MUKHOPADHYAY 1999

7/31/2019 MUKHOPADHYAY 1999

1/6

Proc. Natl. Acad. Sci. USAVol. 96, pp. 95399544, August 1999Biochemistry

Rapid GTP binding and hydrolysis by Gq promoted by receptorand GTPase-activating proteins

SUCHETANA MUKHOPADHYAY AND ELLIOTT M. ROSS*

Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9041

Communicated by Eva J. Neer, Harvard Medical School, Boston, MA, June 23, 1999 (received for review May 11, 1999)

A BSTR ACT Re cept or-pro mote d GTP bi nd ing andGTPase-activating protein (GAP)-promoted GTP hydrolysisdetermine the onset and termination of G protein signaling;they coordinately control signal amplitude. The mechanismswhereby cells independently regulate signal kinetics andsignal amplitude are therefore central to understanding Gprotein function. We have used quench-flow kinetic methodsto measure the rates of the individual reactions of the agonist-stimulated GTPase cycle for Gq during steady-state signaling.Gq and m1 muscarinic cholinergic receptor were co-

reconstituted into proteoliposomes with one of two GAPs:phospholipase C (PLC)-1, the major Gq-regulated effectorprotein, and RGS4, a GAP commonly thought to be aninhibitor of Gq signaling. In this system, the rate constant forGAP-stimulated hydrolysis of Gq-bound GTP at 30C was912 s1 for PLC-1 and 2227 s1 for RGS4. These rates are1,000- to 2,000-fold faster than in the absence of a GAP andfar faster than measured previously. Gq can thus hydrolyzebound GTP w ith deactivation half-times of 2575 ms at 30C,commensurate with physiological rates of signal termination.GDP/GTP exchange, which reactivates Gq, was the principalrate-limiting step for the GTPase cycle and was also fasterthan previously thought. At physiological concentrations ofGTP, exchange was limited by the rate of dissociation of GDPfrom the receptorG

qcomplex, with a maximal rate of 1.8 s1

at 30C. Comparison of activation and deactivation rates helpexplain how GDP/GTP exchange balance rapid GTP hydro-lysis to maintain steady-state signal amplitude.

The initiation, amplitude, and termination of G protein sig-naling are all determined by a tightly controlled cycle ofactivation and deactivation. GTP binding, the activation step,is promoted by G protein-coupled receptors; hydrolysis ofbound GTP, and consequent deactivation is accelerated byGTPase-activating proteins (GAPs). GAPs perform two fun-damentally different functions in G protein signaling. First, aGAP can simply inhibit signaling by decreasing the fraction ofG protein that is in the active state during the GTPase cycle.Several GAPs function physiologically as signaling inhibitors(13), and overexpression of GAPs can inhibit G proteinpathways in many cells [reviewed in (4 6)]. Alternatively,GAPs can accelerate signal termination on removal of agonistwithout substantially inhibiting steady-state signaling. GAPsthereby enhance the temporal acuity of the signaling processwithout attenuating the signal itself (4, 7). For example,expression of two Gi GAPs, RGS4 or RGS8, acceleratedpotassium channel deactivation on agonist removal more than20-fold but caused little if any decrease in net conductanceduring agonist stimulation (811). How GAP-stimulated de-activation is balanced with receptor-promoted activation togive rapid turn-off with minimal inhibition of signal amplitudeis not well understood mechanistically.

G protein GAPs include two functional groups of proteins,effectors and regulator of G protein signaling (RGS) proteins,whose GAP activ ities are commonly thought to fulfill differentpurposes. The first G protein GAP to be identified wasphospholipase C (PLC)-1, which is both the principal Gq-regulated effector and an active, Gq-specific GAP (12). Sim-ilarly, p115 rhoGEF is both a G13 GAP and a G13-regulatedeffector protein (13, 14), and type V adenylyl cyclase hasrecently been reported to have Gs GAP activity (15). Thephysiological function of GAP activity in an effector protein is

not well understood, but it is clearly important in allowingrapid termination of downstream signaling when agonist isremoved. Without such GAP activity, the decay of G proteinsignaling on termination of input from receptor would take upto 10 s at physiological temperatures, far longer than usuallyobserved physiologically and much too slow for many signalingevents (synaptic transmission, response to light, secretion,contraction, etc.) (4, 7, 16).

RGS proteins, which are GAPs for the Gi and Gq families,are not known to act as G protein-regulated effectors and arecommonly assumed to be inhibitors of G protein signaling (5,6). An exception to this assumption is RGS9, the major RGSprotein in mammalian retinal photoreceptor cells, whose prin-cipal physiological function is thought to be the rapid deacti-

vation of Gt after illumination (7, 17). The GAP activity ofRGS9 is potentiated by the subunit of cGMP phosphodies-terase, the Gt effector, and its GAP activity may be moreanalogous to that of the effectors mentioned above (17).

