THE OF BIOLOGICAL CHEMISTRY Vol. 259, No. 6, Issue of ... fileThe nonhydrolyzable analog of GTP,...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 19&1 by The American Society of Biological Chemists, Inc

Vol. 259, No. 6, Issue of March 25. pp. 35763585 1984 Printed in Li.9.A.

The Inhibitory Guanine Nucleotide-binding Regulatory Component of Adenylate Cyclase SUBUNIT DISSOCIATION AND GUANINE NUCLEOTIDE-DEPENDENT HORMONAL INHIBITION*

(Received for publication, August 15, 1983)

Toshiaki KatadaS, John K. Northup*$, Gary M. BokochST, Michio Uill, and Alfred G. GilmanS From the $Department of Phurmology, University of Texas Health Science Center a t D a l h , D&, Texas 75235 and the 11 Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Hokknido University, Sapporo, Japan

The inhibitory and stimulatory guanine nucleotide- binding regulatory components (Gi and G.) of adenylate cyclase both have an a.B subunit structure, and the B (35,000 Da) subunits are functionally indistinguishable. Gi and G. both dissociate in the presence of guanine nucleotide analogs or A13+, M e , and F- in detergent- containing solutions. Several characteristics of Gi- and G.-mediated regulation of adenylate cyclase activity have been studied in human platelet membranes.

The nonhydrolyzable analog of GTP, guanosine-5’- (3-0-thio)triphosphate (GTPyS) mimics GTP-dependent hormonal inhibition or stimulation of adenylate cyclase under appropriate conditions. This inhibition or stimulation follows a lag period. The combined addition of epinephrine or prostaglandin El with GTPyS results in the immediate onset of steady state inhibition or activation. The effects of the GTP analog are essentially irreversible. Fluoride is also an effective inhibitor of prostaglandin El-stimulated adenylate cyclase, while it markedly stimulates the basal activity of the enzyme. The addition of the resolved 35,000-Da subunit of Gi to membranes results in inhibition of adenylate cyclase, and the resolved 41,000-Da subunit has a stimulatory effect on enzymatic activity. The inhibitory action of the 35,000-Da subunit is almost completely abolished in membranes that have been irreversibly inhibited by GTPyS plus epinephrine; this irreversible inhibition is almost completely relieved by the 41,000-Da subunit.

Detergent extracts of membranes that have been treated with GTPyS plus epinephrine contain free 35,000-Da subunit. The 41,000-Da subunit of Gi con- tained in such extracts has a reduced ability to be ADP- ribosylated by islet-activating protein (IAP), which implies that this subunit is in the GTPyS-bound form. The irreversible inhibition of adenylate cyclase caused by GTPyS (plus epinephrine) in membranes is highly correlated with the liberation of free 35,000-Da subunit activity and is inversely related to the 41,000-Da IAP substrate activity in detergent extracts prepared therefrom. The increase in free 35,000-Da subunit activity in extracts and the inhibition of adenylate

* This work was supported by United States Public Health Service Grant NS18153 and by American Cancer Society Grant BC240D. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “oduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address, Department of Pharmacology, University of Calgary Faculty of Medicine, 3330 Hospital Drive, N. W., Calgary, Alberta, Canada.

ll Supported by National Institutes of Health Postdoctoral Fellow- ship GM08399.

cyclase activity in GTPyS (plus epinephrine)-treated membranes are both markedly inhibited by treatment with IAP. We hypothesize that hormone-induced inhibition of adenylate cyclase results from dissociation of the subunits of Gi in the membranes. Such inhibition is explicable by the interaction of the 35,000-Da subunit of Gi with the 45,000-Da subunit of G.. It is likely that ADP-ribosylation of the 41,000-Da subunit of Gi by IAP prevents guanine nucleotide- and inhibitory hormone-induced dissociation of dimeric GI.

In the first report in this series, we described methods for purification of the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase (Gi)’ from rabbit liver plasma membranes. Purified Gi consists of two predominant polypeptides with molecular weights of 41,000 and 35,000: The 41,000-Da subunit contains the site for NAD-dependent ADP-ribosylation catalyzed by IAP, and this covalent modi- fication results in attenuation or abolition of guanine nucleotide- and receptor-mediated inhibition of adenylate cyclase. The 41,000-Da subunit also contains the guanine nucleotide- binding site, identified both by photoaffinity labeling and by binding of radiolabeled guanine nucleotide analogs to the resolved polypeptide (1-3). The 35,000-Da subunit of Gi is indistinguishable from the 35,000-Da subunit of the stimulatory guanine nucleotide-binding regulatory component of adenylate cyclase, G.. The two 35,000-Da polypeptides have identical amino acid compositions (4), yield identical patterns of peptides during proteolysis (4), and are functionally inter- changeable (3-5).

The resolved 45,000-Da subunit of G. activates the catalyst of adenylate cyclase (6) . The 35,000-Da subunit of G, or Gi

The abbreviations used are: GB and Gi, the stimulatory and inhibitory guanine nucleotide-binding regulatory components of adenylate cyclase; C, the catalytic subunit of adenylate cyclase; IAP, islet-activating protein (pertussis toxin); 8-N3GTP, 8-azido guanosine-5’-triphosphate; GTPrS, guanosine-5’-(3-O-thio)triphosphate;

AlC13, 6 mM MgCl2, and 10 mM NaF; PGEI, prostaglandin El; solution Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonate; AMF, 20 p~

A, 50 mM sodium Hepes (pH 8.0), 1 mM sodium EDTA, and 1 mM dithiothreitol; solution B, 20 mM Tris. HCl (pH 8.0), 0.1 mM sodium EDTA, 1 mM dithiothreitol, 100 mM NaC1, 1% sodium cholate, and 10 mM MgSO,; solution c , 50 mM sodium Hepes (pH 8.01, 1 mM sodium EDTA, 1 mM dithiothreitol, 10 mM MgSO4, 1% sodium cholate, and 100 mM Na2SOI; solution D, 20 mM Tris.HC1 (pH 8.01, 1 mM sodium EDTA, and 1 mM dithiothreitol; HAM buffer, 150 mM sodium Hepes (pH 8), 0.75 mM ATP, 6 mM MgC12, 9 mM potassium phosphoenolpyruvate, 30 pg/ml of pyruvate kinase, and 0.3 mg/ml of bovine serum albumin.

