.GTP Stimulates and Inhibits Adenylate Cyclase in Fat Cell Membranes through Distinct Regulatory Processes* (Received for publication, March 23, 1977) HIROHEI YAMAMURA,S PRAMOD M. LAD, AND MARTIN RODBELL From the Section on Membrane Regulation, Laboratory of Nutrition and Endocrinology, National Znstitute of Arthritis, Metabolism and Digestive Disease, Bethesda, Maryland 20014 CTP and hormones activate, synergistically, adenylate cyclase in purified plasma membranes from rat adipocytes. Addition of chelating reagents (EDTA or ethylene glycol bis(J?-aminoethyl ether)-XflJV',N'-tetraacetic acid) or thiol-reducing reagent8 (dithiothreitol or t-mercaptoetha- nol) nrult~ In marked Inhlbltlon ot'enzymc activity without altering the synergistic stimulatory effects of GTP and hormones. The inhibitory effects of the reagents required the presence of GTP, indicating that inhibition invoives a CTP-dependent proces.5. This process is separate from the CTP-dependent process responsible for activation of the enzyme since it is nelectively abolished by pretreatment of fat cell membranes with trypsin. It is suggested that inhibi- tion and activation of fat cell adenylate cyclase by GTP occur through distinct regulatory processes. that the inhibitory process is due to a regulatory protein which is distinct from the regulatory components responsible for GTP and hormonal activation of adenylate cyclase. Mareriofr -ACTHa-" I (Synacthen) was a gift of Ciba-Geigy. Cry+ talllne porcine glucagon was obtained from Eli Lilly and Co. Secretin wae a gin of Dr. V. Mutt (Karolinska Inalitute, Stockholm). Epi- nephrine bitartrata. epinephrine/HCI, dithiothreitol, and 2.mercap toethanol were purchased from Sigma. Trypsin (266 units/n@ and soybean trypein inhibitor (1 mg inhibits 1.53 mg of trypsin) were purchased from Worthington. All radioactive maLeI lale and other reagenrr were obtained from sources previously described (4). Preparofion of Fat Cell Membranes- Fat cell membranes were prepared M deacribed previously (I). The final membrane pellet was suspended in 1 mw EDTA and 20 my TrislHCl, pH 7.5, Lo give a concentration of 1 mglml of membrane protein. Aliquob were frozen and stored in liquid nitrogen. Several studies have shown that CTP or Cpp(NH)p exert both stimulatory and inhibitory effects on the multireceptor adenylate cyclase system in fat cells (I-4). Both effects of the nucleotides were found to be sensitive to assay conditions, including temperature, magnesium ion concentrations, pH, and hormones. Aa an explanation for the effects of the nucleo- tides, it was proposed recently (I), that the enzyme system is accessible to three states having differing V,,, and K, for uncomplexed ATP (HATPJ- or ATPV, postulated to bs a potent inhibitor at the active site (5,6). The guanine nucleo- tides, acting through a common site, affect the distribution of the states such that, depending on the incubation conditions tpH, Mff'+, hormone, temperature), the activity expressed will be inhibitory or stimulatory relative to the basal state of the enzyme. Alsay of Adenylatc Q&se-The method of Salomon tf al. (7) was used for aesaying the production of cyclic AMP (2). The standard assay medium contained in 100 pl, 30 mM TrislHCI, pH 7.5, 0.05% bovine aerum albumin, 1 my cyclic AMP, 0.1 mM [u-`~PIATP (50 to 200 cpmlpmol), 5 my crearine phosphate, 50 units/ml tf erostine phoaphokinase, 1 my ascorbic acid, and, except when indic- 4, I: rnL( MgCI,. Reactions were initiated by the addition of mel&loranes to give a final concentration of 3 to 5 pg of protein/ml. EDTA present in the membrane suspenrion was no higher than IO PM in chc final aeeay medium. Incubarions were carried our for 8 min a(. 