is observed for PLC-beta in native membranes (see (38) for a review or (39) for an example). We have not explored the interaction of muscarinic stimulation and Ca further, however, because agonist-stimulated PLC activities at micromolar Ca concentrations were very high and became nonlinear after a few seconds.


Figure 3: Effect of Ca on G(q)-stimulated PLC-beta1 activity. m1AChR and G(q) were co-reconstituted with [^3H]PIP(2), and the activity of added PLC-beta1 was measured at various free Ca concentrations. The data plotted are the initial rates determined from time courses of PLC activity conducted at each Ca concentration in the presence of 10 µM GTP and either 1 mM carbachol (filled circles) or 10 µM atropine (empty circles). Reactions contained 0.35 nM m1AChR, 3.0 nM G(q), 0.33 µM accessible PIP(2), and 1.0 nM PLC-beta1.




Figure 4: Dependence of carbachol-stimulated PLC activity on concentration of PIP(2). m1AChR and G(q) were co-reconstituted in vesicles that contained various concentrations of PIP(2). PLC activity was measured in the presence of 10 nM free Ca, 10 µM GTP, 1 nM PLC-beta1, and either 1 mM carbachol or 10 µM atropine, Each data point represents the initial rate of carbachol-stimulated PIP(2) hydrolysis derived from a time course performed with a different batch of vesicles. The abscissa is the concentration of PIP(2) accessible to PLC-beta1, as described under ``Experimental Procedures.'' The concentration of G(q) in the 12 batches of vesicles varied between 0.6 and 0.8 nM. Although other experiments in this study were frequently performed at molar concentrations of PIP(2) higher than those shown here, the mole fraction of PIP(2) in the vesicles was maintained in the range shown here (1.1-5.5 mol %). The discrepancy reflects experimental scale. The data come from multiple small-scale reconstitutions wherein lipid/protein ratios were decreased by half to conserve materials and to maximize yield during reconstitution.




Figure 5: GTPase and PLC activities at increasing concentrations of PLC-beta1. m1AChR-G(q) vesicles were mixed with increasing concentrations of PLC-beta1 and assayed for both GTPase (filled circles) and PLC activity (empty circles) under identical conditions. Assays contained 0.27 nM m1AChR, 1.7 nM G(q), 0.44 µM accessible PIP(2), 10 µM GTP, 10 nM Ca, and either 1 mM carbachol or 10 µM atropine. Data show increases in activity caused by carbachol. Assay times were adjusted to ensure linear reactions.



Carbachol-stimulated PLC activity increased linearly with the concentration of PIP(2) in the vesicles up to 6 mol % of total phospholipid (Fig. 4). The routine concentration of PIP(2) was held at 3 mol %, which is not unreasonable with respect to the concentrations of PIP(2) in the inner leaflet of animal plasma membranes(40, 41) . Much higher concentrations of PIP(2) decreased recovery of m1AChR during reconstitution and may inhibit m1AChR-G(q) coupling (as measured by agonist-stimulated GTPS binding) (data not shown). PLC activity also increased linearly with increasing amounts of total vesicles added to the assays (data not shown). Added PLC-beta1 presumably has access only to PIP(2) in the outer leaflet of the vesicle bilayer, and prolonged incubation of the vesicles with 100 nM GTPS, 1 mM carbachol, and 1 µM Ca resulted in hydrolysis of only about half of the PIP(2). We have therefore defined PLC-accessible PIP(2) as that which can be hydrolyzed in the presence of these activators after 40 min at 30 °C. PLC assays were linear with time so long as <15% of the accessible PIP(2) was hydrolyzed.

Note that carbachol-stimulated GTPase activity, measured in either the presence or absence of PLC-beta1, was not altered by the presence of Ca or by the concentration of PIP(2) in the vesicles (data not shown).

