The Fat Cell Adenylate Cyclase System CHARACTERIZATION AND MANIPULATION OF ITS BIMODAL REGULATION BY GTI" (Received for publication, November 13. 19%. and in rev&d form, h4sy 3, 1979) Dermot M. F. Cooper, Werner Schlegel, Michael C. Lin, end Martin Rodbell From the Section on Membrane Regulation. National Institute o/Arfhrifin, Mefobolinm. and I>&r&itr Di-wr, National In~tituten of Health. Eethcrda. Motylond 20!205 GTP evok t! a both an activatorv and an inhibitorv rcsponec fro r adipocytc adcnylnte cyclasc. This pap& describes th `peraietence of the bimodal response under a variety of assay conditions. Additionally, manipula- tions are described which eliminate one or other of these actions. Treatment of adipocyte plasma mem- branes with cholera toxin Al pepride and NAD* abol- ishes the inhibitory phase of GTP action while preeerv- inn the activating phase. Treatment of the membranes with phydroxymercuriphenylaulfonic acid eliminates the activatory phase while mdntaining the inhibitory action of the nucleotide. Thus it appears that the two proceseee mediated by GTP in adipocytes normally co- exist and operate through different pathways since either phase can be abolished leaving the other intact. Adenoaine and ita purine-modified analogs inhibit fat cell adenylate cyclaee in the GTP inhibitory phare (Lon- doe, C., Cooper, D. M. F., Schlegel, W., and Rodbell, M. (1978) PJUC. Nat& Acd Sci U. S. A. 75. 5362-5366). When this effect of GTP ie abolished by either cholera toxin or Gpp(NH)p pretreatment, the inhibitory action of adenoeine analogs is also lost. These data sugges, 1 central role for GTP in mediating both activation and inhibition of adenylate cyclase by agents which act through cell surface receptors. The enhancement of hormonal stimulation and the stimu- latory effects of GTP on many adenylate cyclase systems have received widespread attention (see Ref. 1 and references therein). The adenylate cyclase of rat adipocyte plasma mem- branes is unusual in that GTP not only enhances activity but also pauses inhibition of the enzyme (2-6). The potential regulatory significance of this latter, seemingly paradoxical behavior of GTP has become apparent Corn recent findings in this laboratory (7). These studies showed that the p&ent inhibitory actions of adenosine and ita purine-modified ana- logs on cyclic AMP' production in intact adipocytes could be explained by inhibition of adenylate cyclase in the GTP inhibitory phase.' Yamamura et ;;. (6) have suggested that ' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduerfisement" in accordance with 18 USC. Section 17.34 solely to indicate this fact. `The abbreviations used are: cyclic AMP, edenosine 3':5'-mono- phosphate; ACI'H, adrenocorticotropic hormone; Gpp(NH)p. 5'. guanylylimidodiphosphate; Ko.s. the concentration of an agent evoking half of its maxima) effect.. `Throughout this paper, the terms "activatory" and "inhibitory" GTP phases are used aa follows: the activatory phase refers to the progressive increase in activity seen with increasing GTP concentm- tions from xero GTP to the peak activity. The decline in activity from thI peak with increased GTP leveb k referred to M the GTP inhibitory phur. the two effects of CTP are mediated by separak proteins since trypsin treatment of fat cell membranes abolished the inhibitory effect of GTP. It is not clear from the literature whether the biphaaic effects of CTP on aLipocyte adenylate cyclase are always encountered or to what extent assay conditions modify either phase. It has been suggested that at low temperatures or low magnesium concentrations CTP selectively inhibita fat cell cyclase activity (4. 