Regardless of their precise physiological functions, thesteady-state inhibitory potential of a GAP must be reconciledwith its role in sharpening the turn-off process when stimula-tion ends. If GAP activity is to be used for rapid signaltermination without squelching the signal, then rapid GTPhydrolysis must be balanced by commensurately fast activationby GTP binding. Intuitively, this balance is necessary for thoseeffectors that are also GAPs. To explain the potential conflictof rapid turn-off and high steady-state signal output, weproposed that fast GTP hydrolysis allows receptors to remainbound to G proteins throughout the GTPase cycle and thereby

catalyze reactivation more efficiently (18). This stable recep-torG proteinGAP complex would turn over GTP rapidly,but the receptor would be able to keep up with the activity ofthe GAP.

To evaluate the interactive effects of receptors and GAPs onG protein signaling, we have used quench-flow mixing to studythe individual steps in the GTPase cycle in reconstitutedphospholipid vesicles that contain Gq and m1 muscariniccholinergic receptor (m1AChR). We found that PLC-1 ac-celerates the hydrolysis of Gq-bound GTP 1,000-fold andthat RGS4 can accelerate hydrolysis2,000-fold. The lifetimeof the GqGTP complex under these conditions is 2575 ms,

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. 1734 solely to indicate this fact.

PNAS is available online at www.pnas.org.

Abbrev iations used: GAP, GTPase activat ing protein; GTPS,

guanosine 5-[-thio]triphosphate; PLC-1, phospholipase C-1;m1AChR, m1 muscarinic cholinergic receptor; RGS, regulator of Gprotein signaling.*To whom reprint requests should be addressed. E-mail ross@

utsw.swmed.edu.

9539


2/6

well within the range of physiological turn-off rates. Toaccommodate such rapid hydrolysis, receptor-catalyzed GDPrelease achieved a rate of 1.8 s1. These data indicate thatheterotrimeric G proteins can hydrolyze GTP rapidly and thatthe hydrolysis rate is matched by receptor-catalyzed GDP/GTPexchange.

EXPERIMENTAL PROCEDURES

Materials. [35 S]g uano si ne 5-[-thio]triphosphate([35S]GTPS), [-32P]GTP, and [-32P]GTP were purchasedfrom NEN. [-32P]GTP was further purified, and [-32P]GDPwas synthesized and purified as described (18). Sources of allother reagents have been described (18, 19). Wild-typem1AChR, Gq, and G12, and hexahistidine-tagged PLC-1were expressed in Sf9 cells and purified as described (18).Hexahistidine-tagged RGS4 was expressed in Escherichia coliand purified as described (20).

m1AChR-Gq Proteoliposomes. m1AChR and Gq were co-reconstituted into unilamellar phospholipid vesicles (phosphati-dylethanolamine/phosphatidylserine/cholesteryl hemisuccinate,165:98:18) as described (18). The concentration of m1AChR in

the vesicles was measured by [

3

H]quinuclidinyl benzilate binding(19). Receptor-coupled Gq was measured as carbachol-stimulated [35S]GTPS binding as described (19). To minimizebackground, receptor-coupled Gq assays were routinely per-formed at 0.4 M [35S]GTPS, which may underestimate recep-tor-coupled Gq by as much as 50%. Thus, values of kcat Vmax/[E] for steady-state GTPase may be overestimated up to2-fold, and the amounts of binding or hydrolysis in pre-steady-state bursts often appear to be just above the total amount ofcoupled Gq.

Steady-State GTPase Assays. In all experiments,m1AChR-Gq vesicles and GAP, when present, were preincu-bated in assay buffer (20 mM Hepes/0.1 M NaCl/2 mMMgCl2/1 mM EGTA/0.1 mg/ml BSA/1 mM DTT) with either1 mM carbachol or 10 M atropine for 3 min at 30C or 15 min

at 10C. The same assay buffer was also used in all presteady-state experiments described below except as noted. Assayswere initiated by addition of [-32P]GTP at concentrationsshown in the figure legends. Assay times were adjusted toensure linear reactions according to the GTPase activities andthe concentration of substrate. Reactions were quenched witha slurry of cold 5% Norit in H3PO4 (pH 3.0), and [32P]Pi wasmeasured in the supernatant as described (18).