In addition to its activity as a phospholipase, PLC-beta1 stimulates the GTPase activity of G(q)(6) . Maximal stimulation at steady state can exceed 60-fold in the reconstituted vesicles when carbachol is added to promote GDP/GTP exchange (35-fold in Fig. 5). The G(q) GAP activity of PLC-beta1 is saturable, with an EC of 1-2 nM(6) (1.1 nM in Fig. 5). Carbachol-stimulated PLC activity also reached a plateau in this same range of PLC-beta1 concentrations, with half-maximal activity at 0.5 nM PLC-beta1. This behavior was not caused by substrate depletion at high enzyme concentrations; <7% of the accessible PIP(2) was hydrolyzed in the assays shown in Fig. 5. The plateau in activity at high nanomolar concentrations of PLC-beta1 apparently reflects the limiting steady-state concentration of GTP-activated G(q) present during the assay (data not shown).

Dependence of Steady-state PLC-beta1 Activity and G(q) GTPase Activity on Carbachol and GTP

The GAP activity of PLC-beta1 and the GDP/GTP exchange activity of the receptor combine to determine the response of the reconstituted system to both GTP and carbachol. As reported previously(6) , PLC-beta1 increases the concentration of carbachol needed to stimulate GTP hydrolysis, increasing the EC by 6-10-fold such that the EC becomes approximately equal to the K(d) for agonist (Fig. 6A)(6) . This change reflects a change in the rate-limiting step of the GTPase cycle. In the absence of PLC-beta1, the receptor-independent hydrolysis of G(q)-bound GTP becomes rate-limiting as the rate of receptor-catalyzed GDP/GTP exchange increases, and only a fraction of receptors need be agonist-bound to maintain maximal flux through the GTPase cycle. In the presence of PLC-beta1, where hydrolysis of G(q)-bound GTP is fast, receptor-catalyzed GDP/GTP exchange is rate-limiting even when all receptor is agonist-bound, and the steady-state GTPase rate reflects agonist binding to receptor. Stimulation of PLC activity by carbachol displayed the same concentration dependence as did the GAP-stimulated GTPase reaction, indicating that stimulation of both activities depends on the rapid binding of GTP.


Figure 6: Dependence of steady-state GTPase and PLC activities on concentrations of GTP and carbachol. GTPase and PLC activities were measured under identical conditions using the same batch of vesicles for all assays shown in each panel (except filled squares). The concentration of PLC-beta1 was 10 nM in the PLC assays (filled triangles) and, where added, in the GTPase assays (filled squares and circles). GTPase was also assayed without added PLC (empty circles). Carbachol-stimulated activities are shown. A, activities were assayed in the presence of 10 µM GTP with 0.19 nM m1AChR, 2.0 nM G(q), and 0.52 µM accessible PIP(2). Maximum activities were 93.6 ± 3.2 fmol of P(i)/min without PLC, 976 ± 39 fmol of P(i)/min with PLC, and 8.58 ± 0.37 pmol of IP(3)/min. B, activities were assayed in the presence of 1 mM carbachol with 0.37 nM m1AChR, 2.3 nM G(q), and 0.45 µM accessible PIP(2). Maximum activities were 40.5 ± 5.5 fmol of P(i)/min without PLC, 2610 ± 170 fmol of P(i)/min with PLC, and 4.50 ± 0.31 pmol of IP(3)/min. Filled squares and circles in B are from separate experiments.



As shown in Fig. 6B, addition of PLC-beta1 also increased the K(m) for the agonist-stimulated GTPase reaction from 0.25 µM to 5.1 µM GTP. Such an increase is predicted by classical enzyme kinetic theory for a GAP that increases the rate of hydrolysis of bound substrate without altering affinity for substrate. (^4)The GTP concentration curve for the agonist-stimulated PLC reaction fell about midway between the two GTPase curves in several independent experiments (EC = 1.0 µM in Fig. 6B) (data not shown). This behavior is also qualitatively consistent with a simple analysis of the GTPase reaction, but why the EC is lower than the K(m) for GAP-stimulated GTP hydrolysis is not clear.

