Reprinted from Proc. Nat. Acad. Sci. USA Vol. 71, No. 8, pp. 3087-3090, August 1974 5'-Guanylylimidodiphosphate, A Potent Activator of Adenylate Cyclase Systems in Eukaryotic Cells (hormones/membranes/cyclic AMP/guanine nucleotides/allosteric regulation) CONSTANTINE LONDOS*, YORSM SALOMON*, MICHAEL C. LIN*, JAMES P. HARWOOD*, MICHAEL SCHRAMlM*, J. WOLFF?, AND MARTIN RODBELL* * Section on Membrane Regulation, Laboratory of Nutrition and Endocrinology, and t Section on Endocrine Biochemistry, Clinical Endocrinology Branch, National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Maryland 20014 Communicated by E. R. Stadtman, May 2.2, 1974 ABSTRACT 5'-Guanylylimidodiphosphate (Gpp(NH)- p) stimulates adenylate cyclase [ATP-pyrophosphate- lyase (cyclizing), EC 4.6.1.11 activity in plasma membranes isolated from frog and salmon erytbrocytes, from rat adrenal, hepatic, and fat cells, and from bovine thyroid cells. The nucleotide acts cooperatively with the various hormones (glucagon, secretin, ACTH, thyrotropin, and catecholamines) that stimulate these adenylate cyclase systems with resultant activities that equal or exceed those obtained with hormone plus GTP or with fluoride ion. In the absence of hormones, Gpp(NH)p is a con- siderably more effective activator than GTP, and, under certain conditions of incubation, stimulates rat fat cell adenylate cyclase to levels of activity (about 20 nmoles of 3',5'-adenosine monophosphate mg protein per min) far higher than reported hitherto for any adenylate cyclase system examined. The nucleotide activates frog erythro- cyte adenylate cyclase when the catecholamine receptor is blocked by the competitive antagonist, propranolol, and activates the enzyme from an adrenal tumor cell line which lacks functional ACTH receptors. In contrast, Gpp(NH)p does not stimulate adenylate cyclase in extracts from Escherichia coli B. Gpp(NH)p appears to be a useful probe for investigating the mechanism of hormone and nucleotide action on adenylate cyclase systems in eukary- otic cells. Several studies have shown that GTP enhances the response of adenylate cyclase systems [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.11 to peptide hormones, catecholamines, and prostaglandins (l-6). Kinetic studies of the hepatic glucagon-sensitive adenylate cyclase system indicate that GTP mediates the actions of glucagon on the enzyme system by an interdependent process indicative of an allosteric enzyme system (7). 5'-Guanylylimidodiphosphate (Gpp- (NH)p)$, the terminal phosphate of which appears not to be utilized in phosphotransferase reactions (8), mimics the stimulatory effects of GTP (9). In this paper we report that Gpp(NH)p causes marked stimulation of adenylate cyclase activity in membranes isolated from eukaryotic cells irre- spective of the nature of the receptors coupled to these sys- tems and even in the absence of hormones. Activation by the nucleotide occurs by means of a time-dependent process that is accelerated by hormones. In all cases, the effects of Gpp(NH)p are greater than those of GTP. Combinations of Gpp(NH)p and h'ormones produce activities considerably Abbreviations: Gpp(NH)p, 5'-guanylylimidodiphosphate; cyclic AMP, 3',5'-adenosine monophosphate; ACTHlmZ4, tetracosactide 1-24 corticotropin; DTT, dithiothreitol. $ Supplied by International Chemical and Nuclear Corp. greater than those seen in the presence of fluoride ion; the latter agent, with few exceptions, has been thought previ- ously to stimulate maximally most adenylate cyclase systems (10). Shown in Fig. 1 are typical effects of epinephrine, fluoride ion, and Gpp(NH)p on the time course of adenylate cyclase activity in purified plasma membranes from rat adipocytes. Fluoride ion and epinephrine, at maximally effective concen- trations, elicited immediate stimulatory effects on enzyme activity. In contrast, 1 PM Gpp(NH)p increased activity after a lag of 1 min of incubation. This lag was diminished by addition of the various hormones (secretin, glucagon, ACTH, and epinephrine) that activate the fat cell adenylate cyclase system (data not shown). The lag in onset of activation by Gpp(NH)p, and the effects of hormones thereon, is typical of all adenylate cyclase systems examined in this study. As will be shown in detailed studies to be published later, the initial inhibitory effect of the nucleotide shown in Fig. 1 is abolished when the pH of the incubation medium is increased from 7.6 to 8.5. The latter pH is optimal for the fat cell system. It will be noted in Fig. 1 that the steady state rates achieved with 1 PM Gpp(NH)p are approximately 2-fold higher than observed with 10 mM fluoride ion. Under optimal incubation conditions (1 mM ATP, 50 mM Mg*+, pH 8.5, and 37") for the fat cell adenylate cyclase system, 0.1 mM Gpp(NH)p stimulates activity to approximately 20 nmole.3 of cyclic AMP per min/mg of membrane protein (Fig. 2). This value, ob- tained in the absence of hormones, is the highest activity given by any of the adenylate cyclase systems examined in this study and is only slightly elevated by addition of all the hor- mones that affect the fat cell system. A comparison of the effects of maximal levels of GTP and Gpp(NH)p on the hepatic adenylate cyclase system is shown in Table 1. While GTP increased basal activity by 20$!