Determination of Intermediary Reaction Rates DuringSteady-State GTP Hydrolysis. To measure the kinetics of theindividual reactions that make up the GTPase catalytic cycle(Scheme I), the complete reaction mixture (m1AChR-Gq

vesicles, GAP, agonist, GT P) was first allowed to reach steadystate. Radiolabeled nucleotide was then added, and the reac-tion was terminated at different stages to monitor hydrolysis ofGq-bound GTP (khydrol), GDP dissociation (kdiss), or GDP/GTP exchange (kexch). [We define kexch as the observedfirst-order rate constant for the binding of GTP (or GTPS)to Gq. It is a combined measure of GDP release and GTPbinding.] Because most partial reactions are relatively rapid,assays were usually performed by using a Bio-Logic SFM4/Qfour-syringe, quench-flow mixer in which Kel-F syringes, mix-ing chambers, and delay lines are all under thermostat control.Syringe movement is independently computer-controlled byusing stepper motors to drive the plungers. In typical experi-ments, syringe 1 contained assay buffer that was used to drivereagents through the system as they were mixed. Reactionmixtures were expelled into a final cold quenching solution at

0C as the final step in each experiment. The external quenchwas complete in 17 ms as determined by time-lapse videorecording of the mixing of neutral-pH mock assay volumes thatwere ejected into acidic solutions of pH-sensitive dyes (datanot shown). Quench-flow experimental protocols were de-signed to minimize the required amount of enzyme, back-ground reactions, and cross-contamination among syringesand to maximize recovery of reactants, signal/background

ratio, and final radioisotope signal. We describe all quench-flow experiments in terms of the volume of final reactionmixture that was collected and analyzed for product (boundnucleotide or [32P]Pi). We ignore excesses needed to provideleading and trailing volumes of reactants whose concentrationswere critical or reagents used to f lush mixers and the delaylines before reactions. Detailed mixing protocols are availablefrom the authors. Control experiments indicated that deadtimes were significantly shorter than any incubation time andthat negligible protein denaturation occurred during incuba-tion of vesicles in the syringe and/or during high-speed flowthrough the mixing apparatus (data not shown). Some slowerreactions were measured manually, as noted in the text.

Hydrolysis of Gq-Bound GTP. To measure the rate of

hydrolysis of Gq-bound GTP during steady-state turnover,m1AChR-Gq vesicles, GAP, and agonist were first mixed toallow association of receptor and Gq (18). Vesicles and either44 nM PLC or 9 M RGS4 were incubated with 1 mMcarbachol in a syringe of the quench-flow mixer for 15 minat 10C or 3 min at 30C. An aliquot (20 l) was diluted 1:2with assay buffer that contained carbachol and [-32P]GTP(300700 nM), and the mixture was incubated for 1 min (10C)or 6 s (30C) to initiate steady-state hydrolysis and allowaccumulation of Gq[-32P]GTP without excessive productionof background [32P]Pi. At this point, defined as t 0, further[-32P]GTP binding was quenched by 1:1.5 dilution with 100M nonradioactive GTP and 100 M atropine in assay buffer(to inhibit dissociation of bound [-32P]GTP). This reactionmixture was incubated for the times shown and then quenched

by mixing with 1.8 vol of 5% Norit charcoal in 50 mM H 3PO4(pH 3.0) at 0C. 32P-Labeled orthophosphate was monitored asdescribed (19). Zero-time background radioactivity was sub-tracted from all data. For this and other assays of reactiontransients, data were fitted to single or double exponentialequations (SIGMAPLOT, SPSS, Chicago).

Receptor-Stimulated GDP Dissociation. [-32P]GDP wasfirst bound to reconstituted Gq by incubating m1AChR-Gqvesicles, 1 mM carbachol, and 300 nM [-32P]GDP (70100cpm/fmol) in a syringe of the quench-flow mixer for at least 15min at 10C or 5 min at 30C. Aliquots of this mixture (30 l)were diluted 1:2 with agonist and 0.3500 M unlabeled GTP

at t 0, and the dissociation reaction was allowed to proceedfor various times. The reaction was quenched in cold bufferthat contained detergent and atropine (18), and the amount ofGq-bound [-32P]GDP was measured by nitrocellulose filterbinding (21). Identical results were obtained when [-32P]GDPwas bound to Gq in the presence of a GAP.

Guanine Nucleotide Exchange. Rates of receptor-catalyzednucleotide exchange on Gq were measured by first equilibrat-ing vesicles and agonist and then initiating exchange by theaddition of radiolabeled nucleotide. m1AChR-Gq vesicles (30l) were first incubated in assay buffer plus 1 mM carbachol,either alone or with 44 nM PLC-1 or 7 M RGS4. Exchangereactions were initiated by 1:2 dilution of vesicles with[-32P]GTP, [-32P]GDP, or [35S]GTPS plus carbachol. Finalconcentrations of nucleotides are indicated in the legends.Reactions were quenched by adding 100 l of cold stop buffer

9540 Biochemistry: Mukhopadhyay and Ross Proc. Natl. Acad. Sci. USA 96 (1999)


3/6

(GTP, atropine, detergent; ref. 18), and bound nucleotide wasmeasured by nitrocellulose filter binding (21).