Rapid Activation and Deactivation of PLC-beta1

Regulation of PLC-beta1 activation in the reconstituted system is reasonably rapid. The experiments shown in Fig. 7measure the onset kinetics for PLC activation when one activating ligand, carbachol or GTP, was added to a mixture of m1AChR-G(q)-[^3H]PIP(2) vesicles and PLC-beta1 that had been preincubated with the other ligand. Stimulation of PIP(2) hydrolysis was essentially instantaneous when GTP was added to vesicles preincubated with carbachol, with no observable lag (leq2 s) in multiple experiments. If stimulation was initiated by addition of carbachol, however, full activation displayed a lag of 10-14 s (12 s in Fig. 7B). A 12-s lag corresponds to a rate constant of 5 min. The rate-limiting step in the initial activation of PLC thus involves the interaction of agonist-liganded m1AChR with G(q) followed by the more rapid binding of GTP to the agonist-receptor-G(q) complex.


Figure 7: Kinetics of PLC-beta1 activation and deactivation. Vesicles that contained m1AChR, G(q), and [^3H]PIP(2) were incubated initially in buffer that contained 1 nM PLC-beta1, 10 nM free Ca, and 1 mM carbachol (A), 10 µM GTP (B), or 0.3 mM carbachol plus 10 µM GTP (C). At the times shown by the arrows, 10 µM GTP (A), 1 mM carbachol (Cch; B), or 50 µM atropine (C) was added, and the assay was allowed to continue (filled circles). The linear extrapolations of the initial rates, shown in B and C, are validated by data from at least three other experiments in which reactions were continued both with and without the second addition. The dotted line in B is an extrapolation of the final stimulated rate. Key concentrations were 0.3 (A), 0.6 (B), and 0.2 (C) nM m1AChR; 1.6 (A), 1.2 (B), and 3.3 (C) nM G(q); and 0.35 (A), 0.58 (B), and 0.42 (C) µM accessible PIP(2).



The deactivation of PLC-beta1 upon addition of a muscarinic antagonist is also quite rapid. Carbachol is a low affinity agonist (K(d) 0.1 mM) (25) and dissociates rapidly, such that addition of excess antagonist (50 µM atropine) should block receptors in <1 ms. As shown in Fig. 7C, addition of atropine during agonist-stimulated steady-state PIP(2) hydrolysis inhibited PLC activity with no perceptible lag. Based on the temporal resolution of the earliest data points, we estimate from multiple experiments that the deactivation rate constant in such an experiment is >25 min. Assuming that deactivation requires hydrolysis of G(q)-bound GTP, 25 min is thus also a lower limit on the GAP-stimulated k for G(q). This rate is 30-fold higher than the GTPase k displayed by isolated G(q)(15) .

Rates of Nucleotide Exchange and G(q) Activation by a m1AChR-G(q) Complex

The rapid rate of activation of PLC-beta1 shown in Fig. 7should reflect the initial rate of receptor-catalyzed binding of GTP to G(q), which was originally described as a markedly slower process(15) . As shown in Fig. 8, the rate of carbachol-stimulated binding of either GTP or GTPS to the m1AChR-G(q) vesicles was substantially faster than the low basal rate observed in the presence of atropine, but still too slow to account readily for the PLC activation rates shown in Fig. 7A or for the high steady-state GTPase activities observed in the presence of PLC-beta1. The observed association rate constants in the experiments shown in Fig. 8were 0.41 ± 0.03 min for GTPS and 0.43 ± 0.09 min for GTP (measured with [alpha-P]GTP, which detects both initially bound GTP and the bound GDP hydrolysis product).


Figure 8: Carbachol-stimulated binding of GTPS (A) and GTP (B) to m1AChR-G(q) vesicles. Reactions were initiated by adding vesicles to prewarmed reaction buffers that contained either 1 mM carbachol (filled circles) or 10 µM atropine (empty circles) and either 100 nM [S]GTPS (A) or 500 nM [alpha-P]GTP (B). The dashed lines are the differences between binding measured in the presence of carbachol and atropine. Reactions shown in A and B contained 0.15 or 0.10 nM m1AChR and 2.1 or 0.70 nM G(q), respectively.