&, the increase with Gpp(NH)p was 35Ooj,, resulting in a rate ap- proaching that seen with GTP plus glucagon. As was ob- served with the fat cell enzyme system, activation of the hepatic enzyme system by Gpp(NH)p was preceded by a lag period. For example, with a submaximal concentration of Gpp(NH)p (50 nM), the lag was as long as 34 min, and was reduced to 1 min by the addition of glucagon. This phe- nomenon is of interest in view of the previous finding with the hepatic adenylate cyclase system that GTP abolishes the lag in onset of activation by low concentrations of glucagon (7). It appears that both Gpp(NH)p and GTP mediate the action of the hormone through a common site or process. However, 3087 3088 Biochemistry : Londos et al. Proc. Nat. Acad. Sci. USA 71 (1974) 4or 30 - P p'*.' ..I" Basal 0 I I I 1 0 2 4 6 8 IO MINUTES FIG. 1. Effects of epinephrine, fluoride ion, and Gpp(NH)p on fat cell adenylate cyclase a&ivity. Purified plasma membranes were isolated from fat cells of the rat, by previously described methods (9, 15). In these experiments, fat cell membranes (11 rg of protein per ml) were incubated at 37" in media containing 1 mM [LY-~~P]ATP, 40 mM MgCl,, ATP regenerating system consisting of 10 mM creatine phosphate and 50 units of creatine kinase, 25 mM Tris.HCl, pH 7.6, 1 mM dithiothreitol (DTT), 2.5 mM cyclic AMP and 0.2% bovine serum albumin. The con- centration of Gpp(NH)p was 1 &I; NaF, 10 mM; and epi- nephrine 0.35 mM. With fat cell membranes, and with all other membrane preparations examined in this report, the reaction was stopped and [32P]cyclic AMP was isolated according to a recently described procedure (16). Gpp(NH)p alone activates the enzyme by a time-dependent process suggestive of a slow transition between different states of the enzyme; this transition appears to be accelerated in the presence of hormones and guanine nucleotides. Previous studies have shown the thyrotropin-sensitive adenylate cyclase system from bovine thyroid cells to be only minimally responsive to GTP (3). However, as is seen in Table 2, Gpp(NH)p increased basal activity 1Zfold to give a rate approaching that observed with fluoride ion and consider- ably greater than that seen with thyrotropin. Indicative of the cooperative interaction of hormone and nucleotide on the thyroid enzyme system is the observation that the combina- tion of Gpp(NH)p and thyrotropin resulted in an activity greater than the sum of activities obtained when each ligand was tested separately. Table 3 shows a comparison of the effects of Gpp(NH)p, ACTH, and fluoride ion on adenylate cyclase activity in mem- branes prepared from rat adrenal and from two mouse adrenal tumor cell lines: Y-l, which is `responsive to ACTH, and 200 r I I I 1 0 10-7 10-G 10-S 10-4 [GPPWWPI. M FIG. 2. Concentration dependency of Gpp(NH)p-stimulation of fat cell adenylate cyclase activity. Fat cell membranes (10 H of membrane protein per ml) were incubated for 10 min at 37" in medium containing 1 mM [,x-~*P]ATP, 50 mM MgCl,, 1 mhl DTT, 2.5 mM cyclic AMP, 0.2y0 bovine serum albumin, and 25 mM Tris. HCl, pH 8.5. OS3, which does not respond to the hormone (11). The en- zyme from rat adrenal was activated by Gpp(NH)p, and the nucleotide acted cooperatively with the hormone to increase activity to a level considerably greater than that observed with fluoride ion; activation of basal and hormone-stimulated activities by Gpp(NH)p was twice that observed with GTP (data not shown). The enzyme system from the ACTH-re- sponsive tumor cell line, Y-l, also exhibited the cooperative interaction of nucleotide and hormone, whereas that from the OS3 cell line, while completely unresponsive to hormone, was activated by Gpp(NH)p. Thus, stimulation by Gpp- (NH)p occurs even in the apparent absence of a functional hormone receptor. It will be noted in Table 3 that, while little difference was observed between fluoride-stimulated activity in the two adrenal tumor cell enzymes, basal and