RESULTS

Steady-State Gq GAP Activities of PLC-1 and RGS4. BothPLC-1 and RGS4 stimulated the steady-state GTPase activityof m1AChR-Gq vesicles from the basal rate of 0.81.0 mol ofGT Pmin1mol1 of Gq to ov er 40 mol of GT Phydrolyzedmin1mol1 of Gq (Fig. 1). PLC-1 typically in-creased activity 20-fold, although 60-fold stimulations havebeen observed (18) (confirmed in this study). RGS4 usuallystimulated GTPase activity to a maximum 1.5- to 2.5-foldgreater than that of PLC-1, although its EC50, 200300 nM,is well above the 2 nM EC50 of PLC-1. Different preparationsof vesicles with m1AChR/Gq ratios of 0.10.3 yielded consis-tent EC50 values for both GAPs, but maximum turnovernumbers increased up to 2-fold with increasing receptor/G q

ratios. Both PLC-1 and RGS4 increased the Km of thereceptor-Gq vesicles from about 100 nM GTP to 13 M GTP(Fig. 2). The elevated Km accompanied by the large increasein Vmax suggests that both GAPs accelerate steady-state hy-drolysis primarily or exclusively by increasing the rate ofhydrolysis of Gq-bound GTP. None of the parameters shownin Fig. 1 except for Vmax were substantially different at 10C or30C.

PLC- and RGS4 Accelerate Hydrolysis of Gq-Bound GTP1,000- to 2,000-Fold. To measure the effect of GAPs on therate of hydrolysis of Gq-bound GTP during the steady-stateGTPase cycle (khydrol), we allowed agonist-liganded receptorand Gq to associate in the vesicles (18), added [-32P]GTP fora brief period to allow it to bind to Gq, and then quenchedfurther binding with excess unlabeled GTP. We then moni-tored release of [32P]Pi from the preformed pool of Gq[-32P]GTP (Fig. 3). A substantial fast and monoexponentialburst of [32P]Pi release occurred in the presence of eitherPLC-1 or RGS4. At 30C, values ofkhydrol in the burst phasewere 9 12 s1 for PLC-1 and 2227 s1 for RGS4 (12 s1 and27 s1 in Fig. 3). This maximum rate is 2,000-fold faster thanthe basal value ofkhydrol previously measured for Gq, 0.013 s1

(12), confirmed in this study, and corresponds to a t1/2 of 2575ms for the deactivation of Gq by GTP hydrolysis. The relativedifference in khydrol between PLC-1 and RGS4 is about equalto the difference in their abilities to stimulate steady-statehydrolysis (Fig. 2). At 10C, hydrolysis of Gq-GTP wassubstantially slower. PLC-1 and RGS4 increased khydrol from0.0019 s1 to 0.80.9 s1 and 1.21.6 s1, respectively. We havenot tried to measure the hydrolysis of GqGTP at 37C, but weestimate from the data described above that the rate ofGAP-stimulated hydrolysis would be about 25 and 65 s1 forPLC-1 and RGS4, yielding deactivation half-times of about30 ms and 10 ms, respectively. These are well within thedeactivation lifetimes reported for G protein signaling path-ways in vivo, suggesting that GAP-stimulated GTP hydrolysiscan fully account for the termination of signaling on removalof receptor agonist.

The burst of Gq[-32P]GTP hydrolysis was only observed invesicles where the m1AChR/Gq molar ratio was at least 0.3.The amount of [32P]Pi released in the burst was reproduciblefor each batch of vesicles. The magnitude of the burstincreasedwith the concentration of [-32P]GTP in the binding reactionin a pattern consistent with the Km for steady-state GTPase

FIG. 1. GAP activity of PLC-1 and RGS4 during steady-stateGTP hydrolysis. The carbachol-stimulated GTPase activity of proteo-liposomes that contained m1AChR and Gq was assayed at 30C in thepresence of increasing c oncentrations of PLC-1 (F) or RGS4 ().Vesicles were preincubated with 1 mM carbachol and either GAP for

3 min before addition of 10 M [-32P]GTP to initiate the reaction.Data shown are representative of several complete titration curves(n 2 for PLC-1, n 3 for RGS4) and multiple other experiments.