Since the relatively slow nucleotide binding reactions described above might reflect an initial delay, we measured agonist-stimulated GDP/GDP exchange under equilibrium conditions in which there was no hydrolysis and slow association of proteins had had time to occur. For this experiment, vesicles were first incubated for 10 min in the presence of both agonist and unlabeled GDP. The rate of GDP binding was then monitored at equilibrium by addition of a tracer amount of [alpha-P]GDP. Subsequently, excess unlabeled GDP was added to measure the rate of dissociation of labeled nucleotide (still at equilibrium). As shown in Fig. 9, both the binding and dissociation of GDP were very rapid under these conditions. Although the association and dissociation time courses were best fit by biphasic functions, the major kinetic components for both the binding and release of GDP displayed rate constants of geq20 min. These rates are probably underestimates because they are calculated according to only a few data points at the limits of resolution of the manual binding assay. Because the rate of binding of GDP is well below the rate predicted by diffusion-controlled association and because the rate of GDP binding equals the rate of GDP release, dissociation of bound GDP is probably rate-limiting for association of a second GDP molecule. According to these data, the reconstituted m1AChR-G(q) vesicle system can achieve agonist-stimulated guanine nucleotide exchange rates commensurate with the high steady-state GTPase rates observed in the presence of agonist and PLC-beta1. Data from preliminary experiments similar to those shown in Fig. 9indicate that PLC-beta1 does not alter the kinetics of agonist-stimulated GDP/GDP exchange.


Figure 9: Receptor-stimulated equilibrium GDP/GDP exchange. Binding and release of GDP were measured at a saturating concentration of free nucleotide. A, a suspension of m1AChR-G(q) vesicles was incubated for 10 min at 30 °C in 1.8 ml of binding assay buffer that contained 500 nM unlabeled GDP and 0.3 mM carbachol. At time 0, 5.4 times 10^7 cpm [alpha-P]GDP (36 µl) was added, and 50-µl aliquots were withdrawn at the indicated times for measurement of bound radiolabeled GDP. These data were best fit by the sum of two exponential binding functions, one with a rate constant of 22 ± 11 min that accounted for 44% of the total (31 fmol) and a second with a rate constant of 2.1 ± 0.5 min that accounted for the remainder. B, at 12.25 min, concentrated unlabeled GDP (8.5 µl) was added to the mixture, and bound radiolabeled GDP was measured at the indicated times as described for A. GDP dissociation was also biphasic; 67% of the total (46 fmol) dissociated rapidly with a rate constant of 20 ± 7 min, and the remainder dissociated more slowly with a rate constant of 1.4 ± 0.3 min. The data shown in A and B were derived from the same binding assay mixture; each 50-µl aliquot contained 0.35 nM (17.5 fmol) m1AChR.



To correlate the high equilibrium nucleotide exchange rates shown in Fig. 9with the slow initial onset of agonist-stimulated GTP binding (Fig. 8) and PLC activation (Fig. 7B), we measured the rate of formation of the presumed complex of agonist, m1AChR, and G(q) that accounts for the faster rates. Vesicles were first incubated with carbachol for increasing times, and GTP binding was then initiated by simultaneously adding both [alpha-P]GTP and atropine. The reaction was then allowed to proceed for 60 s to selectively monitor the rapid component of GTP binding. As shown in Fig. 10, initial exposure of the vesicles to carbachol created a species that bound GTP during the subsequent 60-s incubation. Formation of this putative carbachol-m1AChR-G(q) complex occurred with a rate constant of 1.6 min, somewhat slower than the rate of stimulation of PLC by added carbachol (Fig. 7B) and faster than the initial nucleotide exchange rate measured by exposing m1AChR-G(q) vesicles to agonist (Fig. 8). The relationship of these rates is discussed below.