* Gi may contain an additional subunit with M, = 10,000. This is discussed in the first paper in this series.

~~ ~~

3578

Gi Subunit Dissociation and Inhibition of Adenylate Cyclase 3579

inhibits such activation (7), stimulates deactivation of the 45,000-Da subunit (3, 5, 6) , and inhibits membrane-bound adenylate cyclase (3). The 41,000-Da subunit of Gi appears to be a relatively weak inhibitor of the catalyst in its GTPyS- bound state (3). Guanine nucleotide analogs and fluoride cause dissociation of the subunits of both G, and Gi in detergent-containing solution (3, 6, 8). We thus hypothesize that hormone-induced inhibition of adenylate cyclase may result from dissociation of the subunits of Gi in the bilayer. Such inhibition is explicable by the interaction of the 35,000-Da subunit of Gi with the 45,000-Da subunit of G,. The 41,000- Da subunit of Gi may also inhibit catalytic activity directly or competitively.

In the present report, we have examined the effects of hormones and guanine nucleotides on the state of membrane- bound Gi. Several characteristics of G,- and Gi-mediated regulation of adenylate cyclase activity and the mechanism of IAP-induced alteration of the function of Gi are also discussed.

EXPERIMENTAL PROCEDURES Preparation of Human Platekt Membranes-Human platelet mem-

branes were prepared from outdated platelets and, when applicable, were treated with IAP as described in the preceding paper (3).

Treatment of Membranes with GTPyS f Epinephrine; Preparation of Cholate Extracts of Membranes-Membranes (5 mg of protein/ml) were first incubated for 2 min (unless otherwise indicated) at 30 "C with or without GTPyS * epinephrine in a mixture containing adenylate cyclase assay reagents (see below), except for exclusion of 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidione and [CY-~~P]ATP. The reaction mixture was rapidly diluted with 20 volumes of ice-cold solution containing 25 mM Na Hepes, pH 8.0, 1 mM EDTA, 0.1 mM dithiothreitol, 25 mM NaCl, and 2 mM MgC12 and was centrifuged at 18,000 rpm for 20 min at 2 'C in a Beckman JA-20 rotor. The pellets were washed twice with the diluting buffer and suspended in the same buffer at a final concentration of 10 mg of protein/ml. Aliquots of the suspension were assayed for adenylate cyclase activity, and a 5% volume of 20% sodium cholate was added to the remainder to prepare a detergent extract. The suspension containing 1% sodium cholate was shaken for 60 min on ice and was centrifuged at 50,000 rpm in a Beckman 70 Ti rotor for 30 min at 2 "C. The supernatant was used as the cholate extract.

Assays for Adenylate Cyclase, the 35,000-Da Subunit, and 41,000- Da Subunit IAP Substrate Activity-Adenylate cyclase assays were performed as described in the preceding report (3).

Quantitative assay for the 35,000-Da subunit in cholate extracts from membranes was performed by examination of the rate of deactivation of G., as described earlier (5). G. (10 pl), which had been activated with AMF in solution A containing 0.2% Lubrol (3, 5), was diluted to 100 pl with a mixture of the extract to be assayed (usually 10 pl ) and solution A containing 5 mM EDTA and 0.2% Lubrol 12A9; these samples were incubated at 21 "C. In order to construct the G. deactivation progress curves, 10-p1 aliquots were withdrawn at different times, and G. activity was measured by reconstitution of adenylate cyclase activity in membranes of the S49 cyc- cell line (5,8). Since a number of substances have been found to alter the rate of deactivation of G., the activity of the 35,000-Da subunit was expressed as a k value, calculated from the initial rate of deactivation; within the relatively narrow range of k values utilized, there was a close linear correlation of the k value with the amount of 35,000-Da subunit added to the assay as an internal standard. (The concentration of the 35,000- Da subunit in pmol/lO pl of extract = 20. A k ) .

IAP substrate activity of the 41,000-Da subunit of Gi in cholate extracts was estimated by quantitation of IAP-catalyzed [32P]ADP- ribosylation of the 41,000-Da protein with [3ZP]NAD. Radiolabeling of detergent-solubilized 41,000-Da protein was carried out as described previously (2).

Purification of G., Gi, and Their Subunits-G., Gi, and their subunits were purified from rabbit liver plasma membranes as described (2,3,5,6).

Miscellaneous-Other procedures are described in the preceding manuscripts (2,3).

RESULTS Dual Effects of GTPyS on Adenylate Cyclase of Human

Platelet Membranes-Activation of adenylate cyclase by GTP+ has been well characterized (7-9). A lag is observed before steady state velocity is obtained; however, the combined addition of the GTP analog and a stimulatory hormone results in immediate activation. These effects were readily observed in human platelet membranes. Fig. 1 shows a time course of cyclic AMP formation in the presence of GTPyS f PGEl at different concentrations of M T . The rate of GTPyS-stimulated cyclic AMP synthesis was very low at the start of incubation but increased progressively. The simulta- neous addition of PGEl eliminated this lag. Specific activities assayed with 12 mM M P were much higher than those determined with 2 mM M P , particularly in the absence of

We next examined the effect of GTPyS on forskolin- stimulated adenylate cyclase activity, since we (3) and others (10 , l l ) have observed that guanine nucleotide-induced inhibition of adenylate cyclase is well maintained when the enzyme is activated with the diterpene. Forskolin markedly activated adenylate cyclase with no apparent lag; the stimulation was about 100- and 50-fold at 2 and 12 mM MgC12, respectively (Fig. 1). Addition of GTPyS to the forskolin- stimulated enzyme resulted in a strong inhibition, following an apparent lag period, particularly if activity was assessed at

PGEI.