FIG. 2. Dependence of agonist-stimulated steady-state GTPase activity on GTP concentration. The carbachol-stimulated GTPase activity ofm1AChR-Gq vesicles was assayed with either 15 nM PLC-1 or 4 M RGS4 in the presence of increasing concentrations of GTP. (Left) 30C,PLC-1 (F) or RGS4 (). (Center) 10C, PLC-1 (E) or RGS4 (). (Right) No GAP, 30C (s) or 10C (). Data are means from two separateexperiments.

Biochemistry: Mukhopadhyay and Ross Proc. Natl. Acad. Sci. USA 96 (1999) 9541


4/6

(Fig. 2), although we were unable to demonstrate saturationbecause of assay background at high [-32P]GTP concentra-tions. At 30C, the burst was followed by a second slower phaseof GTP hydrolysis with an apparent rate constant of0.35 s1

(Fig. 3 Insets). This is about equivalent to rates observed invesicles that contained too low a m1AChR/Gq ratio to display

a measurable burst. We interpret this slower second phase torepresent a combination of receptor-promoted binding of GTPto Gq and subsequent hydrolysis. We have not pursued itfurther.

GDP Dissociation. Because hydrolysis of Gq-bound GTPwas found to be so fast, we reexamined the activating steps ofthe GTPase cycle,GDP release, and GTP binding. To measurethe dissociation of GDP from Gq, we allowed [-32P]GDP tobind Gq in a syringe of the quench-flow mixer by incubatingm1AChR vesicles either with agonist and [-32P]GDP itself orwith agonist, GAP, and [-32P]GTP. We then diluted themixture into excess unlabeled GTP and, after appropriatetimes, quenched the mixture and measured remaining bound[-32P]GDP (Fig. 4). The average GDP dissociation rate

constant kdiss was 1.5 s1 at 30C (1.8 s1 maximum), about4-fold faster than estimated previously by using manual mixing(18). This value was consistently somewhat higher than kcat(Vmax/[E]) for the steady-state GTPase reaction (Figs. 1 and 2),

suggesting that receptor-promoted dissociation of GDP is theprincipal rate-limiting step in the GTPase cycle when GAP,agonist, and GTP are all present at saturating concentrations.The value of kdiss did not vary whether [-32P]GDP itself wasbound to Gq in the absence of a GAP or it was generated onGq in the presence of either PLC-1 or RGS4 (data not

shown). At 10C, kdiss was 0.14 s1, also in agreement with kcatat that temperature (Figs. 1 and 2). The amount of [-32P]GDPreleased was appropriate for the amount of Gq in the reactionmixture and its fractional saturation. Dissociation wasmonophasic at 10C, but a small, very slow second phase couldbe detectedat 30C. Neither the rate nor magnitude of the slowphase could be measured accurately. GDP dissociation wasindependent of the concentration of free GTP present duringthe dissociation reaction (data not shown).

Receptor-Catalyzed GDP/GTP Exchange. Because recep-tor-promoted dissociation of GDP from Gq appeared to berate-limiting at high GTP concentrations and was muchsmaller than khydrol, we measured the rate of GDP/GTPexchange, a combination of GDP release and GTP binding, to

test the role of GTP binding to nucleotide-free Gq in theGTPase cycle. Vesicles, with or without a GAP, were incu-bated with agonist and then mixed with radiolabeled nucleo-tide, either [-32P]GTP or [35S]GTPS. Exchange reactions

FIG. 3. GAP-stimulated hydrolysis of Gq-bound GTP. m1AChR-Gq vesicles and either 44 nM PLC or 9 M RGS4 were incubated with 1 mMcarbachol for at least 15 min at 10C or 3 min at 30C. Aliquots were then diluted 1:2 with carbachol and [-32P]GTP (300700 nM) and allowedto incubate for 1 min at 10C or 6 s at 30C. At t 0, this mixture was diluted 1:1.5 further with buffer that contained unlabeled GTP and atropineand incubated for the times shown. Solid lines are fits to first-order rate equations to yield the following rate constants (khydrol): PLC-1, 30C,11.9 s1; PLC-1, 10C, 0.84 s1; RGS4, 30C, 27 s1; RGS4, 10C, 1.2 s1. Data obtained at 30C are fit to two components, that listed aboveand a slower one visible in the insets (see Results). Vesicles contained the following amounts of receptor and Gq, in the above order: 68 fmol and187 fmol; 106 fmol and 260 fmol; 106 fmol and 260 fmol; and 150 fmol and 432 fmol. Each data set is representative of at least two experimentsusing different m1AChR-Gq vesicle preparations.