Figure 10: Formation of agonist-m1AChR-G(q) complex that rapidly binds GTP. A, m1AChR-G(q) vesicles were pre-equilibrated for 5 min in binding assay buffer at 30 °C and then exposed to 0.3 mM carbachol for the times shown. At each time point, 300 nM [alpha-P]GTP and 50 µM atropine were added, and the incubation was continued for 60 s before quenching for measurement of bound nucleotide. The zero time point was determined in vesicles that were equilibrated, exposed to atropine and [alpha-P]GTP for 60 s, and then quenched before carbachol was added. The concentrations of m1AChR and G(q) were 0.2 and 2.2 nM, respectively. The experimental background (no added vesicles) was subtracted from all data.



We have not studied the stability of the m1AChR-G(q) complex in detail, but initial evidence suggests that it is not stable over long periods of time. Although 50% of the total G(q) was able to exchange GDP rapidly in experiments of the sort shown in Fig. 9, only 10% of the total G(q) could be trapped as a rapidly binding species in experiments of the sort shown in Fig. 10. For the complex that does remain stable in this experiment, however, the observed decay rate after addition of antagonist was only 1.3 min (data not shown), suggesting that the average lifetime of the complex is equivalent to >30 rounds of the GTPase cycle. The lifetime of the complex could of course be considerably longer in the continued presence of agonist, and we do not know the molecular event(s) that initiates its decay.

Estimation of the Rate of PLC-stimulated GTP Hydrolysis

The high rate of nucleotide exchange shown in Fig. 9(geq20 min) is comparable to the lower limit for the rate of PLC-stimulated hydrolysis of G(q)-bound GTP (previously estimated to be geq40 min(6) or geq25 min according to the data of Fig. 7C). While we have been unable to measure the hydrolysis rate directly, it can be calculated according to the concentrations at steady state of G(q)-bound GTP and G(q)-bound GDP if the rate of GTP binding is known. To measure the binding of GTP or GDP to G(q), m1AChR-G(q) vesicles were incubated for 5 min with or without PLC-beta1, with either carbachol or atropine, and with either [alpha-P]GTP or [-P]GTP. Bound radioactivity from [-P]GTP measures bound GTP, and bound radioactivity from [alpha-P]GTP measures the sum of bound GDP and GTP. Binding reactions were quenched rapidly at 0 °C and applied to nitrocellulose filters immediately. Table 1shows the results of one such experiment. In the presence of agonist, PLC-beta1 had little if any effect on the total amount of GDP plus GTP bound at steady state, but diminished the fraction of bound GTP from 62% to 12%. Thus, PLC reduced the amount of activated G(q) by 80%. Rate constants for hydrolysis of bound GTP can be calculated from these data and the GTP binding rates using a simplified steady-state reaction scheme. According to the data shown in Table 1, PLC-beta1 increases the rate of hydrolysis of G(q)-bound GTP >100-fold, from 0.7 min(15) to 100 min. This experiment was performed three times with similar results, except for variability in the fraction of GTP bound in the presence of both carbachol and PLC-beta1. The calculated values for the hydrolysis rate constants in the other two experiments were 66 and 400 min. We believe that the variability of these results reflects the difficulty of ``instantaneously'' quenching hydrolysis and chose for Table 1the results of the experiment with the fastest quench and the smallest variation among replicate samples. While not a precise value, we believe that 100 min is a good first estimate of the rate at which G(q)-bound GTP is hydrolyzed under the influence of PLC-beta1. Regardless of imprecision, stimulation of the hydrolysis rate is certainly on the order of 100-fold and perhaps severalfold faster. The data of Table 1also show that in the presence of atropine, either steady state was not achieved or there was considerable unliganded G(q) because the concentration of bound GTP plus GDP was only 20% of the total G(q). This result was confirmed in two other experiments. As expected, little GTP was bound to G(q) in the presence of both atropine and PLC-beta1 because the rate of hydrolysis was far greater than the rate of GTP binding.