Without Forskolin

2.5

1 - With 10uM Forskolin / /%

- II/ I I I I I 1 )

0 2 4 6 8 1 0 1 2 0 2 4 6 8 I C 1 12 Incubation Time ( m i d

FIG. 1. Time course of GTPyS-induced activation or inhibition of human platelet adenylate cyclase; effect of PGEl or epinephrine. Platelet membranes (0.5 mg of protein/ml) were assayed for adenylate cyclase activity at 30 "C with (C and D ) or without (A and B ) 10 p~ forskolin at 2 mM MgCl2 (A and C) or 12 mM M&lZ ( B and D ) in the presence of the reagents listed no further addition (O), 1 p~ GTPyS for C and D or 10 p~ GTPyS for A and B (A), 10 p~ GTPyS plus 10 p~ PGEI for A and B or 1 p~ GTPyS plus 25 p~ epinephrine for C and D (CI), 50 p~ GTP (V), or 50 p~ GTP plus 25 p~ epinephrine (V). Epi, epinephrine.

3580 Gi Subunit Dissociation and Inhibition of Adenylate Cyclase

low concentration of M e (Fig. IC). Inhibition caused by GTPyS was blocked by the addition of 50 p~ GTP (data not shown), although this concentration of GTP by itself inhibited adenylate cyclase activity only slightly. The lag period that preceded the onset of steady state inhibition by GTPyS was quite similar to that observed for activation by the nucleotide. Furthermore, the combined addition of epinephrine and GTPyS immediately inhibited the cyclase to a steady state level similar to that induced by GTPyS alone, without an apparent lag phase. When GTP replaced GTPyS, epinephrine inhibited the enzyme to the same extent, When forskolin- stimulated adenylate cyclase activity was assessed at 12 mM M e , GTPyS f epinephrine-induced inhibition was much less than that observed at 2 mM M$+, although the kinetic characteristics of the inhibitory effects were similar (Fig. 1D).

Fig. 2 shows the dual effects of GTPyS on control and forskolin-stimulated adenylate cyclase at 2 and 12 mM MgC12. The steady state rates of activity were measured by monitor- ing cyclic AMP formation as shown in Fig. 1. Half-maximal and maximal stimulation of adenylate cyclase in the absence of forskolin was observed at about 1 and 10 p~ GTPyS, respectively, at either low or high M e . However, elevation of the concentration of M e increased GTPyS-induced stimulation of the enzyme from about 5-fold at 2 mM MgClz to approximately 10-fold at 12 mM MgC12.

When 10 p~ forskolin was included, GTPyS had a biphasic effect, i.e. inhibition occurred at low concentrations of the nucleotide, followed by an increase in activity at higher concentrations (especially when assayed at high M e ) . GTPyS- induced inhibition of adenylate cyclase was more prominent at low concentrations of Mg+; maximal inhibition was about 60% at 2 mM M e and 25% at 12 mM M e . Half-maximal and maximal inhibition was observed at about 0.1 and 1 p M GTP+, respectively. Thus, the inhibitory effect of GTPyS is observable at 10-fold lower concentrations than is the stimulatory action.

Dual Actions of Fluoride on Adenylate Cyclase-Fluoride promotes dissociation of the subunits of both Gi and G, (2,3,

41600

GTPyS ( p M )

FIG. 2. Stimulatory and inhibitory effects of GTPrS on adenylate cyclase activity. Membranes (0.5 mg of protein/ml) were incubated with (0. A) or without (0, A) 10 HM forskolin in the

6). When membranes were assayed with increasing concentrations of NaF, there was an increase in adenylate cyclase activity, followed by a slight decrease in activity at high concentrations (Fig. 3). The NaF-induced activation of the enzyme was greater at 12 mM M&lz than at 2 mM Mgf& When NaF was added in the presence of PGEI, there was a marked inhibition of adenylate cyclase. The dose-dependent inhibitory effect of NaF was also more prominent at high concentrations of M e .

Irreversible Inhibition of Adenylate Cyclase by GTPyS- Nonhydrolyzable guanine nucleotide analogs stimulate adenylate cyclase irreversibly (9); this is also true for the inhibitory effects of GTPyS (3, 10) (Fig. 4). Human platelet membranes were incubated with GTPyS f epinephrine for 2 min; membranes were then washed thoroughly, followed by assay with forskolin in the presence or absence of GTPyS f epinephrine. When membranes that had been treated with both GTPyS and epinephrine were assayed for adenylate cyclase, enzymatic activity observed without further addition of hormone or nucleotide was much lower than that of control membranes, and there was no additional inhibitory effect of GTPyS f epinephrine (compare Fig. 4, A and C). There was a partial, irreversible inhibition of adenylate cyclase in membranes that had been treated with GTPyS alone (Fig. 4B). The inhibitory effect of GTP + epinephrine was totally reversible (data not shown).

The extent of irreversible inhibition of adenylate cyclase as a function of the concentration of GTPyS is shown in Fig. 40. Half-maximal inhibition was observed at about 0.1 or 0.5 p~ GTP+ in the presence or absence of epinephrine, respectively.

Effect of the Sduni ts of Gi on Inhibited Adenylate Cyclase- We have shown previously that the 35,000-Da subunit of Gi markedly inhibits forskolin-stimulated adenylate cyclase at low concentrations of M e (3). The characteristics of this inhibition are similar to those of the irreversible inhibition caused by GTPyS plus epinephrine. We thus studied the effect of the 35,000-Da subunit on membranes that had been treated with GTPyS + epinephrine (Fig. 5) . The addition of the 35.000-Da subunit to control membranes resulted in a

L “ -J

presence of increasing concentrations.of GTPGS, and cyclic AMP synthesis was measured over a period of 15 min as shown in Fig. 1. The steady state rates of activity obtained at each concentration of GTPyS were plotted against the concentration of GTP-yS used. The concentration of M&1, was 2 mM (0,O) or 12 m M (A, A). ~ ~~~ ~ ”

. , . NoF (mM)

FIG. 3. Fluoride-induced stimulation and inhibition of adenylate cyclase. Membranes (0.3 mg of protein/ml) were incubated with (0, A) or without (0, A) 10 p~ PGE, in the presence of increasing concentrations of NaF, and adenylate cyclase was assayed. The concentration of MeCl, was 2 mM (0.0) or 12 mM (A, A).