5). Furthermore, Yamamura et al. (6) recently showed that chelators and sulfhydryl reagenL; corn- monly included in the assay of adenylate cyclaae, modify the actions of GTP on the fat cell enzyme. A further complication arises from the contamination of many commercial ATP preparations with GTP (8) such that the effects of low GTP concentrations are obscured. In the present study using a simplified incubation medium and purified ATP we have shown that the biphaaic effecta of GTP are present under a wide variety of conditions and should be considered to represer.t the normal response of the fat cell enzyme to the nucleotide. In addition, we have established procedures whereby either the activating or inhibitory phase can be examined separately. When the inhibitory process is absent, the enzyme is no longer sensitive to inhibition by purine-modified adenosine analogs. The regulatory impor- tnnce and the molecular basis for this behavior is discussed. MATERIAIS AND METHODS The sources of materials used in the assay of adenylate cyclase have been reported (9). L-isoproterenol-D-bitartrate and calf intestinal adenosine deaminase (230 units/ml) were purchased from Sigma. ACTHI-*' (Synacthen) was a gift of C&a-Geigy. h"-PhenyIiipropyl- adenosine was from Dr. J. N. Fain, Brown Univenrity. _ `ethyl-3- ieobutyl xanthine was bought from Aldrich Chemical Co. The ATP used in these studies was either purified according to the method ol Klmura et al. (8) or wan :ne Sigma product (A-2383) prepared by phosphorylation of adenosine. Preparation of Fat Cell Memb-s-Pl- membranea wert isolated by a simplification of the method of Avruch and Wallach (10). suspended in I mM EDTA containing IO rnM `Itis-HCl. pH 7.5, and stored in liquid Nr aa described by Harwootl cl of. (3). Adenylate Cyclase Assay-AdenyIate cyc;s :tiTity was scisayed by the method of Salomon cl aL (9) in a rnediu..l atdini 0.1 trY ATP, 1 PCi of [a-"PIATP, 4 nu( MgCl,. 0.1 rrul cyclic AMP. 2 mu creatine phosphate. creatine phosphokinaJe at 25 units/ml. r) mu Tris-HCI. pH 7.5. and 0.1% crystalline bovine serum albumin. I&%- tions were initiated by the addition of approximately 1 pg of mem- brane protein to give a total volume of 0.1 ml. Incubations were carried out for 30 min at 24'C, apart from the exceptions 1.6xd in the text. Experiments were performed in dupIicate or triplicate 5 L :;~n or more batches of fat cell plasma membranea. Replicates agccd to within 5% intraexuerimental variation was within SO to 120% of the valued shown. Trtahrunf with Cholem Toxin-The A, peptide of choler toxin wan prepared as described previously (II). Fat cell membruns (200 M) were incuhtad with 40 mu Ms-HCl, pH 7.5.1 my dithiothrcitol. 1 mg/tnl of bovine ncmm albumin. 2 mu NAD'. and 20 pa of AI a921 89'23 Bimodal Reguiation of Adenyiate Cyclase peptide of cholera toxin in a final volume of 166 ~1. After 5 min at 30oC. 2 ml of 10 m" Tris-HCl (pH 7.5) was added. The snrnple was then centrifuged at 36.000 X g (15 min. 4'Cl and the pellet was resusnended in 20 mM Tris-HCI containing 1 mg/ml of bovme serum alburkn. Treatment with Mercurial--Fat cell membranes UOO )I& were incubated with 40 mM Tris-HCI. pH 7.5. and 0.3 rnH p-hydroxymer- curlphenylsulfonic acid (Sigma) in II final volume of 100 pl. After 10 mm at OoC, 2 ml of Tris-HCI. pH 7.5 containing 0.2 rnH dithiothreitol was added; the membranes were sedimented W.ooO X g, 15 min. 4oC) in 26 rnH Trls-HCI contair.:ng 1 mg/ml of bovine serum albumin and resuspended in thz same medium prior to adcnylate cyclase astray. RESULTS Ejfecfs of GTP, ITP, and 2'.Deoxy-GTP-A relatively symmetrical biphasic (activating and inhibitory) relationship is apparent between the concentration of the three nucleotides and hormone-stimulated activity (Fig. 1). CTP is the most potent compound in terms of both its maximum activation and its effective concentration range. Stimulation of activity can be obtained by 2 nM GTP while maximal activity is evoked by 30 nr+t GTP. The relative steepness of both sides of ,hese curves should be noted, the concentration range in going from 16 to 90% (or 90 to 19%) of the maximal effect is only approximately lo-fold in both c-sees. Such behavior is strongly suggestive of positive co,operative interactions (12). Effect o/ Tewerahre-The effect of increasing CTP con- centration on basal, ACTH, and isoproterenol-stimulated ade- nylate cyclase activity is compared at 24oC and 36'C in Fig. 2? Previous studies (3.5) had shown that the inhibition caused by GTP at 25oC could be either reduced or altered to activa- tion by raising the temperature to 37oC. Indeed Pairault (51 had suggested that the only