Mechanism of action of choleragen andE. coli heat-labile enterotoxin: activation of adenylate cyclase by ADP-ribosylation

Summary Choleragen exerts its effects on cells through the activation of adenylate cyclase. The initial event appears to be the binding of the B subunit of the toxin to ganglioside G_M1 on the cell surface, following which there is a delay prior to activation of adenylate cyclase. Patching and capping of the toxin on the cell surface, perhaps involved in the internalization of the enzymatically active subunit, may be occuring during this time. The activation of adenylate cyclase, which is catalyzed by the A₁ peptide of choleragen, does not require the B subunit or ganglioside G_M1. The A₁ peptide catalyzes the transfer of ADP-ribose from NAD to an amino acid, probably arginine, in a 42 000 dalton membrane protein. This protein appears to be the GTP-binding component (or G/F factor) of the adenylate cyclase system and is cruical to the regulation of cyclase activity by hormones such as epinephrine. ADP-ribosylation of the G/F factor is enhanced by GTP and, in some systems, by a cytosolic factor. GTP is also required for stabilization and optimal catalytic function of the choleragen-activated cyclase. Calmodulin, a calcium-binding protein, is necessary for expression of catalytic activity of the toxin-activated adenylate cyclase in brain and other tissues. The ADP-ribosyltransferase activity required for activation of the cyclase is an intrinsic property of the A₁ peptide of choleragen which is expressed only after the peptide is released from the holotoxin by reduction of a single disulfide bond. In the absence of cellular components, choleragen catalyzes the ADP-ribosylation of small guanidino compounds such as arginine as well as peptides and proteins that contain arginine. It is assumed, therefore, that the site of ADP-ribosylation in the natural acceptor protein is an arginine or similar amino acid. When guanidino compounds are not present as ADP-ribose acceptors, choleragen hydrolyzes NAD to ADP-ribose and nicotinamide at a considerably slower rate. E. coli heat-labile enterotoxin (LT) is very similar to choleragen in structure and function. It consists of two types of subunits, A and B, with sizes comparable to those of the A and B subunits of choleragen. Binding of LT to the cell surface is enhanced by prior incorporation of G_M1 but not other gangliosides; the oligosaccharide of G_M1 specifically interacts with LT and its B subunit. The A subunit of LT exhibits ADP-ribosyltransferase activity following activation by thiol to release the A₁ peptide. The A subunit of LT can be isolated in an ‘unnicked’ form and thus requires, in addition to reduction by a thiol, proteolytic cleavage to generate the active A₁ peptide. Like choleragen, LT uses guanidino compounds as model ADP-ribose acceptors and catalyzes the ADP-ribosylation of a 42 000 dalton protein in cell membrane prepatations. ADP-ribosyltransferases that use arginine as ADP-ribose acceptors are not restricted to bacterial systems; such an enzyme has been purified to apparent homogeneity (>500 000-fold) from turkey erythrocytes. Based on a subunit molecular weight of 28 000, its turnover number with arginine as the ADP-ribose acceptor is considerably higher than that of either toxin. Although with low molecular weight guanidino derivatives the substrate specificity of the enzyme is similar to that of choleragen, with protein substrates it clearly differs. The physiological role of the turkey erythrocyte transferase remains to be established.     

Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain

Choleragen (cholera toxin) activates adenylate cyclase by catalyzing ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide-binding protein. It was recently found (Tsai, S.-C., Noda, M., Adamik, R., Moss, J., and Vaughan, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5139-5142) that a bovine brain membrane protein known as ADP-ribosylation factor or ARF, which enhances ADP-ribosylation of Gs alpha, also increases the GTP-dependent NAD:arginine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase, and auto-ADP-ribosylation activities of choleragen. We report here the purification and characterization of two soluble proteins from bovine brain that similarly enhance the Gs alpha-dependent and independent ADP-ribose transfer reactions catalyzed by toxin. Like membrane ARF, both soluble factors are 19-kDA proteins dependent on GTP or GTP analogues for activity. Maximal ARF effects were observed at a molar ratio of less than 2:1, ARF/toxin A subunit. Dimyristoyl phosphatidylcholine was necessary for optimal ADP-ribosylation of Gs alpha but inhibited auto-ADP-ribosylation of the choleragen A1 subunit and NAD:agmatine ADP-ribosyltransferase activity. It appears that the soluble factors directly activate choleragen in a GTP-dependent fashion. The relationships of the ARF proteins to the ras oncogene products and to the family of guanine nucleotide-binding regulatory proteins that includes Gs alpha remains to be determined.     