7 L L I

lncubotion Time (min)

FIG. 4. GTPrS-induced irreversible inhibition of adenylate cyclase. Membranes (5 mg of protein/ml) were first incubated for 2 min at 30 "C either without (A) or with 1 p~ GTPyS ( B ) or 1 p~ GTPyS plus 25 p~ epinephrine (C); reaction mixtures were diluted with 20 volumes of ice-cold buffer containing 25 mM Na Hepes, (pH 8.0), 1 mM EDTA, 0.1 mM dithiothreitol, 25 mM NaCl, and 2 mM MgC12, followed by centrifugation. Membranes washed by repeated dilution and centrifugation were further incubated without (0) or with 1 p~ GTP-yS (A) or 1 p~ GTPyS plus 25 p~ epinephrine (B) in the presence of 10 p~ forskolin, and cyclic AMP synthesis was quantitated. D, Membranes were first incubated for 2 min at 30 "C with different concentrations of GTPyS in the presence or absence of 25 p~ epinephrine, and the incubation was terminated by dilution and centrifugation. Washed membranes were further incubated with forskolin for 15 min at 30 "C to assay adenylate cyclase activity.

1 30 t

0 1 3 10 30 100

35K (oa) or 41K ( *A)Subuni t (pg/ml )

FIG. 5. Effect of the 35,000- and 41,000-Da subunits of Gi on adenylate cyclase in control and GTPrS plus epinephrine- treated membranes. Purified 35,000-Da subunit (0, A) or 41,000- Da subunit (0, A) (prepared by hydrophobic chromatography in the presence of A13+, F-, and M P ) was diluted on ice to the concentrations indicated on the abscissa with solution A containing 0.05% Lubrol. Samples (10 pl) were then mixed with 30 pl of control (0 ,O) or GTPyS plus epinephrine-treated (A, A) membranes and 20 pl of HAM buffer containing 50 p~ GTP, and the mixtures were incubated at 30 "C for 15 min. Assay reagents (40 pl) were added and adenylate cyclase activity was measured at 30 "C for 15 min. The membranes were treated with (A, A) or without (0, 0) 1 p~ GTPyS plus 25 p~ epinephrine for 2 min at 30 "C, as shown in Fig. 4.

concentration-dependent inhibition of adenylate cyclase, as observed previously (3). However, the inhibitory action of the protein was almost completely abolished in membranes that had been treated with GTPyS plus epinephrine; these inhib-

itory effects were thus not additive (see also Fig. 7). The effect of the purified 41,000-Da subunit was next

examined (Fig. 5). This polypeptide clearly increased the adenylate cyclase activities of both control and treated membranes in a dose-dependent manner. This stimulatory action of the 41,000-Da subunit was observed to a much greater extent in treated membranes than in the control, since the maximal specific activities attained were similar in both cases. Thus, the irreversible inhibition caused by GTPyS and epinephrine was almost completely relieved by the 41,000-Da subunit when high concentrations of the protein were used. When the GTPyS-bound 41,000-Da subunit was utilized, control membranes were inhibited at high concentrations of the protein (3).

It is of interest to note that the 41,000-Da subunit also relieved epinephrine-induced inhibition of adenylate cyclase when the protein was added to an assay mixture containing forskolin, GTP, and epinephrine (3).

These results suggest that treatment with GTPyS and epinephrine causes irreversible dissociation of Gi in the bilayer into the GTPyS-bond 41,000-Da subunit and the free 35,000- Da subunit. Either or both of these two polypeptides are candidates as mediators of the inhibitory effects that are observed. However, the striking ability of the unliganded 41,000-Da subunit to relieve this inhibition (Fig. 5) suggests that the 35,000-Da subunit is the dominant inhibitor. While it is possible that the free 41,000-Da subunit might compete with the GTPyS-bound form of the polypeptide to block its inhibitory effect, it seems more logical to suggest that the action of the unliganded 41,000-Da subunit results from its ability to interact with the free 35,000-Da subunit in the membrane.

Enhancement of 35,000-Da Subunit Actiuity in Extracts from Membranes Treated with GTPyS and Epinephrine-We have shown previously that examination of the rate of deactivation of the fluoride-activated state of G, is a useful method for assay of free 35,000-Da subunit and that the sensitivity of the assay allows detection of the polypeptide in detergent extracts of membranes. The 35,000-Da subunit of S49 cell and rabbit liver membranes appears to be associated with other proteins, such as the 41,000-Da subunit of Gi and the 45,000-Da subunit of G.. Detectable levels of the free 35,000- Da polypeptide were observed only after extracts of these membranes were treated with high concentrations of Mg2+ at 30 "C (to denature the GTP-binding subunits of Gi and G,) or after membranes were treated with Mg2+ and GTPyS (5). The experiments shown in Figs. 6 and 7 were designed to test the possibility that GTPyS k epinephrine might dissociate Gi into its component subunits in human platelet membranes. Membranes were incubated at 30 "C for 2 min either with or without GTPyS k epinephrine and were extracted with sodium cholate after washing by dilution and centrifugation. A sample of each extract was assayed by the activity of the free 35,000-Da subunit. In agreement with data obtained with S49 cells (5), there was no apparent difference in the initial rate of deactivation of G, following addition of extract from un- treated membranes when compared with the control rate. When extracts were incubated with Mg2+ at 30 "C for 1 h, free 35,000-Da subunit activity was liberated. When assays were performed on extracts from membranes that had been treated with GTPyS, a stimulation of the initial rate of deactivation of G. was readily observed. The combined addition of epinephrine and GTPyS to the membranes resulted in an apparent increase in this activity. When GTPyS was replaced by GTP, free 35,000-Da subunit activity was not detectable, even in the presence of epinephrine (data not