action of GTP was inhibition at low temperatures which was changed to activation at high temperatures. The basis for these observations can be appre- ciated by reference to Fig. 2, where it is apparent that the entire biphasic curve is present at both temperatures but is shifted 3- to 4-fold to the right on elevating the temperature. The total dependence of ACTH and the relative independence of isoproterenol on GTP for stimulation of activity, which had been previously pointed out by Yamamura et al. (6). is main- tained at both temperatures. Effects of [Mgz'J and [Mn2'] on GTP Inhibition-Rodbell (4) previously showed that at high magnesium concentrations the inhibition caused by 0.1 mM GTP (3) was changed to activation. Fig. 3 demonstrates that raising [Md'] from 4 to 20 mM caused no selective effect on either the inhibitory or the activatory GTP phase. Londos and Preston (13) have shown Mn*' to be 50 to 100 times more potent than MS+ in activating hepatic adenylate cychtse. When 2 mM Mn'+ wat included with 20 mru Mg' in the incubation medium, the inhibitory phase of GTP nction on the fat ceU cyclase system was eliminate1 and activation by GTP was reduced to the extent that ACTH stimulation was at F-Rt 10% above basal activity (Fig. 3). These results suggest that full activation of adenylate cyclase at a putr tive metal ion site (13) renders the enzyme relatively insensitive to regulation by GTP, as has been noted previously for hormonal activation of the adipo- cyte adenylate cyclase system (14). Effects of p-Hydroxymercuriphenylsulfonate-Generally, mercurials inhibit adenylate cyclase in a manner that can be reversed by thiol reagents (15-18). However, pretreatment of fat ceU membranes with p-hydroxymercuriphenylsulfonate J'lbe GTP curve obtained at 36oC in the present study is consid- erably more sensitive than that previously reported (6). We attribute this difference to the use of purified ATP and to the exclusion of aseorbate from the asray. We fiid that aecorbate can diminish the GTP inhibitory phase under /.me circum&ancea. o GTP 0 2:deoxy GTP 1+* 1 -.---< -9 -8 -7 -6 -5 LOG [nucleotide], M FIG. 1. Effecte of nucleotide triphosphntes on isoproterei.ol- stimulated fat cell adenylate cyclase activity. Adcnylate cyrlase activity wss assayed in the presence of I JIM iRoproterenol under standard conditions described under "Materials and Methods." followed by neutralization with dithiothreitol resulted in a 3- fold stimulation of basal adi nylate cyclase activity (Fig. 4). Accompanying the stimulation of basal activity by the mer- curial was complete loss of the stimulatory effects of GTP on the enzyme system. Instead, CTP caused frank inhibition of enzyme activity even at concentrations as low as 2 nM (Fig. 4); note that the apparent K"s for GTP was decreased by mtr- curia1 treatment relative to that seen in nontreated mem- branes (Fig. 1). `l'he unique effects of p.hydroxymercuriphen- ylsulfonic acid pretreatment will be dealt with in detail in a future report. Cholera Toxin Treatment-Following pretreatment of fat cell membranes with cholera toxin (the A-l subunit) and NAD', the typical biphasic effects of GTP were converted largely to a monophasic activating relationship both in the absence and presence of hormones (Fig. 5). NAD' was re- quired for this effect of the A, subunit. It can be .seen thet the stimulatory effect of GTP was enhanced by toxin treatment as has been reported for other cyclase systems (11, 19-21). It has been reported that the toxin inhibits the breakdown of GTP at a specific GTPase associated with the nucleotide activation process and that this effect of the toxin explains its activating effects (21). The finding that the toxin abolishes the GTP inhibitory effect on the fat cell cyclase system raises the possibility that the toxin also may influenrc' ' :lase activ- ity by eliminating a competing GTP-depender.. ahibitory process. Adenosine Action-Adenosine and analogs such as N"- phenyliaopropyladenosine inhibit fat ceU adenylate cyclase through a receptor that reacts competitively with methylxan- thines; with isolated membrane preparations, inhibitior by N"-phenylisopropyladenosine is observed only in the preset., of inhibitory concentrations of GTP (Ref. 7 and Table I).' Mercurial treatment, shown above to convert GTP action to a purely inhibitory mode, does not affect the ability of the adenosine analog to inhibit cyclase activity (Table I). How- ' No effect of A'"-phenyliaopropyladenosine wan observed at low (activatory range; 6 X 10e9 Lo GTP concentratiorrn following any of the treatmenta described in Table I. Rimodal Regulation o/./idenylate Cyclase 8929 300 o BASAL 24' `-- I -- _._- -- - _ -.