Biochimica et Biophyswa Acta, 1092 (1991) 391-396 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03..50 ADONIS 016748899100171R

391

BBAMCR 12907

Characterization of high affinity GTPase activity correlated to fl-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes Y u k i h a r u H i r a m a t s u , I n d u S. A m b u d k a r a n d Bruce J. B a u m Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD (U.S.A.)

(Received 28 September 1990)

Key words: Parotid: GTPase: fl-Adrenoreceptor: Adenylyl cyclase: G protein: Signal transduction: Cholera toxh'

O-Adrenergic receptor stimulation of adenylyl cyelase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The ~-adrenergic agonist isoproterenol (10 laM) increased the Vma~of the high affinity GTPase component approx. 50% from 90 to 140 p m o l / m g protein per min, but did not change its K m value ( --- 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5'-(3-O-thio)triphosphate (GTP~S), isoproterenol increased cAMP formation to the same extent as that observed with AIF4-. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins ( = 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the 'turn-off' step for the adenylyi cyclase activation seen following O-adrenergie stimulation of rat parotid glands.

Introduction In the rat parotid gland, fl-adrenergic receptor stimulation leads to a marked increase in intracellular cAMP levels, cAMP is considered an important intracelhdar messenger for the function of this exocrine cell [1] and elevated cAMP has been causally associated with secretion [2,3]. Based on studies with many cell types, it appears that activation of adenylyl eyclase by/~-adrenergic receptor stimulation is mediated via a specific guanine nucleotide binding regulatory protein (G-protein, termed Gs), which can bind GTP and display high affinity GTPase activity [4-6]. Although the exact mechanisms involved in such hormone-induced stimulation of adenylyl cyclase are not yet fully established, Abbreviations: Gs, stimulatory GTP-binding regulatory protein (Gprotein). Correspondence: B.J. Baum, CIPCB, NIDR, NIH. Building 10, Room 1N-I13, Bethesda, MD 20892, U.S.A.

data obtained from reconstitution experiments, and with mutant cell lines, indicate that occupation of the receptor by a stimulatory hormone promotes the exchange of GDP by GTP at the Gs regulatory site and subsequent G-protein subunit dissociation (Gsa-GTP, ~ , ) [7,8]. The activated Gsa subunit, in its GTP-bound form, can then activate adenylyl cyclase. According to the regulatory GTPase cycle first proposed by Cassel and Selinger, this agonist-induced activation is turned off by the hydrolysis of the bound GTP to GDP and P~ by a high affinity GTPase activity integral to the Gsa subunit [9,10]. In the present report, we have characterized GTPase activities in rat parotid gland membranes and studied the effects of the fl-adrenergic agonist isoproterenol, and cholera toxin, on adenylyl cyclase and the GTPase activities in these membranes. Both cAMP production and high affinity GTPase activity are stimulated by isoproterenol. Conversely, cholera toxin treatment results in attenuation of the high affinity GTPase, while augmenting GTP dependent cAMP formation. The aggregate data demonstrate that the high affinity

392 GTPase component studied here is associated with the inactivation of the Gs mediated stimulation of adenylyl cyclase following fl-adrenergic receptor activation of rat parotid glands. Materials and Methods

reaction was terminated by the addition of 9 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.4). For experiments analyzed by gel electrophoresis, ADP-ribosylation was carried out similarly except that the incubation medium contained 0.2 mM GTP to decrease nonspecific labelling and that to terminate the reaction, an equivalent volume of sample buffer was added.

Materials (-)-Isoproterenol bitartrate, sodium fluoride, aluminium chloride, GTP, GTPTS, 5'-adenylylimidodiphosphate (AppNHp), ATP, creatine phosphokinase, and phosphocreatine were obtained from Sigma. Cholera toxin was from List Biologicals. [32P]NAD (30 C i / retool) was obtained from New England Nuclear Research, while [),-~-'P]GTP (30 Ci/mmol) was from Amersham. A cyclic AMP assay kit and ACS scintillation cocktail were also from Amersham. All other reagents used were of the highest grade commercially available.

Analytical gel electrophoresis and autoradiography SDS-PAGE was performed according to Laemmli [13] using 16% acrylamide separation gels containing 0.1~ SDS. Stacking gel contained 5.5% acrylamide. Electrophoresis was performed overnight at 3-4 V / c m in electrophoresis buffer containing 25 mM Tris-HCl (pH 8.3), 192 mM glycine, 0.1~ SDS. After electrophoresis, the proteins in the gel were transferred on to nitrocellulose sheets by the method of Towbin et al. [14] using the following buffer~ 25 mM Tris-HCl (pH 8.3), 192 mM glycine, 20% methanol. After drying, the nitrocellulose sheets were exposed to Kodak X-ray films.