LL 'V I I I I

0 5 10 15 Time of Deactivation (min)

FIG. 6. Stimulation of 35,000-Da subunit activity in detergent extracts of membranes treated with GTPyS and epinephrine. Membranes (5 mg of protein/ml) were incubated at 30 "C for 2 min without (0, 0) or with either 1 p~ GTP-yS (A, A) or 1 p~ GTP-yS plus 25 p~ epinephrine (Epi) (0, R). The incubation was terminated by dilution and centrifugation. Membranes (washed twice) were then extracted (10 mg of protein/ml) with 1% sodium cholate, and the resultant extracts were assayed for 35,000-Da subunit activity as described under "Experimental Procedures." Both control (0, A, 0) and IAP-treated membranes (0, A, R) were studied. Cholate extracts from control membranes were incubated at 30 "C for 1 h and then assayed for 35,000-Da subunit activity (V). The rate of deactivation of G. in the presence of extraction buffer containing 1% sodium cholate is also shown (V). When AMF was added to these reaction mixtures after 15 min there was rapid recovery of G. activity and there were no differences between the samples. G/F, stimulatory GTP-binding regulatory component of adenylate cyclase.

shown). This is presumably due to the reversible nature of the inhibition caused by GTP + epinephrine.

Extracts of membranes that had been treated with IAP and NAD were also assayed for 35,000-Da subunit activity, since the inhibition of adenylate cyclase caused by GTPyS f epinephrine is largely attenuated by IAP (Fig. 6; see also Fig. 9). Extracts from IAP-treated membranes had no detectable 35,000-Da subunit activity (unless the membranes were incubated with GTPyS), indicating that ADP-ribosylation of the 41,000-Da subunit of Gi does not cause dissociation of the 35,000-Da subunit. Treatment with GTPyS f epinephrine did result in the appearance of free 35,000-Da subunit activity in extracts of IAP-treated membranes. However, these activities were much lower than those in the corresponding extracts from membranes that were not exposed to the toxin. It is unlikely that the extracts from IAP-treated membranes con- tained a factor that inhibited the assay for the 35,000-Da subunit, since the addition of internal standards revealed the appropriate activity (data not shown).

The 35,000-Da subunit activity in all of these extracts was completely abolished by heating at 90 "C for 10 min and was inactivated by alkylation with 5 mM N-ethylmaleimide (data not shown). These characteristics are consistent with those described earlier (5).

Fig. 7 shows time courses of cyclic AMP formation catalyzed by platelet membranes under various assay conditions and the activity of the free 35,000-Da subunit in extracts prepared therefrom. As the time of exposure of membranes to GTPyS was prolonged from 1 to 4 min, there were parallel and progressive increases in both 35,000-Da subunit activity and the level of inhibition of adenylate cyclase. The combined addition of epinephrine and GTPyS accelerated both of these effects. The effect of epinephrine was completely blocked by

~ ~ 1 1 1 1 1 1 1 1 1 / 0

0 2 4 6 8

Incubofion Time (rnin)

FIG. 7. Time course of the effects of GTPyS 2 epinephrine on adenylate cyclase activity and 35,000-Da subunit activity. Two sets of membranes were incubated simultaneously at 30 "C for assay for both cyclic AMP formation ( A ) and 35,000-Da subunit activity ( B ) . A, membranes (0.5 mg of protein/ml) were incubated with 10 p~ forskolin (0) or with, in addition to forskolin, either 1 p~ GTP-yS (A), 1 p~ GTP-yS plus 25 p~ epinephrine (R), 1 JLM GTP-yS plus 25 p~ epinephrine plus 2.5 p~ yohimbine (A), 2 pg/ml of purified 35,000-Da subunit (O), or 1 p~ GTP-yS plus 25 pM epinephrine plus 2 pg/ml of 35,000-Da subunit (0). Aliquots (100 pl) were withdrawn for assay of [s2P]cyclic AMP formation at the indicated times. E , membranes (5 mg of protein/ml) were incubated without (0) or with the reagents indicated above in the absence of forskolin, [3ZP]ATP, or 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidione, and aliquots (0.5 ml) were withdrawn at the indicated times. The samples were diluted, centrifuged, and extracted with sodium cholate for assay of 35,000- Da subunit activity, as described in Fig. 6. The solid bar on the ordinate in B shows the value obtained with extraction buffer alone in the G. deactivation assay.

the a2-adrenergic antagonist yohimbine, but the effects of GTP+ were not. The addition of purified 35,000-Da subunit to the membranes markedly inhibited cyclic AMP formation without any apparent lag period. The inhibitory effect of the protein was not additive with that caused by GTPyS and epinephrine. Thus, the inhibition of membrane-bound adenylate cyclase by GTPyS + epinephrine appears to be closely related to and probably reflects the release of the 35,000-Da subunit from Gi.

Reduction of IAP-catalyzed ADP-ribosylation of the 41,000- Da Subunit of Gi in Extracts from GTPyS-treated Mem- branes-The GTPyS-bound form of the 41,000-Da subunit is no longer a substrate for ADP-ribosylation catalyzed by IAP (1, 2). Membranes that had been incubated with different concentrations of GTPyS in the presence of epinephrine were washed and assayed for adenylate cyclase activity, and extracts from such membranes were also assayed for 41,000-Da IAP substrate activity (with IAP and radiolabeled NAD), as well as for 35,000-Da subunit activity.