-._ a -9 8 -7 -a .5 0 -9 -a -7 -6 LOG [GTP]. M I' 2.9 a .7 .a LOG IGTP], M ,J' z-9 a -7 a H I I I 1 K lo-' 104 1v lo-' CONCENTRAllON OF GTP, M FIG. 4. Effecta of mercurial pretreatment on the CTP titra- tion of baaal adenylate cyclw activity. Cyclase activity was determined under standard conditionn with membranes which had been pretreated with p-hydroxymercwiphenyl sulfonate (0) or not (0) under conditions deecribed under "Mat.eriale and Methods" FIG. 2. Effect of temperature on the CTP dependency of bd lad hormone-stimulated o ctjvity. Stan- dard -ym were performed for 30 min al 24'C (lrfi panrf) and for 7% min at WC (right panet) in the o taence (0) or presence of either 5 pu isoprotewol IISOI (0) or I ,UM ACTH (AL FIG. 3. Effects of metal ion con- centration on the CW titration. Ade- nylate cyelase activity wan deternuwd h the almence (0) or preaencc of either 10 PM isoproterennl (A) or 2 FLU ACW (0). Stindard condition8 were employed using A. 4 mM Md': B, m mY Md'; C. 20 mM Md' plus 4 mu Mn". FIG. 5. Effecter of cholera toxin pretreatment on the biiodal actiona of GTP. Aliquots (1 pg) of memb- which had been pretreated with either cholem toxin and NAD' (4 A) or NAD' alone (*, 0) were assayed under stmdard cmditiuna in the absence (0. A) or presence (0. A) of 0.4 (ur ieoproterenol. Pretreatment with NAD' done had no efkct on control activity (results not shown). 8930 Bimodal Regulation of Adenylalr Cyclase TABLE I Modification o/ ~`".phcnylinc~propylucfenosine inhibitiort of Jot cell adenylate cyclone activity by clarinus procedures The adenylate cyclane activity of 05 to 1.5.pg atiquota of variously treated membranes wan determined under rtanderd assay conditions in the presence of 2.5 unita/mt of adenoaine deaminaae lo reduce endogenous adenosine levels. Where indicated, CTP and A'"-phenyl- b,opropyladenoeine (PIA) were present at final concentrationa of 9 CM and 0.4 PM. reapeclively. _-_. .._-- ~. _ .- .._..- ___- Pnlrcatmml or con. lul,.mtcrmol -GTI' i(;TI' tG'r1' &lion of my ' -1'1.4 -I'IA +I'IA ___e- ~._.-.-__.- ------ __~. w nmol q-h- A.til'/mg/:ll) mm Normal None 1.75 1.55 0.39 (25)" 0.4 2.67 4.60 2.52 (4Hl Cholera toxin pre- None 1.70 4.51 3.97 IHH) treated* 0.4 3.73 7.33 6.36 (H6) GppNHp preacti- 0.4 24 57 25Rn 23.49 (92) vated Mn" (2 mu). M& 0.4 10.49 14.34 14.09 l9H) (20 mM) in as- MY" Mercurial treat- None 2.01 0.39 O.lH (4H) ment* __.-. -~~-~~~ n Values in bracketa express the percentage of activity in the presence of PIA compared IO that in i& ahsencc. The inhibition caused by PIP. could in all cases be rrvewd by 250 )IH I-methyl-3- iwbutyl xanthine (resulti not shown). ' Pretreatment of mcrnhrancs with cholera toxin A, peptide plus NAI) and the mercurial MI'S an described under "MaterinlR' and Methd." `Membranes (58 pg) were incubated for 0 min al 24'C in the presence of IO JIM Gpp(NtZ)l' a(.:! all adenylate ryclaxe avsay rom- ponentn except [n-"P]A?`I'. `i')~e activity of ahquota (1.4 pg) of this material was then determined over IO min by its nddition to a mixture containing [a-"P]ATP. CT]`, isoproterenol. and PIA as indicated. " Activities were determined in the presence of 20 mxi Mg'* con- taining 2 mM Mn" under the conditions indicated in the table. ever, any treatment which caused loss of GTP inhibition, including cholera toxin treatment, incubation with 2 mM Mgl' and 20 mru Mn" or preincubation with Gpp(NH)p." resulted in loaa of the inhibitory effects of both GTP and N"..phenyli- aopropyladenosine. The.se findings indicate an intimate link- age between the GTP inhibitory proceaa and the process through which adenosine inhibits