Preparation of plasma membranesfrom rat parotid glands Male Wistar rats, fed ad iibitum, were killed by diethyi ether anesthesia and subsequent cardiac puncture. Glands from two rats were dissected free of fat, connective tissues and lymph nodes. All subsequent steps were performed at 4°C. Glands were minced and homogenized twice, each for 10 s, in 12 ml of 50 mM Tris-HC! buffer (pH 7.4) with a Polytron PT-10 homogenizer (Brinkmann Instruments) at setting 5. The homogenate was centrifuged for 10 rain at 3000 × g. The supernatant was filtered through four layers of cheese cloth and then re-centrifuged for 10 min at 40000 × g. The resulting supernatant was generally discarded except for cholera toxin substrate localization studies for which this supernatant was centrifuged at 200000 × g for 60 rain. The 40000 x g pellet was re-suspended in 20 ml of Tris-HCI buffer, re-homogenized once for 10 s, and re-centrifuged as above. After discarding this supernatant, the pellet was re-homogenized once more for 10 s in 10 ml Tris-HCI buffer and used as the membrane source for assays of GTPase and adenylyl cyclase activities.

ADP-ribosvlation of membranes by cholera toxin Cholera toxin catalyzed ADP-ribosylation was carried out using a modification of methods previously described [11,12]. Cholera toxin was preactivated in 20 mM Tris-HCI (pH 7.5), 2 mM EDTA, 20 mM dithio. threitol and 1 mM ATP for 30 rain at 30 ° C. ADP-ribosylation was initiated by the addition of membranes (0.75 mg/ml) in 100 mM Tris-HCl (pH 8.0), 10 mM thymidine, 2 mM ATP, 1 mM EDTA, and 2.5 /~M [3a P]NAD (or NAD for non-radiolabeUing experiments), unless otherwise described. The reaction mixture was incubated for 30 rain at 30°C in the presence or absence of preactivated cholera toxin (0.2 mg/ml). The

Measurement of GTPase activity GTPase activity was measured by monitoring the release of [32P]Pi from [~,-32p]GTP according to the methods described originally by Cassel and Selinger [9,15]. In brief, membrane protein (1.5-3 #g) was added to 100 pl of GTPase assay medium containing 20 mM Tris-HCl (pH 7.8), 10 mM MgCl 2, 1 mM EDTA, 1 mM ATP, 1 mM AppNHp, 1 mM dithiothreitol, 5 mM creatine phosphate, 70 units/ml creatine phosphokinase and 50-100 nM h,-32p]GTP. For kinetic experiments, an appropriate amount of unlabelled GTP was included to yield the indicated concentrations. Assays were performed at 30 ° C for 5 rain in the absence or presence of various agents, unless otherwise described. Reactions were terminated by the addition of 1 ml of ice-cold phosphate buffered saline (pH 7.2) containing 5~ (w/v) activated charcoal. The samples were kept on ice for at least 10 min and then centrifuged for 10 min at 4000 x g. 400/~l of the supernatant were transferred into scintillation vials containing 10 ml of acidified ACS and radioactivity was determined by liquid scintillation spectrometry. The hydrolysis of [-t-a2p]GTP increased linearly with the amount of added membrane 9rotein for at least 5 rain and the spontaneous relea~.- in the absence of membrane was about 5% of added [y32p]GTP. Calculation of the high affinity GTPase component was based on earlier reports which showed that measured GTPase activity (total) consists of a high affinity component, which is saturable with increasing [GTP], and a low affinity component, which increases linearly with increasing [GTP] [15,16]. To obtain the high affinity GTPase at 100 nM GTP (the concentration used in most experiments), the low affinity GTPase activity was estimated by extrapolation from the GTP hydrolysis

393 measured at 100/zM GTP (based on a linear increase in GTP hydrolysis, see Results). This value was then subtracted from the total GTP hydrolysis measured at 100 nM GTP. High affinity GTPase activity was analyzed by a non-linear least-square regression of the values (total hydrolysis) at various GTP concentrations and kinetic constants determined using the MichaelisMenten equation.

Measurement of adenylyl cyclase activity Adenylyl cyclase activity was determined under conditions similar to those described above for measurement of GTPase activity with the exception that the AppNHp was omitted and incubations were for 30 rain at 37°C. Reactions were terminated with the addition of the same volume of ice-cold 8 mM EDTA as that of reaction mixture. The reaction mixtures were centrifuged at 4000 x g for 10 rain and the cAMP present in the resulting supernatants was measured by using a commercial cyclic AMP assay kit. Results

Demonstration of Gs in parotid membranes To study adenylyl cyclase regulation and Gs associated GTPase activity, we obtained a parotid membrane

1

97.4 kDa

2

3

4

5

6

7

TABLE ! Cyclic A M P accumulation in rat parotid membranes

Assays were performed at 37°C for 30 min as described in the text. Results are the mean _+S.E. of three separate experiments. Addition