Mechanism of action of choleragen. Evidence for ADP-ribosyltransferase activity with arginine as an acceptor.

Choleragen catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide; nicotinamide production was dramatically increased by L-arginine methyl ester and to a lesser extent by D- or L-arginine, but not by other basic amino acids. Guanidine was also effective. Nicotinamide formation in the presence of L-arginine methyl ester was greatest under conditions previously shown to accelerate the hydrolysis of NAD by choleragen (Moss, J., Manganiello, V. C., and Vaughan, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4424-4427). After incubation of [adenine-U14C]NAD and L[3H]arginine with coleragen, a product was isolated by thin layer chromatography that contained adenine and arginine in a 1:1 ratio and has been tentatively identified as ADP-ribose-L-arginine. Parallel experiments with [carbonyl-14C]NAD have demonstrated that formation of the ADP-ribosyl-L-arginine derivative was associated with the production of [carbonyl-14C]nicotinamide. As guanidine itself was active and D- and L-arginine was equally effective in promoting nicotinamide production, whereas citrulline, which possesses a ureido rather than a guanidino function, was inactive, it seems probable that the guanidino group rather than the alpha-amino moiety participated in the linkage to ADP-ribose. Based on the assumption that the ADP-ribosylation of L-arginine by choleragen is a model for the NAD-dependent activation of adenylate cyclase by choleragen, it is proposed that the active A protomer of choleragen catalyzes the ADP-ribosylation of an arginine, or related amino acid residue in a protein, which is the cyclase itself or is critical to its activation by choleragen.     

Amino acid-specific ADP-ribosylation. Sensitivity to hydroxylamine of [cysteine(ADP-ribose)]protein and [arginine(ADP-ribose)]protein linkages

Hydroxylamine stability has been used to classify (ADP-ribose)protein bonds into sensitive and resistant linkages, with the former representing (ADP-ribose)glutamate, and the latter, (ADP-ribose)arginine. Recently, it was shown that cysteine also serves as an ADP-ribose acceptor. The hydroxylamine stability of [cysteine([32P]ADP-ribose)]protein and [arginine([32P] ADP-ribose)]protein bonds was compared. In transducin, pertussis toxin catalyzes the ADP-ribosylation of a cysteine residue, whereas choleragen (cholera toxin) modifies an arginine moiety. The (ADP-ribose)cysteine bond formed by pertussis toxin was more stable to hydroxylamine than was the (ADP-ribose)arginine bond formed by choleragen. The (ADP-ribose)cysteine bond apparently represents a third class of ADP-ribose bonds. Pertussis toxin ADP-ribosylates the inhibitory guanyl nucleotide-binding regulatory protein (Gi) of adenylate cyclase, whereas choleragen modifies the stimulatory guanyl nucleotide-binding regulatory protein (Gs). These (ADP-ribose)protein linkages are identical in stability to those formed in transducin by the two toxins, consistent with the probability that cysteine and arginine are modified in Gi and Gs, respectively. Bonds exhibiting differences in hydroxylamine-stability were found in membranes from various non-intoxicated mammalian cells following incubation with [32P]NAD, which may reflect the presence of endogenous NAD:protein-ADP-ribosyl-transferases.     

GTP stabilization of adenylate cyclase activated and ADP-ribosylated by choleragen

Choleragen activates adenylate cyclase in human skin fibroblasts by catalyzing the ADP-ribosylation of the 42,000 and 47,000 dalton guanyl nucleotide-binding regulatory components (G) of adenylate cyclase. The ADP-ribose linkage to 42,000 and 47,000 dalton proteins was stable at 30°C for 1 h with or without GTP, whereas GTP was required to stabilize activity of the G proteins. In human erythrocytes, choleragen catalyzed the ADP-ribosylation of only a 42,000 dalton G. The ADP-ribosyl-protein linkage was stable for 1 h at 30°C whether or not GTP was present, despite a rapid loss of G activity in the absence of GTP. Inactivation of choleragen-activated G in both the human fibroblast and human erythrocyte is, therefore, not secondary to the de-ADP-ribosylation of specifically labeled G subunits.     