DISCUSSION

In the G(q)-mediated regulation of PLC-beta, receptor-promoted activation of G(q) is opposed by the GAP activity of the PLC-beta effector. Although this acceleration of GTP hydrolysis allows rapid termination of signaling upon removal of agonist, accelerated deactivation must be balanced by rapid catalysis of GDP/GTP exchange by receptor in order to generate a substantial signal. In general, a G protein must transit the GTPase cycle with markedly different kinetics if its effector protein is or is not a GAP. To address the mechanism of G(q)-mediated signaling, we developed a purified and reconstituted assay system in which the individual steps of activation and deactivation of G(q) and PLC-beta1 can be studied in parallel, either immediately upon activation by agonist or while at steady state.

The reconstituted system described here duplicates several important aspects of natural G(q)-mediated signaling: 1) low basal synthesis of IP(3) in the presence of either agonist or GTP alone, 2) massive activation (50-100-fold) in the presence of both ligands, and 3) rapid onset and termination of signaling. The vesicle system also substantially amplifies its signal, although ``amplification'' is subject to several definitions and is difficult to evaluate in intact cells. In the m1AChR-G(q) vesicles, stimulation by agonist results in synthesis of 200-600 molecules of IP(3)/min/receptor under our standard assay conditions. Such amplification is comparable to that observed in preparations of plasma membranes (see (31) and (42) for examples), and PLC activity in the vesicles can be further increased 3-6-fold by increasing the concentration of Ca within the physiological range (Fig. 3) or by altering phospholipid concentrations (Fig. 4).

The m1AChR-G(q) vesicles are obviously not an exact duplicate of any specific plasma membrane, and plasma membranes vary enormously among different cells. However, the composition and behavior of the vesicles fall within a well described biological range. The phospholipid composition of the vesicles mimics the inner monolayer of the plasma membrane only approximately, but the concentration of PIP(2) (2-4 mol %) is reasonable, and the 10 nM Ca concentration is well within the range described for the cytoplasm of resting cells. The ratio of m1AChR to G(q), usually 0.1-0.5, is also within the likely physiological range. We varied the concentration of PLC-beta1 broadly to ask mechanistic questions, and the actual cytoplasmic concentration of PLC-beta near the plasma membrane is unknown. Most important, 100-fold GTP-dependent muscarinic stimulation of PLC activity indicates that the basic signaling pathway has been efficiently reconstituted. Thus, the m1AChR-G(q) vesicles appear to be a valid model system in which to study the mechanism of G protein signaling when an effector is a G protein GAP. Since the system is active and well regulated in the absence of other proteins suspected of involvement in IP(3) signaling (phosphatidylinositol exchange protein (43, 44) or actin- and PIP(2)-binding proteins(45) ), it should also allow the evaluation of how these factors modulate receptor-G(q)-PLC-beta signaling.

Rigorous interpretation of results from this system depends on the assumption that the PLC activity of PLC-beta1 is strictly a reflection of the concentration of GTP-activated G(q) and of the affinity of the two proteins for each other. This assumption appears to be valid. The concentration ranges over which GTPS-bound G(q) activates PLC-beta1 and PLC-beta1 stimulates the GTPase activity of G(q) are identical, with EC values of 1-3 nM(6) . This value, an apparent K(d) for the binding of activated G(q) to PLC-beta1, is consistent with the data of Fig. 5(although we have not been able to test the relationship over a wide range of vesicle concentrations). The similar dependence of GTPase and PLC activities on agonist concentration (Fig. 6) also argues for this assumption. Qualitatively, these findings suggest strongly that the complex of proteins that displays the high GTPase activity is also responsible for the stimulated PLC activity.