37'. All experimenle were carried 0111 with at least two preparations of far cell plarma membranea and each assay was run in duplicate or triplicate. Other Detcrminotions- Protein was determined by the method of Lowry et al. (8) using bovine serum albumin as standard. During the course of investigating the stimulatory effects of GTP under conditions which minimized the inhibitory erects of the nucleotide, we found that the addition of thiol- reducing agents or chelators resulted in decreases in enzyme activity. The effect-s of these agents were dependent on the concentration of CTP in the medium and the type and concen- tration of divalent cation present in the incubation medium. This report describes these findings and provides evidence Effecfs of GTP on Basal and Hormone-slintulated Acfivi- ties - Fig. 1 illustrates the stimulatory effects of GTP on both basal and hormdne-stimulated activities; half-maximnl stim- ulation required about 0.1 PM GTP in all cases. Previously determined (1) saturating concentrations of the hormones were added in these experiments. Note that, with the excep- tion of epinephrine, stimulation by hormones was marginal in the absence of GTP. These results demonstrate the syner- gistic effects of GTP and hormones on the fat cell adenylate cyclase system under the conditions of assay (0.1 mM ATP, 10 mM Mg'+, pH 7.6, 37") employed in this study. o The costa of publicaGon of thin article were defrayed in part by the r yment of page charges. Thil article muet lhsreforc be hrnby mar ed "odr.rli~rmml" in accordance with 18 U.S.C. Section 1734 aoldly to indicate thir fad. I 7964 Eflects of Chelators in Presence of GTP and Hortttonex- As shown in Fig. 2, EGTA inhibited both basal and hormonr- #.Recipirnl of Public Health Service lntcmalional Fellowship FB5TW-0221-62. 1 The abbreviations used are: ACTH. ndrenocorticotropic hor- mone; EGTA. ethylene glycol bin(p-aminoethyl ether)-N,h'.N'.N'- tetraacetic acid. BXPIRIMENTAL PROCEDURES RESULTS GTP Stimulates and Inhibits Adenylcrle Cyclase 7965 / I I I 1 I -8 -7 -8 -6 -4 GTP lkq MI GTP kg M) Fta. 1 (k/I). Etfects of GTP on basal and hormone-stimulated Fta. 2 trig&). Dependence of ECTA inhibition of adenylate cy- adenylate cyclase activities. Adenylata cyclase activity was assayed clase on concentration of GTP. Adenylata cyclase activity was under the standard conditions described in the text. The concentra- assayed under standard assay conditions in the absence or prteence tionr of hormones are 10 pu ACTH, 10 CM epinephrine. 20 @t of 1 my EGTA and the indicated concentrations of GTP. Concentra- secretin. and 2 FM glucagon. tions of hormones are the same as in the legend to Fig. `. $Ty- 0 -8 -0 GTP WQ Ml Fro. 3 t/eflI. Ijcpendcncy of dithiothreitol inhibition on the con- trypsin for 6 min at 26 . . ..~~_ _. r. The incubation medium (0.5 ml) contained centration of GTP. Adenylate cyclase activities were assayed under the conditions described in the legend to Fig. 1. The concentration of dtthlothreitol tD2T) wu 1 my. Fro. 4 trighf). ENeda of trypsln pretreatment on the actions of GTP and dithiothraitol. Fat call membranes t106 pglml) were incubated in the absence (controlt or presence of 0.2 w/ml of 0.1% bovine serum albumin and 20 my TridHCI. pH 7.6. Soybean trypsin inhibitor was added to both control and trypain-treated membranes at a ltnal concentmlion of 2 &ml. Adcnylate cyelaae activities wem assayed under standard assay conditions in the plrecna of 1 my dithiothreitol. 