adenylate cyclase. DISCUSSION The activating and inhibiting phaaea of GTP regulation of fat cell adenylate cyclaae, which are particularly pronounced in the presence of hormones and adenoaine, have been clearly established. A striking feature of the CTP titration curves ia that in most instances each phase occurs over a narrcs-; concentration range (c/. Fig. l), which might indicate positive cooperative interactions (12). However, such indications dia- appear when one process ia abolished ot masked. Thus, when the activating phase ia abolished by mercurial treatment, the inhibitory effects of GTP occur over a broader concentration range and the sensitivity of GTP action ia enhanced (cf. Fig. 4). Conversely, when the inhibitory phase is obscured by cholera toxin treatment, the stimulatory effect of GTP is enhanced. Such findings lead us to conclude that, in unper- tubed conditions, both processes are active and mutually repressing, poaaibly through competition for some limited catalytic activity. Thus, although aaaay conditions (tempera- ture, divalent metal ions, and type of hormone) may modify ' Gpp(NH)p inhibits basal adenytate cyclaae in short incubations; however with longer incubations (~10 mm), Cpp(NH)p activates at all concentrations (4.22). Folowing a @l-mitt preincubation of fat cell membrana with 10 plc Cpp(NH)p. adenylate cyclaae becomes per- siatttly activated and insensitive to CTP inhibition (unpublished the .s.ensitivity, or the amplitude of the CTP effects. or both, hoth phases nomtally co-exist. Alt!lough the precise mechanism remains ohscure, the im- portance of the bimodal behavior of GTP action is bec.)ming incre&ngly apparent in terms of the regulatory flexibility it provides. The activatory phase amplifies homlonal stimula- tion (Figs. 2 and 3). In the inhibitory phase. adenosine and purine-modified adenosine analogs, acting through ao.ralled R-type receptors inhibit both cyclic AMP production and lipolysis in the fat cell (7). Here we have shown that ttw inhibitory actions of a potent R-site adenosine analog ..rc abolished when the CTP inhibitory pha.se h aholbhrd or obscured by pretreatment with Gpp(NH)p. cholera toxin, or by incubating in the presence of high concentrations of diva- lent cations. Such total dependence of the analog's action on the integrity of the CTP inhibitory phase underlines the regulatory importance of the latter and elevates it to the .same degree of importance as the role of CTP in the stimulatory actions of hormones such as catecholamines, ACTH, and glucagon on the fat cell system. The facility of GTP to regulate an enzyme system such that in one mode it is insensitive to regulation by adenosine and iu another mode extremely sen- sitive to such regulation hints that the bimodal effects of the nucleotide are mediated by rreparate proteins. Consistent with this notion is the tinding of Yamrmura el nl. (6) that t,rypsin treatment of fa,t cell membranes abolished the GTP inhthttory proceaa. leaving the CTP atimulatory process intact. A similar bimodal regulation of adenylatc cyclase by G'I'P may exist in other cells. Ir human platelets, prostaglandins stimulate adenylate * ,ase activity whereas zatecholamines, acting through an o-type receptor, inhibit cycla.se activity. Roth actions require GTP in the incubation medium when membrane fragments containing cyclaae activity are examined for the effects of prostaglandins and catecholamines (2.7). Thus, it is possible that those extracelluhr agenta which inhibit the production of cyclic AMP in cells may do so. in part, through a CTP-dependent process that inhlbtts adenyl- ate cyclase. As a model, the apparent paradoxical birnodal responses of the fat cell adenylate cyclase system to CTP may ye: prove to be the key for understanding how hormones or other extacellular agents can promote either increased or decreased levels of cyclic AMP in their target cells. Acknou$edgnwnl-We wish to exprerrq our thanks to I> . C. I~mdon for his careful reading of the manuscript and helpful RUI eations. 1. 2. 3. 4. 5. 6. 7. a. 9. IO, 11, HEFEHKNCKS Hodbell. M., Lin, M. 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