When membranes were incubated with increasing concentrations of GTPyS in the presence of epinephrine, there was a progressive decrease in the 41,000-Da IAP substrate activity in extracts prepared therefrom (Fig. 8B) . The degree of reduction of IAP substrate activity was inversely correlated with the increment in 35,000-Da subunit activity (Fig. 8A) . Moreover, these changes were closely correlated with the

G; Subunit Dissociation and Inhibition of Adenylate Cyclase 3583

G T P y S ( p M )

FIG. 8. Reduction of IAP-catalyzed ADP-ribosylation of the 41,000-Da subunit in extracts from membranes treated with GTPrS plus epinephrine. Membranes (5 mg/ml) were incubated for 2 min at 30 'C with increasing concentrations of GTPyS and 25 p~ epinephrine, and the incubation was terminated by dilution and centrifugation as described in Fig. 6. Aliquots of the washed membranes were assayed for adenylate cyclase activity (with 10 p~ forskolin, C ) , and other aliquots were extracted with sodium cholate. The 35,000-Da subunit activity ( A ) and 41,000-Da IAP substrate activity (B) in the extracts were assayed as described under "Exper- imental Procedures." The solid bar on the ordinate in A shows the value obtained with extraction buffer alone in the G. deactivation assay.

degree of irreversible inhibition of adenylate cyclase (Fig. 8C). Finally, we studied the effects of IAP on the following three

parameters: 41,000-Da IAP substrate activity, 35,000-Da subunit activity, and adenylate cyclase activity (Fig. 9). In order to obtain membranes with varying extents of ADP-ribosylation of the 41,000-Da subunit, different concentrations of nonradioactive NAD were included when membranes were first incubated with IAP. The membranes were next incubated in the presence (0) or absence (0) of GTPyS plus epinephrine; after washing, membranes were assayed for adenylate cyclase activity and extracts of the membranes were assayed for 41,000-Da IAP substrate activity and free 35,000-Da subunit activity. Increasing concentrations of NAD in the first incubation resulted in progressive reduction of the subsequent radiotabeling of 41,000-Da protein in detergent extracts of the membranes (Fig. 9Z3, 0). This reduction implies that the 41,000-Da protein was already ADP-ribosylated by nonradioactive NAD during the first incubation. Maximally, about 60% of the membrane-bound 41,000-Da protein was ADP- ribosylated at 2.5 mM NAD under these experimental conditions. When the IAP-treated membranes were incubated with GTPyS and epinephrine, there was also a progressive decrease in 41,000-Da IAP substrate activity in the extracts prepared therefrom as the concentration of NAD was increased (Fig. 9B, 0). In agreement with Fig. 6, treatment with IAP also inhibited the GTPyS plus epinephrine-induced stimulation of 35,000-Da subunit activity. The dependence of this inhibition on the concentration of NAD was similar to that for inhibition of 41,000-Da IAP substrate activity. In parallel with the decreases in the activities of both subunits, GTPyS plus epinephrine-induced irreversible inhibition of adenylate cyclase was relieved by IAP in similarly NAD-dependent

: - O L d 0 20 $ 0 0 xx) 2500

N A D I p M I

FIG. 9. Effect of IAP-catalyzed ADP-ribosylation of the 41,000-Da subunit of GI on GTPyS plus epinephrine-induced inhibition of adenylate cyclase and stimulation of 35.000-Da subunit activity. Membranes were first incubated at 30 "C for 30 min with IAP (20 pg/ml) and the concentrations of NAD indicated on the abscissa, followed by dilution and centrifugation as described under "Experimental Procedures." Membranes were next treated with (0) or without (0) 1 pM GTPyS plus 25 p~ epinephrine at 30 "c for 2 min, and the treatment was terminated by dilution and centrifugation. Washed membranes or cholate extracts therefrom were assayed for adenylate cyclase (with 10 p~ forskolin; C ) , 35,000-Da subunit activity ( A ) , and 41,000-Da IAP substrate activity (B), as described in Fig. 8. The solid bar on the ordinate in A shows the value obtained with extraction buffer alone in the G. deactivation assay.

I

2 1 Y

0 0.2 0.4 0.6 0.8 4 1 K I A P Substrate

Activity (Aprno1/10pl)

0 I 2 3 4 5 6 0.0 0.2 0.4 0.6 0.S 35K Subunit Activity 41K IAP Substrata

( A h X 1 0 0 ) Actlvity ( A p d / I O p l 1

FIG. 10. Relationship between GTPyS plus epinephrine-induced increment of 36,000-Da subunit activity, reduction of 41,000-Da IAP substrate activity, and reduction of adenylate cyclase activity. A, GTPrS plus epinephrine-induced increment of 35,000-Da subunit activity is plotted against the reduction of the 41,000-Da IAP substrate activity. B and C, the increment of the 35,000-Da subunit (B) or the reduction of the 41,000-Da IAP substrate activity (C) is plotted against adenylate cyclase activity, which is expressed as a percentage of the control activity. Data are from Fig. 8 (0) and Fig. 9 (A). The correlation coefficient for the line drawn in A is 0.86. While separate lines can be drawn through the data points from each of the experiments shown, interpretation of the modest difference seems unwarranted.

fashion. Thus, IAP-catalyzed ADP-ribosylation of the 41,000- Da subunit of Gi appears to inhibit dissociation of the subunits of Gi in parallel with blockade of inhibition of adenylate cyclase.

The relationships between these parameters are illustrated in Fig. 10, in which the values obtained in Figs. 8 and 9 are replotted. Reduction of 41,000-Da IAP substrate activity caused by GTPyS + epinephrine occurred in direct proportion to the increment of 35,000-Da subunit activity (Fig. 10A). The extent of irreversible inhibition of adenylate cyclase


activity was well correlated with the increment of 35,000-Da subunit activity (Fig. 10B) or the reduction of the 41,000-Da IAP substrate activity (Fig. 1OC). The ability of guanine nucleotide analogs and inhibitory hormones to inhibit membrane-bound adenylate cyclase activity appears be to closely related to and probably reflects the extent of dissociation of Gi into its component subunits.