10 pi CTP. and 1 my epinephrine. stimulated activities. Inhibition by thtc chc!ntor required the presence of CTP at concentrations in excess of 0.1 PM. With the exception of AC'I'H, EGTA did not selectively inhibit basal or hormone-stimulated activities. The sekctive loss of ACI'H reqxmse hith EGTA has been noted previously (9, 10) and has been attributed to removal of calcium ion which is thought to be essential for the actionr of ACTH. Addition of 10 CM cakium ion in the presence of 10 CM EGTA resulted in I I 1 I I ' I 1 I I I -8 -7 -6 -6 -4 GTPIIOIJM) near complete recovery of AGTH action and abolished the inhibitory effect of EGTA on both basal activity as well as the activitioa obtained with the other hormones (data not ahownk A similar pattern of inhibition was obaerved with EDNA but IO-fold higher concentrationn were required to give inhi- bition comparable IO that oboe14 with EGTA. Baaed on the &ability constanta for MgEDTA and MgEGTA (114 the sfMa of the chelatora on enzyme activity cannot be explained by 7666 GTP Stimulates and Inhibits Adenylate Cyclase chelation of sulllcient Mg" to altar the concentration8 of free Mg'*, MgATP (as substrate), or of uncomplexed form8 of ATP (ATP-, HATP'I Effkcfs of Thiot-reducing Agents-As observed with the chelators, dithiothreitol also inhibited both basal and hor- mone-stimulated adivitiea provided that CTP was present in the medium at conccntratione exceeding 0.1 PM (Fig. 3). Half-maximal inhibition required 0.6 mM dithiothreitol under all incubation conditions, indicating that the thiol reagent did not selectively inhibit beeal or hormone-stimulated activi- tiee. Similar inhibitory effects were observed also with 2- mercaptoethanol; half-maximal inhibition required 1 my 2- mercnptoethanol. It should be noted in Fig. 3 that the thiol reagents did not alter the ability of GTP and hormones to nynergidically rtimulate adenyiate cyclaee activity. Effkctr of Difhiothrrifol and Chelators in Pnsence of Mn**-The degree of inhibition in the presence of GTP, chelators, and thiol reagent8 wa8 dependent on the type and concentration of divalent cation present in the incubation medium. In all experiments reported above, inhibition wa8 observed in the presence of 10 mM Mg*+. However, increasing the concentration of Mg*+ to 50 mM aboliehed the inhibitory effect of dithiothreitol (data not shown, but 8ee Ref. 4). When 10 rnM Mg'+ wa8 replaced with 3 my Mn'*, only slight inhibition was observed even with 10 rnM dithiothreitol in the presence of CTP alone (basal) or plus ilormones (epinephrine could not be tested because of oxidation of catecholamines in the presence of Mn*+). tratione of the nucleotide in excess of 0.1 mM. With the exception of the effects of EGTA on ACTH action, the reagents did not alter the ability of CTP and hormone8 to activate the enzyme. Earlier it had been suggested that the biphasic effects of CTP could be accounted for by the formation of a transition state which is highly susceptible to inhibition by HATPa- (4). However neither the chelators (present below I rnM) or thiol- reducing agents (which do not form atable complexes with Mg" or other cations) are likely to affect the concentration of HATPa- under the incubation condition8 described here. Other explanations for the effects of CTP must therefore be sought. The finding that trypsin treatment of the membranes re- eulta in 1088 of inhibition by GTP (in the presence of dithio- threitol) without affecting the synergistic response of ade- nylate cyclaee to GTP and hormones provide8 strong evidence that the inhibitory process is distinct from !he GTP-regulatory component involved in hormonal activation of the enzyme. The fact that the inhibitory process is trypsin-sensitive sug- gests that it is a protein that interacts with GTP. Previous etudies (1) have ahown that Gpp(NH)p mimics the stimulatory