Gpp(NH)p-stimulated activities were considerably lower with the enzyme from the TABLE 1. Effects of Gpp(NH)p, GTP, and glucagon on hepatic adenylate cyclase Adenylate cyclase activity pmoles of cAMP per min/mg of protein Additions Basal Glucagon (1 &I) None 8 33.6 GTP, 0.1 mM 9.8 39.2 Gpp(NH)p, 0.1 ml1 31 50.4 Purified plasma membranes from rat liver were prepared as described previously (17). Membranes, 0.72 mg of protein per ml, were incubated at 30" for 5 min in medium consisting of 5 mM MgCl,, 1 mM CAMP, and 2.3 m&I Tris. HCl, pH 7.3. Substrate was 0.1 mM [u-~~P] 5'-adenylylimidodiphosphate (1). Proc. Nat. Acad. Sci. USA `71 (1974) V-Guanylylimidodiphosphate TABLE 2. Adenylate cyclase activity in bovine thyroid membranes: e$ects of thyrotropin, Gpp(NH)p, and jluoride TABLE 4. Activation of frog erythrocyte adenylate cyclase by Gpp(NH)p in absence and presence of a'soproterenol: lack of inhibition by propranolol of Gpp(NH)p egect Adenylate cyclase activity (pmoles of cyclic AMP per mg of protein per min) Additions None GPPCNWP, 10fiM Thyro- Basal tropin Fluoride 60 239 1060 729 1648 - Thyroid membranes were purified &s described previously (18). Adenylate cyclase assay medium contained 1 mM [cz-~~P]ATP, 5 mM MgCl,, 1 mM cyclic AMP, 5 mM phosphocreatine, 50 U/ml of creatine phosphokinase, and 25 mM Tris. HCI, pH 7.6, in a total volume of 0.1 ml. Reaction time was 10 min at 37" with the use of 0.16 mg/ml of membrane protein. Thyrotropin concentration WBS 200 mu/ml, and fluoride, 10 mM. Adenylate cyclsse activity pmoles of cyclic AMP per min/mg Additions of protein None 110 Fluoride 2450 Isoproterenol 2440 Isoproterenol + propranolol 110 GPP(NH)P 1300 Gpp(NH)p + isoproterenol 6820 Gpp(NH)p + propranolol 1360 Gpp(NH)p + isoproterenol + propranolol 1360 OS3 cells than that from the Y-l cells. These differences ap- pear to be related to incubation temperature. For example, at 30o Gpp(NH)p exerts little stimulatory effect on the enzyme from the OS3 cells but markedly stimulates the Y-l enzyme. However, at, 43' Gpp(NH)p-stimulated activity with the OS3 enzyme was somewhat greater than with the Y-l enzyme. The basis of the differences in temperature sensitivity of the adenylate cyclase systems in OS3 and Y-l adrenal tumor cells is under investigation. Activation of adenylate cyclase systems was observed also in hormone-sensitive cells obtained from amphibia and fish. Table 4 shows the cooperative effects of Gpp(NH)p and iso- proterenol on the frog erythrocyte adenylate cyclase system. Gpp(NH)p and isoproterenol increased activity lo- and 20- fold, respectively. When the nucleotide and the hormone were combined, the activity increased 60-fold and attained a rate three times higher than that produced with fluoride ion. Similar effects of Gpp(NH)p and isoproterenol were obtained with the adenylate cyclase system in purified plasma mem- branes from salmon erythrocytes. Frog erythrocyte plasma membranes were prepared according to Rosen and Rosen (20) with the following modificat,ions. After cell lysis, membranes obtained from 100 ml of frog blood were sedimented by centrifugation for 20 min at 12,000 X g. The pellet was suspended in 40 ml of Tris. HCl buffer, pH 8.1, containing 1 mM DTT, and centrifuged at 24,000 X g for 10 min. The pellet was washed twice as described above and stored in the Tris. DTT buffer under liquid nitrogen at a concentration of about 5 mg of protein per ml. Adenylate cyclase activity was measured under the same incubation conditions described in Table 2 for the thyroid adenylate cyclase system. Incubations were carried out with 33 rg of membrane protein per ml for 5 min at 30". Final concentration of isoproterenol, propranolol (Sigma), and Gpp- (NH)p was 10 pM. Fluoride ion was 10 mM. increased activity in an adrenal tumor cell line which lacks a functional receptor, and by the fact that, Gpp(NH)p causes near maximal activation of the fat cell enzyme system in the absence of hormones. Propranolol, an agent which blocks &receptors in adenylate cyclase systems, inhibited the stimulatory effect of isoproter- enol, but not that of Gpp(NH)p (Table 4). These findings demonstrate that Gpp(NH)p stimulates adenylate cyclase activity by a mechanism independent, of receptor occupation, a conclusion reinforced by the observation that the nucleotide Gpp(NH)p did not alter the activity of adenylate cyclase in extracts of Escherichia coli B under incubation conditions comparable to those employed with the eukaryotic adenylate cyclase systems. Adenylate cyclase in E. coli B appears to play an important regulatory role in the phenomenon of glucose repression of enzyme induction (12). The lack of ef- fect of Gpp(NH)p on E. coli B adenylate cyclase suggests that regulation of bacterial and eukaryotic adenylate cyclase systems may differ with respect to their nucleotide require- ments. 