Functional homology between signal-coupling proteins. Cholera toxin inactivates the GTPase activity of transducin

Both the light-stimulated cGMP phosphodiesterase of retinal rod outer segments (ROS) and hormone-stimulated adenylate cyclase are regulated by guanine nucleotide-binding regulatory proteins (N). Transducin serves as the signal-carrying regulatory protein in ROS, and the N protein (also called G or G/F) performs this role in the adenylate cyclase system. The GTP form of these regulatory proteins activates the corresponding enzyme, whereas the GDP form does not. Both transducin and the N protein possess a GTPase activity that restores the regulatory protein to the unstimulated state. Cholera enterotoxin catalyzes the transfer of ADP-ribose from NAD+ to the N protein, which inhibits its GTPase activity and activates adenylate cyclase. We report here that the toxin also catalyzes ADP-ribosylation of the alpha-subunit of transducin in ROS membranes. This modification of the guanine nucleotide-binding subunit of transducin is markedly enhanced by the bleaching of rhodopsin and by the addition of guanosine-5'-(beta, gamma-imino)triphosphate. In contrast, GDP, GTP, and guanosine-5'-(3-O)thiotriphosphate inhibit the reaction, while GMP and ATP have no effect. Under optimal conditions, toxin catalyzes labeling of 0.7 mol of the alpha-subunit of transducin/mol of bound [3H]guanosine-5'-(beta, gamma-imido)triphosphate and causes 70% inhibition of the light-dependent GTPase activity of transducin in ROS. These results indicate close functional homology between transducin of ROS and the N protein of adenylate cyclase.     

Release of guanyl nucleotides from the regulatory subunit of adenylate cyclase

Choleragen and beta-adrenergic agonists, both of which activate turkey erythrocyte adenylate cyclase, have been reported to accelerate release of bound [3H]guanyl nucleotides from turkey erythrocyte membranes. We have now obtained evidence that choleragen- or isoproterenol-stimulated release reflects a change in the affinity of the regulatory subunit (G/F) of adenylate cyclase for guanyl nucleotides. Solubilized preparations of turkey erythrocytes that had bound radiolabeled GTP were chromatographed on Ultrogel AcA 34. The protein from which guanyl nucleotide was released upon incubation with choleragen or isoproterenol was co-eluted with G/F activity. Furthermore, this protein appears to be the same size as the complex containing the 42,000-dalton peptide, ADP*-ribosylated by choleragen, which is presumably a subunit of G/F. ADP ribosylation of the 42,000-dalton subunit of G/F by choleragen occurred with a half-time of about 5 min, whereas choleragen-stimulated release of guanyl nucleotides was much slower (t1/2 greater than or equal to 60 min). When membranes were treated with choleragen and NAD, the delay in activation of adenylate cyclase by guanylyl imidodiphosphate was decreased but not abolished, a finding consistent with the idea that release of endogenously bound nucleotide (and subsequent binding of the nonhydrolyzable GTP analog) occurs only slowly following ADP ribosylation. In contrast, activation of the adenylate cyclase of either toxin-treated or untreated membranes in the presence of isoproterenol and guanylyl imidodiphosphate was very rapid. These data support the hypothesis that isoproterenol and choleragen may activate adenylate cyclase, at least in part, by increasing the rate of release of guanyl nucleotides from G/F.     

NAD-dependent ADP-ribosylation of arginine and proteins by Escherichia coli heat-labile enterotoxin.

Escherichia coli heat-labile enterotoxin (labile toxin, LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide and the ADP-ribosylation of arginine (Moss, J., and Richardson, S.H. (1978) J. Clin. Invest. 62, 281-285). Analysis of the product of the ADP-ribosylation of arginine by nuclear magnetic resonance spectroscopy indicated that the reaction was stereospecific and resulted in the formation of alpha-ADP-ribosyl-L-arginine. This reaction product rapidly anomerized to yield a mixture of the alpha and beta forms. In the presence of [adenine-U-14C]NAD, E. coli enterotoxin catalyzed the transfer of the radiolabel to proteins; the ADP-ribosylation of proteins was inhibited by arginine methyl ester, an alternative substrate. Digestion of the 14C-protein with snake venom phosphodiesterase released predominantly 5'-AMP. No product was obtained with a mobility similar to that of 2'-(5'-phosphoribosyl)-5'-AMP. This result is consistent with the covalent attachment by the enterotoxin of ADP-ribose rather than poly(ADP-ribose) to protein. Thus, LT is catalytically equivalent to choleragen, an enterotoxin of Vibrio cholerae, and activates adenylate cyclase through a similar stereospecific ADP-ribosylation reaction.     