5/6

were quenched at appropriate times, and the accumulation ofbound nucleotide was fit to first-order rate equations to yieldthe rate constant, kexch (Fig. 5 Upper). Binding of [-32P]GTPand [35S]GTPS yielded similar exchange rates over a rela-tively wide concentration range, and these rates were unalteredby the presence of either PLC-1 or RGS4 (Fig. 5 Lower).

Thus, neither GAP acts as an exchange catalyst regardless ofwhether the nucleotide bound can be hydrolyzed.

GDP/GTP exchange was relatively slow over the range ofGTP concentrations studied, withkexch significantly belowkdiss.Although we were not able to demonstrate saturation of kexch,its value depended on the concentration of free nucleotide ina pattern similar to the rate of the overall GTPase cycle, with

kexch approximately equal to the molar turnover number overthe accessible range of GTP concentrations (compare Figs. 5

Lowerand 2). At saturating GTP (Fig. 2), kcat kdiss for GDP.The rate of GTP binding to Gq, itself limited by GDPdissociation, thus appears to be rate-limiting for the GTPasereaction at all concentrations of GTP.

Because kdiss is not altered by the concentration of free

nucleotide, the observed dependence of kexch on the GTPconcentration reflects rate-limiting GTP binding at theseconcentrations (Fig. 5). This idea is corroborated by theobservation that allowing Gq to bind either IDP or XDP beforethe assay did not alter kexch (data not shown), even thoughthese nucleotides bind Gq with much lower affinity than doesGDP and also dissociate much faster (22). We can thereforeuse the dependence of kexch on GTP concentration to deter-mine the rate constant, kassoc, for GTP binding to nucleotide-free Gq from kexch to be 1 105 M1s1 at 30C. [A plot of

kexch vs. the concentration of GTP is a line with slope 1 105

s1M1. Because kdiss is much greater than the measuredvalues ofkexch over this range of GTP concentrations, this slopeapproximates kassoc, GTP.] Based on this value, increasing the

concentration of GTP should increase kexch until it surpasseskdiss for GDP, 1.5 s1, at about 15 M GTP. Thus, GTPaseactivity reaches saturation at high GTP concentrations whenGDP dissociation becomes rate-limiting.

DISCUSSION

G protein GAPs allow rapid termination of a signal on removalof agonist, but can also substantially inhibit signaling in thepresence of agonist by shortening the activation lifetime of theG protein during the GTPase cycle. The data presented heredescribe how the individual steps in the GTPase cycle combineto produce both robust activation by agonist and rapid deac-tivation on agonist removal. Such balance is particularly im-portant for G protein-regulated effectors, such as PLC-1, thatuse intrinsic GAP activity to modulate the kinetics of their ownactivation.

The most striking outcome of this study was the speed ofGAP-stimulated hydrolysis of Gq-bound GTP, with an average

khydrol of 25 s1 for RGS4 and 15 s1 for the effector PLC-1at 30C. This represents a 2,000-fold increase over the basal

FIG. 4. Receptor-stimulated GDP dissociation. m1AChR-Gq ves-icles were incubated with 300 nM [-32P]GDP and 1 mM carbachol forat least 3 min at 30C. Aliquots (10 fmol m1AChR, 26 fmol Gq) were

then diluted 1:2 with 500 M nonradioactive GTP and 1 mMcarbachol. Mixtures were quenched at the times shown. Bound[-32P]GDP was determined by nitrocellulose filter binding. The solidline represents a fit to a bi-exponential function with a principaldissociation rate constant kdiss of 1.84 s1 (55% of total) and a slowercomponent (0.35 s1). Data are from one of five different experimentsthat yielded similar fast (1.6 s1 mean) and slow (0.21 s1 mean)dissociation rates.

FIG. 5. Receptor-stimulated guanine nucleotide exchange. (Upper)Time course of binding of GTPS to m1AChR-Gq vesicles.m1AChR-Gq proteoliposomes and 1 mM carbachol were incubated at30C for at least 3 min. Aliquots (12 fmol m1AChR, 47 fmol Gq) werediluted 1:2 with 500 nM [35S]GTPS for the times shown beforequenching and measurement of bound [35S]GTPS. The solid lineshows a fit to a first-order rate equation with kexch 0.135 s1. (Lower)Dependence of exchange rates on the concentration of free nucleotide.Nucleotide exchange was measured as described above in the presenceof increasing concentrations of radiolabeled nucleotide at either 30C(solid symbols) or 10C (open symbols). Values of kexch are plotted vs.the concentration of free nucleotide. At 10C, exchange was relativelyslow and was measured manually by using a protocol identical to thatdescribed for the quench-flow mixer except that the preincubationtime was at least 15 min. Labeled nucleotides were [35S]GTPS in the

presence of either 15 nM PLC-1 (F,E) or 4 M RGS4 () or withoutGAP (s, ), or [-32P]GTP with PLC-1 present () or absent ().