Cyclic AMP accumulation (nmol/mg protein)

None GTP(100/~M) GTPvS (100 laM) AIF4- (10/zM)

control

+ isoproterenol (10/~M)

0.572_+ 0.188 0.965_+ 0.320 * 9.771 -+ 3.342 * 35.335_+20.323 *

1,285_+ 0.458 * 8,070_+ 2.563 * 28,316-+ 12.523 *

* Differences which are statistically significant by a paired Student's t-test, P < 0.01.

fraction enriched in Gs using cholera toxin-induced ADP-ribosylation as a probe for the presence of Gs. Membranes were incubated with [~-32p]NAD and cholera toxin, as described earlier. Two proteins, which could be ADP-ribosylated specifically in the presence of cholera toxin, were identified only in the 40000 × g pellet fraction (Fig. 1). Their molecular masses were approx. 45 and 51 kDa, respectively. Similar molecular masses of Gs forms have been observed in other tissues [17,18]. Essentially no Gs-like cholera toxin substrates were observed in parotid membranes sedimented at 200000 × g or in the soluble parotid fraction (200000 x g supernatant). However, the latter fraction contained a cholera toxin substrate (molecular m a s s - 26 kDa) which we have not yet characterized. Accordingly, we used the 40000 x g pellet as the plasma membrane source for our studies.

,,

High Affinity GTPaseActivity 100

66.2 kDa

f.r,4

" i

•P ~

.F4 m

,,~ o

a.

5O

,as

,~L

31.0 kDa

~

lID

0

0.0

0.5

Concentration of GTP 21.51d)a

~

"' ' ~

14.4 kDa Fig. l . ADP-ribosylation by cholera toxin. Membranes were incubated without (lane 2, 4 and 6) or with (lane 3, 5 and 7) the preactivated cholera toxin (20/~g in 100 ~al) at 3 0 ° C for 30 rain, as described in the text to determine ADP-ribosylation suhstrates. (Lane 1, molecular weight standards; lanes 2 and 3: 40000x g pellet; lanes 4 and 5: 200000x g pellet; lanes 6 and 7:200000 x g supernatant).

t.0

(pHi

Fig. 2. GTPase activity in parotid plasma membranes. Enzymatic activity was studied in the presence of increasing GTP concentrations. Values for high affinity GTPase activity were calculated by subtracting the hydrolysis due to low affinity GTPase activity (i.e., that observed in the presence of 100 ~M GTP) using a non-linear leastsquare regression program• Assays were performed in the absence (o) or in the presence (O) of 10/~M isoproterenol at 30°C for 5 min. Increasing amounts of unlabelled GTP were added to 50 nM [~,32pIGTP to give the final concentrations indicated on the abcissa. Results shown are the mean+S.E, of seven separate experiments. Differences in Vma~ between the two conditions are statistically significant (t =1.96; P < 0.05).

394

High Affinity GTPase Activity Induced by Zsoproterenol 5.0

T 'PQ

C

(D

~Z 0,0 Centr'ol

Proprenolol

Fig, 3. Effect of propranolol on isoproterenol-induced high affinity GTPase activity. High affinity GTPase activity was determined at 30°C for 5 rain, as described in Table L in parotid membranes, treated with 10 ~tM isoproterenolin the absence(control, open bar) or in the presence(hatched bar) of 20 itM propranolol, Results shown are the mean+S.E, of four separate experiments and have been corrected for basal levels of GTP hydrolysis, i.e,, that without isoproterenol, Differences observed in GTPase activity between the conditions are statisticallysignificant (t = 3.57; P < 0.025). GTPase activity in the Gs-enriched membrane fraction The hydrolysis of [y-32p]GTP by parotid membranes could be resolved into at least two components with high and low affinities for GTP. The high affinity GTPase represented only a small fraction of the total GTP hydrolytic activity associated with parotid plasma membranes (= 15~, Table II). Release of [32p]Pi from [V-32p]GTP by the high affinity GTPase was saturable, giving an apparent Km value of approx, 450 nM and a V,,~ of about 94 pmol/mg protein per min (Fig. 2). Conversely, low affinity GTPase activity increased linearly with the concentration of added GTP up to 10 mM (not shown). The ~-adrenergic agonist isoproterenol (10/~M) had no effect on the low affinity GTPase (not shown), but significantly increased the high affinity GTPase (Fig. 2). lsoproterenol treatment did not alter the K m of the high affinity GTPase, but significantly increased the Vm,~ by about 50~, from 94 pmol/mg protein per rain to 143 pmol/mg protein per min ( P < 0.05). The fl-adrenergic antagonist, propranolol (20 ~tM) had no effect on high affinity GTP hydrolysis in the absence of isoproterenol, but markedly reduced the isoproterenol-stimulated increase in high affinity GTPase activity (---75%, P