DISCUSSION

There are many similarities in the characteristics and mechanisms of hormonal stimulation and inhibition of adenylate cyclase. (a) Receptor-mediated stimulation and inhibition of the enzyme are both dependent on guanine nucleotides. The two systems are, however, differentially sensitive to divalent cations (12, 13), proteases (14-16), and sulfhydryl reagents (16-18). These differences have implied the existence of independent guanine nucleotide-binding regulatory proteins, G. and Gi. (b) Nonhydrolyzable analogs of GTP, such as GTPyS, mimic GTP-dependent, hormonal inhibition or stimulation of adenylate cyclase under certain conditions. The inhibition or stimulation usually reaches steady state levels after a lag period. The combined addition of an inhibitory or a stimulatory hormone with the GTP analog results in the immediate onset of steady state levels of inhibition or activation. The effects of GTP analogs are essentially irreversible. (c) Fluoride is also an effective inhibitor of hormone-activated adenylate cyclase, while it functions as a powerful stimulator of the basal enzyme. Thus, regulation of membrane-bound adenylate cyclase activity by both G. and Gi is under the influence of guanine nucleotides and fluoride. These results are consistent with the finding that the subunits of both G, and Gi dissociate in solution in the presence of guanine nucleotide analogs or fluoride (1-3, 6-8).

There are, however, interesting quantitative differences between Gi- and G,-mediated regulation of adenylate cyclase activity. GTPyS (plus inhibitory hormone)-induced inhibition of activity is observed at lower concentrations of nucleotide than is activation of the enzyme; inhibitory effects are also more prominent at lower concentrations of M F . These phenomena appear to be explained by the greater intrinsic affinity of purified Gi (compared to G.) for guanine nucleotide analogs and for Mg2+ (2, 7). This greater affinity of Gi for guanine nucleotides and Mg2+ appears to allow relatively specific release of the 35,000-Da subunit of Gj.

Release of the 35,000-Da Subunit of G; As a Mechanism of Guanine Nucleotide- and Hormone-induced Inhibition of Ad- enylate Cyclase-GTPyS inhibits adenylate cyclase irreversibly under certain conditions, and the nucleotide binds to the dissociated 41,000-Da subunit of Gi with very high affinity in the presence of M e . By analogy with the mechanism of G.- induced activation of the catalyst, one might expect inhibitory effects of the GTPyS.41,000-Da subunit complex, and, in fact, such are seen (3). However, relatively high concentrations of the complex are required. In contrast, the 35,000-Da subunit of Gi is a more potent inhibitor. Furthermore, its effects, as well as the inhibitory effects of GTPyS, can be relieved by the unliganded 41,000-Da subunit. Relief of the inhibitory effect of GTPyS by the 41,000-Da subunit implies that the inhibition is caused by the 35,000-Da subunit, free in the bilayer. Finally, the inhibitory effect of GTPyS + epinephrine is not additive with that of the 35,000-Da subunit. It thus seems very likely that at least a major fraction of the inhibition caused by GTPyS -t epinephrine is the result of the action of the 35,000-Da subunit, rather than the GTPyS. 41,000-Da subunit complex.

We showed previously that enhancement of the rate of

deactivation of activated G, by the 35,000-Da subunit could be utilized to assay the polypeptide and that the method is sensitive only to the free 35,000-Da subunit of Gj or G, but not to the dimers ( 5 ) . The assay has thus allowed detection of the 35,000-Da subunit in detergent extracts of plasma membranes. In addition, IAP-catalyzed ADP-ribosylation of the 41,000-Da subunit of Gi has provided us with a sensitive assay for Gi in detergent extracts of membranes; this method was utilized to monitor Gi during its purification (1, 2). However, the GTPyS-bound form of the 41,000-Da subunit is not a substrate for ADP-ribosylation by IAP (1,2), and loss of 41,000-Da IAP substrate activity can thus be utilized to assess the binding of GTPyS to Gi in membranes. The experimental results in the present study clearly indicate that the irreversible inhibition of membrane-bound adenylate cyclase caused by GTPyS and epinephrine is highly correlated with the liberation of 35,000-Da subunit activity and is inversely related to the 41,000-Da IAP substrate activity in detergent extracts prepared from treated membranes. As shown in Fig. 10,50% inhibition of membrane-bound adenylate cyclase was associated with a A k value of 0.05 for the 35,000-Da subunit (-1 pmol/lO pl of extract) and 0.8 pmol/lO pl of extract for the 41,000-Da IAP substrate activity. These values correspond to about 0.3 pg of subunit/mg of membrane protein in the extract. This agrees well with the value (0.5 pg of 35,000-Da subunit/mg of membrane) that was obtained for 50% inhibition of platelet adenylate cyclase by purified 35,000-Da subunit from rabbit liver plasma membranes under similar conditions (3). Of significance is that fact that 50% inhibition caused by the GTPyS .41,000-Da subunit was obtained only with considerably higher concentrations of protein (3).

Other results indicate, however, that the inhibitory effect of the 41,000-Da subunit may be relevant. These include in particular the fact that the forskolin-stimulated adenylate cyclase activity of the cyc- S49 cell can be inhibited by guanine nucleotide analogs (19-21) and by somatostatin + GTP (21, 22). Since the activity of the 45,000-Da subunit of G. is absent in this variant (23, 24), the 35,000-Da subunit is not an effective inhibitor. These relationships will be described in detail in a future r e ~ o r t . ~

The Mechanism of Action of IAP-Guanine nucleotide and hormone-induced inhibition of adenylate cyclase is markedly attenuated or abolished by IAP in human platelet membranes and in other cell types (see Ref. 3). The action of IAP is associated with the ADP-ribosylation of a membrane-bound 41,000-Da protein (25-28), and this 41,000-Da protein is one of the subunits of Gi (1, 2). This suggests that the IAP- catalyzed ADP-ribosylation of the 41,000-Da subunit impairs the function of Gi. We have observed that increases in 35,000- Da subunit activity in extracts from GTPyS (plus epinephrine)-treated membranes was markedly prevented by treatment with IAP. Unlike abolition of guanine nucleotide- or hormone-induced inhibition of adenylate cyclase, inhibition caused by the 35,000-Da subunit of Gi is well preserved in IAP-treated membranes (3). This excludes the possibility that the interaction of the 35,000-Da subunit with G. is impaired in IAP-treated membranes. It thus is likely that ADP-ribosylation of the 41,000-Da subunit of Gi by JAP prevents or impairs the guanine nucleotide- and inhibitory hormone- induced dissociation of dimeric Gi. Characterization of ADP- ribosylated Gi, using the purified protein, will provide more direct evidence.