Biochemistry: Mukhopadhyay and Ross Proc. Natl. Acad. Sci. USA 96 (1999) 9543


6/6

rate of 0.013 s1 (12), comparable to the 10,000-fold effect ofras GAP on p21ras (23). Moreover, G protein GAPs acceler-ate GTP hydrolysis by an allosteric mechanism, whereas GAPsfor small monomeric GTP-binding proteins provide a catalyticarginine residue to the active site in addition to any confor-mational influence (24).

The GTP hydrolysis rates determined here agree well withcellular rates of signal termination on removal of agonist.

PLC-1-stimulated hydrolysis occurred with a half-time of5075 ms at 30C, commensurate with that for closure ofG-regulated K channels (4, 8) or for termination of theGq-mediated photoresponse in Drosophila (25). Although wedid not measure khydrol at 37C, extrapolation from the valuesat 10C and 30C suggest that the PLC-1-stimulated turn-offrate at 37C will be about 25 s1, a deactivation half-time ofabout 25 ms, and thus accounts well for physiological turn-offrates without recourse to other desensitizing mechanisms.

Both the maximal rate of GAP-stimulated hydrolysis ofGq-bound GTP observed here, 27 s1, and the relative stim-ulation above the basal rate are notably greater than thosedescribed previously for heterotrimeric G protein GAPs, eventhough basal khydrol for Gq is only about 25% that for Gi, Go,

or Gs (12). For comparison, members of the Gz GAP subfamilyof RGS proteins (RGSZ1, GAIP, RET-RGS1) elevated khydrolabout 400-fold for Gz in solution (26). Arshavsky et al. (27)reported that RGS4-stimulated Gt hydrolyzed GTP at a rate of2.8 s1 at 22C, and Posner (28) reported a rate of 2.0 s1 forsoluble RGS4-stimulated Go at 8C. The vesicle system usedhere may contribute to the high GAP activities that weobserved. Effects of both PLC-1 and RGS4 on Gq were muchlarger here than in a study of detergent-solubilized RGS4 anda mutant Gq (29), and the GAP activities of RGS2 andseveral RGS4 mutants are greater in vesicles than in detergentsolution (28, 30).

The rates determined here for receptor-promoted GDPdissociation and overall GDP/GTP exchange, kdiss and kexch,are also significantly faster than previously determined for

purified receptors and G proteins. More importantly, kexch isadequate to activate Gq substantially despite rapid GTPhydrolysis. Based on the values of khydrol and kexch, m1AChRcan maintain 15% of total Gq in the activated state in thepresence of saturating agonist and PLC-1.[The fraction ofGqactivated at steady-state is equal to kexch/(kexch khydrol)].Presumably, a cell can do somewhat better, but even cellularsignaling systems are typically much more active in the pres-ence of nonhydrolyzable GTP analogs than with GTP itself.Although it is not feasible to measure these individual reac-tions in intact cells or native cell membranes, the valuesreported here should approximate physiological parametersreasonably well.

Two considerations indicate that the GDP/GTP exchange

rate is the principal rate-limiting step in the GTPase cycle inthe presence of both agonist and GAP. First, the steady-stateturnover number is only slightly greater than kexch over therange of GTP concentrations where it could be measured. Inaddition, the maximum steady-state rate (kcat) saturates at avalue approximately equal to the experimentally determinedvalue of kdiss, which is necessarily the upper limit of kexch.Although we could not directly measure the second-order rateconstant for GTP binding to the complex of receptor andunliganded Gq, its calculated value is 1 105 s1M1. Atcellular concentrations of GTP (200500 M) (31), the rate ofbinding of GTP to unliganded Gq would substantially exceedthe rate of GDP dissociation, and dissociation from thereceptorG protein complex will be the essentially rate-limiting step for GDP/GTP exchange. Thus, the rate ofactivation of Gq and its level of steady-state activation will bedetermined by how quickly the receptor drives GDP dissoci-

ation. We propose that the t wo principal determinants ofreceptors efficacy will be the value of kdiss that it can achieveand the stability of its binding to G protein during multipleturnovers of the GTPase cycle.

We thank Karen Chapman for expert and careful technical assis-tance throughout this study and Harold Brooks, Gloria Biddlecome,and Yves Dupont for discussion and suggestions. This work was

supported by National Institutes of Health Grants GM30355 andCA66187 and by R. A. Welch Foundation Grant I-0982.