3089 TABLE 3. Adenylate cyclase activity in membranes from rat adrenal tumor cells: effects of ilCTH1-t4, Gpp(NH)p, and jluoride Adenylate cyclase activity (pmoles of cyclic AMP per mg of protein per 5 min) GPP(NH)P + Enzyme source Basal GPPWWP ACTHI-*" ACTHI-*' NaF Rat adrenals 144 708 957 2468 1466 Adrenal tumor cells Y-l cells 34 163 155 437 1065 OS3 cells 13 93 16 94 925 Adrenals from 150 g male Sprague-Dawley rats and mouse tumor cells grown in monolayer (11, 19) were homogenized with six strokes in a ground glass homogenizer in 250 mM sucrose, 1 mM DTT, 20 mM Tris.HCl, pH 7.6. Homogenates were centrifuged at 600 X g for 10 min, and the pellets suspended in 20 mM Tris.HCl, pH 7.6, 1 mM DTT, and stored under liquid nitrogen. Adenylate cyclase activity was measured under the same incubation conditions described in Table d for the thyroid adenylate cyclase system. Reaction time WBS 5 min at 37" with the use of approximately 0.2 mg/ml of membrane protein. The concentration of Gpp(NH)p was 10 MM, ACTHI-" 1 &I, and fluoride 20 mM. ACTHI-*" (Synacthen) w&s a gift of CIBA-Geigy. Y-l cells were obtained from the American Type Culture Collection; we gratefully acknowledge the gift of the OS3 cells by Dr. B. P. Schimmer. 3090 Biochemistry : Londos et al. Proc. Nat. Acad. Sci. USA 71 (1974) It has been suggested recently (7) that glucagon regulates hepatic adenylate cyclase activity by inducing the formation of states of the enzyme system that have increased reactivity with GTP. 5'-Guanylyldiphosphonate (Gpp(CH2)p) (1) and Gpp(NH)p, analogs with r-phosphates that are not readily cleaved, substitute for GTP in the activation of the enzyme. Therefore, it is reasonable to suggest that guanine nucleotides do not serve as phosphate donors but as allosteric activators of adenylate cyclase systems. The present study shows that Gpp(NH)p is a potent activator of eukaryotic adenylate cyclase systems, irrespective of the nature of the receptor coupled to these system; the nucleotide is an effective ac- tivator in the absence of hormones, and even in the absence of functional hormone receptors. Activation by Gpp(NH)p is a time-dependent process. Hormones appear to facilitate the rate at which Gpp(NH)p activates the enzyme systems, as evidenced by the finding that addition of hormones diminishes the lag in onset of the Gpp(NH)p effect. In studies to be reported elsewhere, in- creasing the reaction temperature also diminishes the lag in onset of activation by Gpp(NH)p. These findings suggest that the process of adenylate cyclase activation may proceed through different transition states which equilibrate slowly in the absence of hormone; addition of hormone or increas- ing the reaction temperature permits more rapid equilibra- tion between these states. This view is in accord with the con- cepts proposed by Ainslie et al. (13), and by Frieden (14), to explain transient kinetic characteristics of a number of regula- tory enzymes. Whether such changes involve associative reactions between subunits remains to be determined for the complex, membrane-bound adenylate cyclase systems. The basis for the differences in effectiveness of GTP and Gpp(NH)p is unknown. It is possible that the y-phosphate of GTP is hydrolyzed at the activation site, and that the rate of hydrolysis of GTP may be faster than the rate of transition between the various states suggested above. Gpp(NH)p, in contrast, being resistant to hjrdrolysis, may remain bound to the activation site. Studies to be reported elsewhere will show that adenylate cyclase systems remain activated fol- lowing pretreatment with Gpp(NH)p and extensive washing to remove all but bound nucleotide, and that resultant enzyme activity corresponds closely to the amount of nucleotide bound to the membranes. In conclusion, this report shows that Gpp(NH)p stimulates adenylate cyclase activity in plasma membranes obtained from a variety of eukaryotic cells. Gpp(NH)p appears to act through the same process by which GTP mediates hormone action; however, under appropriate incubation conditions, the analogue may stimulate the enzyme systems to maximal catalytic capacity even in the absence of hormones. The cooperative interactions of hormones and nucleotides, origi- nally discovered by the use of GTP, demonstrate the im- portance of the nucleotide function in hormonal regulation of adenylate cyclase systems. 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