In contrast to our previous effort to reconstitute GTP-supported stimulation of PLC-beta1 by m1AChR(15) , this study demonstrates that the combination of receptor and G(q) in phospholipid vesicles that contain PIP(2) allows both efficient stimulation of PLC and the rapid initiation and termination of signaling. The most interesting and informative outcome, however, is how the rate of agonist-stimulated binding of GTP (or GTPS) to G(q), which is relatively slow under usual in vitro assay conditions (Fig. 8)(6, 8, 15) , can balance the rapid hydrolysis of G(q)-bound GTP to account for muscarinic activation of PLC-beta1 in the presence of GTP.

The data of Fig. 8Fig. 9Fig. 10indicate that initiation of receptor-stimulated nucleotide exchange by G(q) is indeed slow, but that a more rapidly exchanging species forms over 2 min. This species, apparently a complex of receptor and G(q), displays a nucleotide exchange rate of geq20-25 min (Fig. 9) that can balance rapid GTP hydrolysis and thereby sustain a small but adequate fraction of G(q) in the active GTP-bound conformation at steady state (Table 1). This mechanism, described by the inner cycle in Fig. 11, contrasts with more widely accepted schemes for the action of G protein-coupled receptors in which receptor is assumed to dissociate from GTP-activated G protein during each cycle. In the more traditional scheme (outer cycle in Fig. 11), association of agonist-bound receptor with G(q) is rate-limiting for the slow activation step ( Fig. 8and Fig. 10A) and is followed by the rapid binding of GTP. This scheme is essentially that proposed for the G(s)-mediated activation of adenylyl cyclase by Levitzki(46) , in which the initial encounter of receptor and G protein in the plasma membrane was suggested to be the actual slow step in the action of agonist. The feature that distinguishes the behavior of m1AChR and G(q) from that described by Levitzki for G(s) is that the complex of receptor and G(q), once formed, is sufficiently stable to remain associated over multiple GTPase cycles (inner cycle in Fig. 11).


Figure 11: Alternative GTPase cycles for stable or unstable association of receptor (R) and G protein (G). Both cycles assume saturating agonist, such that R is always agonist-bound. Activated species are shown with asterisks. In the slow outer cycle, where the effector has no GAP activity and GbulletGTP has a lifetime geq10 s, association of R with GbulletGDP is rate-limiting for regeneration of active GbulletGTP (see (46) ). In the rapid inner cycle, proposed for the agonist-stimulated GTPase reaction catalyzed by G(q) under the influence of a GAP such as PLC-beta1, a complex of R, G, and effector (E) remains intact throughout the cycle. In the rapid cycle, 1) GAP-stimulated hydrolysis is fast under the influence of the GAP activity of E, such that R does not dissociate; 2) GDP dissociation is fast since R is already associated with G; and 3) GTP association is very fast, either diffusion-limited (28, 48) or nearly so(50) , such that E does not dissociate significantly before reactivation of G by GTP. Although steady-state GTPase activity is high, enough GTP-activated G is maintained to mediate signal transduction.



The GAP activity of PLC-beta1 is central to the stability of the receptor-G(q)-PLC complex and its ability to rapidly traverse the inner cycle of Fig. 11. Although m1AChR might be expected to dissociate from the activated G(q)-PLC complex, rapid GAP-stimulated hydrolysis allows the receptor to cause the dissociation of GDP instead. (Receptors displace GDP more efficiently than GTP (47) (confirmed by us for several G proteins(25) .) Since the receptor then maintains the nucleotide-binding site on G(q) in the ``open'' conformation, GTP binding is rapid, and another round of activation is completed before PLC itself would dissociate. We cannot determine whether GTP binding to the carbachol-m1AChR-G(q) complex is actually diffusion-controlled (28, 48) because GDP dissociation rather than GTP binding appears to be rate-limiting (Fig. 9, compare A and B). Regardless, binding is fast.