Dissociation of the Subunits of G. and G; in the Bilayer- Evidence for guanine nucleotide- and fluoride-induced disso-

T. Katada, G. M. Bokoch, M. Smigel, M. Ui, and A. G. Gilman (1984) J. Biol. Chem. 259,3586-3595.


ciation of the subunits of G. and Gi in detergent-containing solution now seems unequivocal. Furthermore, free subunits can be detected in detergent extracts of membranes that have been exposed to GTPyS f epinephrine. We cannot, however, rule out the possibility that such dissociation occurs during extraction, if for no other reason than dilution alone. Spec- troscopic techniques will hopefully allow direct investigation of the proximities of subunits under conditions of hormonal stimulation and inhibition with freely reversible ligands (hormones + GTP). In the absence of such data, we still wish to suggest that subunit dissociation is an important mechanism of regulation of membrane-bound adenylate cyclase activity under physiological conditions. Results from both this and the preceding report that lead us to such a suggestion include in particular the prominent effects of free subunits on hormonal activation and inhibition of enzymatic activity in the presence of GTP.

Acknowledgments-We express thanks to Thomas Rall for his superb technical assistance and to Carol Manning for maintenance of cell cultures. We are indebted to Wendy Deaner for the preparation of the manuscript.

REFERENCES 1. Bokoch, G. M., Katada, T., Northup, J. K., Hewlett, E. L., and

2. Bokoch, G. M., Katada, T., Northup, J. K., Ui, M., and Gilman,

3. Katada, T., Bokoch, G. M., Northup, J. K., Ui, M., and Gilman,

4. Manning, D. R., and Gilman, A. G. (1983) J. Biol. Chem. 258,

5. Northup, J. K., Sternweis, P. C., and Gilman, A. G. (1983) J.

6. Northup, J. K., Smigel, M. D., Sternweis, P. C., and Gilman, A.

7. Northup, J . K., Smigel, M. D., and Gilman, A. G. (1982) J. Biol.

Gilman, A. G. (1983) J. Biol. Chem. 258, 2072-2075

A. G. (1984) J. Biol. Chem. 259, 3560-3567

A. G. (1984) J. Biol. Chem. 259, 3568-3577

7059-7063

Biol. Chem. 258, 11361-11368

G. (1983) J. Biol. Chem. 258, 11369-11376

Chem. 257, 11416-11423

8. Sternweis, P. C., Northup, J. K., Smigel, M. D., and Gilman, A.

9. Ross, E. M., and Gilman, A. G. (1980) Annu. Rev. Biochem. 49,

10. Seaman, K. B., andDaly, J. W. (1982) J. Biol. Chem. 257,11591-

11. Insel, P. A., Stengel, D., Ferry, N., and Hanoune, J. (1982) J.

12. Jakobs, K. H., and Aktories, K. (1981) Biochim. Biophys. Acta

13. Hoffman, B. B., Yim, S., Tsai, B. S., and Lefkowitz, R. J. (1981)

14. Yamamura, H., Lad, P. M., and Rodbell, M. (1977) J. Biol. Chem.

15. Stiles, G. L., and Lefkowitz, R. J. (1982) J. Bid. Chem. 257,

16. Aktories, K., Schultz, G., and Jakobs, K. H. (1982) Nuunyn- Schmiedeberg’s Arch. Pharmacol. 321, 247-252

17. Jakobs, K. H., Lasch, P., Minuth, M., Aktories, K., and Schultz, G. (1982) J. Biol. Chem. 257,2829-2833

18. Harden, T. K., Scheer, A. G., and Smith, M. M. (1982) Mol. Pharmol . 21,570-580

19. Hildebrandt, J. D., Hanoune, J., and Birnbaumer, L. (1982) J. Bwl. Chem. 257,14723-14725

20. Hildebrandt, J. D., Sekura, R. D., Codina, J., Iyengar, R., Man- Clark, C. R., and Birnbaumer, L. (1983) Nature (Lond.) 302,

G. (1981) J. Bwl. Chem. 256, 11517-11526

533-564

11596

Bwl. Chem. 257,7485-7490

676,51-58

Biochem. Bwphys. Res. Commun. 100,724-731

252,7964-7966

6287-6291

706-709 21. Jakobs, K. H., Aktories, K., and Schultz, G. (1983) Nature ( L o n d . )

303.177-178 22. Jakobs, K. H., and Schultz, G. (1983) Proc. Natl. Acad. Sci.

23. Ross, E. M., and Gilman, A. G. (1977) J. Bwl. Chem. 252,6966-

24. Ross, E. M., Howlett, A. C., Ferguson, K. M., and Gilman, A. G.

25. Katada, T., and Ui, M. (1982) J. Bwl. Chem. 257, 7210-7216 26. Burns, D. L., Hewlett, E. L., Moss, J., and Vaughan, M. (1983)

27. Murayama, T., and Ui, M. (1983) J. Bwl. Chem. 258,3319-3326 28. Kurose, H., Katada, T., Amano, T., and Ui, M. (1983) J. Biol.

U. S. A. 80,3899-3902

6969

(1978) J. Biol. Chem. 253,6401-6412

J. Bwl. Chem. 258, 1435-1438

Chem. 258,4870-4875

THE OF BIOLOGICAL CHEMISTRY Vol. 259, No. 6, Issue of ... fileThe nonhydrolyzable analog of GTP,...

Documents

Transcript of THE OF BIOLOGICAL CHEMISTRY Vol. 259, No. 6, Issue of ... fileThe nonhydrolyzable analog of GTP,...