and inhibitory actions of CTP on the fat cell systems; GDP does not inhibit the enzyme. Therefore, it is unlikely that this putative protein ha8 phosphohydrolase or phosphotrans- ferase activities. Efecls of Trypsin Treatment on Actions of GTP-The bipinasic stimulatory and inhibitory effecta of GTP observed in the presence of chelators and dithiothreitol raised the possibility that thelre effects were exerted through independ- ent procesaea. A8 one mean8 of testing this poru,! ility, fat cell membrane8 were pretreated with varying cohcentrationa of trvwin for 5 min at 30". followed by addition of trypsin _. inhibitor u r' 7 the rea&on. Trypsin-treated and &&rot membrane8 .re the.. tested for epinephrine-stimulated ade- nylate cycle<& activity UI der standard a8say condition8 in the prellence of dithiothreitol (1 rnM) and GTP (IO FM). A rypicc.1 result, obtained with an optimal concentration of trypsin (0.2 M/ml), is shown in Fig. 4. The control (nontrypsin-treatel) membrane8 rhowed the typical biphaaic elfects of GTP on adenyla(e cyclars activity in the prewnn of dithiothreltol whereas the trypsin-treated membranes showed only the stimulatory effect of GTP. The8e result.8 demonetrpte that the GTP-inhibitory pnress ie particularly sensitive to trypsin and clearly distinguishes this process from the GTP-etimula- tory procescl observed in the presence of hormones. Since the inhibitory eff't of GTP wa8 not lpecific for a particular hormone and basal activity wa8 affected to about the same extent, it ie unlikely that either hormone receptors or the "coupling" between receptors and catalytic unit are involved. The simplest explanation ie that inhibition occurs through a discrete regulatory protein which alters the cata- lytic unit of the enzyme and that this interaction is a@cted by temperature, pH, metal ions, chelators, and thiol-reducing agents. The selective ef%cts of trypsin treatment on GTP inhibition and the fact that the process involved in the synergistic action8 of GTP and hormone8 can be preserved under all theee conditions now provide a means ofdistinguish- ing between the two GTPdependent effect8 on enzyme activity and of selectively examining the mechanisms underlying these proces8ps. Such studies are in progress. REFERENCES 1. Harwood, J. P.. Ww, H., and Rcdbell, M. (1973)5. Biol. Chcm. 246, 6239-6245 2. Cryer, P. E., Jarett, L., and Kipnis, D. M. (1969) Biochirn. Biophye. Acfa 177. 666-696 3. Yemamura. H.. Rodbell. M.. and Fain, H. M. (1976) Mol. Phormoc~f. Ii. 693-700 Dl8CUBBION Inhibitory eNect8 of GTP have been reported with the fnt cell ryetern in the abwnce of chelators of thiol reagents when the incubation temperature wa8 below 37' (1) or when the pH and the concentration of Mg** in the incubation medium were reduced (4). We have shown in this study that two different type8 of reagenb, thiol-reducing agentn and chelators, cause inhibition of adenylate cycla8e activity in fat cell membranes. Inhibition by both type8 of reagent.8 WM dependent on the presence of GTP and became particularly evident at concen- 4. Rodbell, M. (197&J. Biol. Chcm. 250. 5626-5634 6. de Haan, C. (1974) J. Viol. Chem. 259, 2756-2762 6. Rendell, M.. Salomon, Y., Lin, M. C., Hodbell, M., and Berman, M. (1976) J. Biol. Chcm. 256.4253-4260 7. Balomon, Y.. London, C., and Rodbell, M. (1974)Anol. Biochcm. MI, Ml-548 8. Lowry, 0. H., Rosebrough, N. J.. Farr, A. L.. and Randall. R. J. (1951) J. Biol. Chcm. 193. 265-275 9. Rodbell, M., Bimhaumer. L., and Pohl. S. L. (1970) J. Bid. Chem. 245.716-722 266 10. B&r. H. P., and Heehter, 0. (1969)Proc. Nor!. Acad. Sci. U. S. A. 83,360-356 11. Holloway, J. H., and Reilley. C. N. (1960)Amxl. Chenc. 32. 249-