Two sets of data support this general model. First, although binding of either GTP or GTPS is relatively slow when initiated by addition of agonist (Fig. 8), the rate is 10 times faster after a few minutes of incubation with agonist ( Fig. 9and Fig. 10). A similar but smaller difference was seen in the PLC reaction, which was stimulated only after a substantial delay upon addition of agonist, but which was stimulated at the earliest observable time point when GTP was added after preincubation with agonist. Thus, an initial slow accumulation of a coupled agonist-receptor-G(q) species is followed by the relatively faster binding of GTP, and the complex is then sustained over multiple GTP binding and hydrolytic events. Second, the initial rate of nucleotide binding upon addition of agonist is simply too slow to account for the steady-state turnover of the GTPase reaction. Typical GTPase turnover numbers in this system are usually 10 min in the presence of agonist and PLC-beta1 (20 min has been observed). This is at least 5-fold faster than the observed initial rates of nucleotide binding when the reaction is initiated with agonist. The more rapid nucleotide exchange rate during steady-state hydrolysis is, however, congruent with the binding reactions described in Fig. 9.

We were able to estimate the rate of GAP-stimulated hydrolysis of G(q)-bound GTP from steady-state nucleotide binding data (Table 1) using the rate of receptor-catalyzed GTP binding during steady-state GTP hydrolysis. This calculation is constrained by experimental limitations on the data, but all of the data point to a catalytic rate constant of 100-200 min (1.5-3.0 s), an increase of 150-300-fold in hydrolysis rate over that observed with G(q) alone (0.7 min)(6) . First, this is an enormous increase in k by an allosteric effector, although smaller than the effects of GAPs for small monomeric GTP-binding proteins such as p21(4) . Second, since this rate constant remains 4-12-fold higher than the exchange rate constant, receptor-catalyzed GTP binding remains the slow step in the overall GTPase cycle, as indicated by the dependence on agonist concentration shown in Fig. 6.

Given the scale of the reaction rates and the extent of stimulation of PLC activity observed in the reconstituted system described here, the receptor-G(q)-PLC-beta1 system has the capacity to generate the speed and net activity to account for virtually any described physiological IP(3) release without invoking the need for other modulatory or accessory proteins. The potential function of such proteins is not, of course, ruled out by these data, and this system seems well suited for the study of any such ancillary factors. Because the activation and deactivation rates described here are adequate to account for the fast G protein-mediated responses that have been described electrophysiologically, the search for the G protein GAPs that act in these pathways takes on new interest.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM30355 and Training Grant GM07062 and by Robert A. Welch Foundation Grant I-0982. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Eisai Research Inst., 4 Corporate Dr., Andover, MA 01810-2441.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9041. ROSS{at}UTSW.SWMED.EDU.

(^1)
The abbreviations used are: PLC, phospholipase C; GAP, GTPase-activating protein; m1AChR, m1 muscarinic acetylcholine receptor; IP(3), inositol 1,4,5-trisphosphate; NTA, nitrilotriacetic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PMSF, phenylmethylsulfonyl fluoride; GTPS, guanosine 5`-3-O-(thio)triphosphate; MES, 4-morpholineethanesulfonic acid; PIP(2), phosphatidylinositol 4,5-bisphosphate; BSA, bovine serum albumin; QNB, 1-quinuclidinyl benzilate.

(^2)
G. Berstein, G. H. Biddlecome, J. Wang, and E. M. Ross, unpublished data.

(^4)
For the following simplified reaction,

K= (k + k)/k. Thus, the K for GTP will increase if the G(q) GAP activity of PLC-beta1 increases k above k(49) .


ACKNOWLEDGEMENTS

We thank Karen Chapman for expert technical assistance, Begonia Ho (Medical College of Wisconsin) for construction of the plasmid containing the G(q)alpha cDNA and for preparation of antiserum against G(q), and Tohru Kozasa for the alpha(i)His(6) baculovirus and for sharing purification strategy prior to publication. We also thank Christianne Kleuss for purified Gbeta(1)(2) used in some preliminary experiments, Jorge Iñiguez-Lluhí for Gbeta and G viruses, Susanne Mumby for P-960 antiserum, Alan Smrcka for Z811 antiserum, and Andrew Blatz for the Ca buffering program.


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