1. Dohlman, H. G., Song, J. P., Ma, D. R., Courchesne, W. E. &Thorner, J. (1996) Mol. Cell. Biol. 16, 51945209.

2. Koelle, M. R. & Horvitz, H. R. (1996) Cell 84, 115125.3. Wieser, J., Yu, J.-H. & Adams, T. H. (1997) Curr. Genet. 32,

218224.4. Zerangue, N. & Jan, L. Y. (1998) Curr. Biol. 8, 313316.5. Dohlman, H. G. & Thorner, J. (1997) J. Biol. Chem. 272,

38713874.6. Berman, D. M. & Gilman, A. G. (1998) J. Biol. Chem. 273,

12691272.7. Arshavsky, V. Y. & Pugh, E. N., Jr. (1998) Neuron 20, 1114.8. Doupnik, C. A., Davidson, N., Lester, H. A. & Kofuji, P. (1997)

Proc. Natl. Acad. Sci. USA 94, 1046110466.

9. Saitoh, O., Kubo, Y., Miyatani, Y., Asano, T. & Nakata, H.(1997) Nature (London) 390, 525529.10. Chuang, H.-H., Yu, M., Jan, Y. N. & Jan, L. Y. (1998) Proc. Natl.

Acad. Sci. USA 95, 1172711732.11. Bunemann, M. & Hosey, M. M. (1998) J. Biol. Chem. 273,

3118631190.12. Berstein, G., Blank, J. L., Jhon, D.-Y., Exton, J. H., Rhee, S. G.

& Ross, E. M. (1992) Cell 70, 411418.13. Hart, M. J., Jiang, X., Kozasa, T., Roscoe, W., Singer, W. D.,

Gilman, A. G., Sternweis, P. C. & Bollag, G. (1998) Science 280,21122114.

14. Kozasa, T., Jiang, X., Hart, M. J., Sternweis, P. M., Singer, W. D.,Gilman, A. G., Bollag, G. & Sternweis, P. C. (1998) Science 280,21092111.

15. Scholich, K., Mullenix, J. B., Wittpoth, C., Poppleton, H. M.,Pierre, S. C., Lindorfer, M. A., Garrison, J. C. & Patel, T. B.

(1999) Science 283, 13281331.16. Breitwieser, G. E. & Szabo, G. (1988) J. Gen. Physiol. 91,469493.

17. He, W., Cowan, C. W. & Wensel, T. G. (1998) Neuron 20, 95102.18. Biddlecome, G. H., Berstein, G. & Ross, E. M. (1996) J. Biol.

Chem. 271, 79998007.19. Berstein, G., Blank, J. L., Smrcka, A. V., Higashijima, T.,

Sternweis, P. C., Exton, J. H. & Ross, E. M. (1992) J. Biol. Chem.267, 80818088.

20. Berman, D. M., Wilkie, T. M. & Gilman, A. G. (1996) Cell 86,445452.

21. Brandt, D. R. & Ross, E. M. (1986) J. Biol. Chem. 261, 16561664.

22. Chidiac, P., Markin, V. S. & Ross, E. M. (1999) Biochem.Pharmacol. 58, 3948.

23. Gideon, P., John, J., Frech, M., Lautwein, A., Clark, R., Schef-

fler, J. E. & Wittinghofer, A. (1992) Mol. Cell. Biol. 12, 20502056.24. Scheffzek, K., Ahmadian, M. R., Kabsch, W., Wiesmuller, L.,

Lautwein, A., Schmitz, F. & Wittinghofer, A. (1998) Science 277,333338.

25. Ranganathan, R., Harris, G. L., Stevens, C. F. & Zuker, C. S.(1991) Nature (London) 354, 230232.

26. Wang, J., Ducret, A., Tu, Y., Kozasa, T., Aebersold, R. & Ross,E. M. (1998) J. Biol. Chem. 273, 2601426025.

27. Nekrasova, E. R., Berman, D. M., Rustandi, R. R., Hamm, H. E.,Gilman, A. G. & Arshavsky, V. Y. (1997) Biochemistry 36,76387643.

28. Posner, B. A., Mukhopadhyay, S., Tesmer, J. J., Gilman, A. G. &Ross, E. M. (1999) Biochemistry 38, 77737779.

29. Chidiac, P. & Ross,E. M. (1999)J. Biol. Chem. 274, 1963919643.30. Ingi, T., Krumins, A. M., Chidiac, P., Brothers, G. M., Chung, S.,

Snow, B. E., Barnes, C. A., Lanahan, A. A., Siderovski, D. P.,Ross, E. M., et al. (1998) J. Neurosci. 18, 71787188.31. Traut, T. W. (1994) Mol. Cell. Biochem. 140, 122.


MUKHOPADHYAY 1999

Documents

Transcript of MUKHOPADHYAY 1999