Evidence for a rabbit luteal ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase.

1. An ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase was identified in a plasma membrane fraction from rabbit corpora lutea and partially characterized by comparing the properties of the luteal transferase with those of cholera toxin. 2. Incubation of luteal membranes in the presence of GTP and varying concentrations of NAD resulted in concentration-dependent increases in adenylyl cyclase activity. 3. Stimulation of adenylyl cyclase by NAD and cholera toxin plus NAD was observed in the presence of GTP but not in the presence of guanosine-5'-O-(2-thiodiphosphate) or guanyl-5'-yl imidodiphosphate. 4. NAD or cholera toxin plus NAD reduced the Kact values for luteinizing hormone to activate adenylyl cyclase 3- to 3.5-fold. 5. NAD or cholera toxin plus NAD increased the extent to which cholate extracts from luteal membranes were able to reconstitute adenylyl cyclase activity in S49 cyc- mouse lymphoma membranes. 6. It was necessary to add ADP-ribose and arginine to the incubation mixture in order to demonstrate cholera toxin-specific ADP-ribosylation of a protein corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (alpha Gs). 7. Treatment of luteal membranes with NAD prior to incubation in the presence of [32P]NAD plus cholera toxin resulted in reduced labeling of alpha Gs. 8. Endogenous ADP-ribosylation of alpha Gs was enhanced by Mg but was not altered by guanine nucleotide, NaF or luteinizing hormone and was inhibited by cAMP. 9. Incubation of luteal membranes in the presence of [32P]ADP-ribose in the absence and presence of cholera toxin did not result in the labeling of any membrane proteins.     

Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin. Relations to the mode of activation by hormones.

The influence of Vibrio cholerae enterotoxin (choleragen) on the response of adenylate cyclase to hormones and GTP, and on the binding of 125I-labeled glucagon to membranes, has been examined primarily in rat adipocytes, but also in guinea pig ileal mucosa and rat liver. Incubation of fat cells with choleragen converts adenylate cyclase to a GTP-responsive state; (-)-isoproterenol has a similar effect when added directly to membranes. Choleragen also increases by two- to fivefold the apparent affinity of (-)-isoproterenol, ACTH, glucagon, and vasoactive intestinal polypeptide for the activation of adenylate cyclase. This effect on vasoactive intestinal polypeptide action is also seen with the enzyme of guinea pig ileal mucosa; the toxin-induced sensitivity to VIP may be relevant in the pathogenesis of cholera diarrhea. The apparent affinity of binding of 125I-labeled glucagon is increased about 1.5- to twofold in choleragen-treated liver and fat cell membranes. The effects of choleragen on the response of adenylate cyclase to hormones are independent of protein synthesis, and they are not simply a consequence to protracted stimulation of the enzyme in vivo or during preparation of the membranes. Activation of cyclase in rat erythrocytes by choleragen is not impaired by agents which disrupt microtubules or microfilaments, and it is still observed in cultured fibroblasts after completely suppressing protein synthesis with diphtheria toxin. Choleragen does not interact directly with hormone receptor sites. Simple occupation of the choleragen binding sites with the analog, choleragenoid, does not lead to any of the biological effects of the toxin.     

Inhibition of the GTPase activity of transducin by an NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes.

The bacterial toxins, choleragen and pertussis toxin, inhibit the light-stimulated GTPase activity of bovine retinal rod outer segments by catalysing the ADP-ribosylation of the alpha-subunit (T alpha) of transducin [Abood, Hurley, Pappone, Bourne & Stryer (1982) J. Biol. Chem. 257, 10540-10543; Van Dop, Yamanaka, Steinberg, Sekura, Manclark, Stryer & Bourne (1984) J. Biol. Chem. 259, 23-26]. Incubation of retinal rod outer segments with NAD+ and a purified NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes resulted in approx. 60% inhibition of GTPase activity. Inhibition was dependent on both enzyme and NAD+, and was potentiated by the non-hydrolysable GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[beta gamma-methylene]triphosphate (p[CH2]ppG). The transferase ADP-ribosylated both the T alpha and T beta subunits of purified transducin. T alpha (39 kDa), after ADP-ribosylation, migrated as two distinct peptides with molecular masses of 42 kDa and 46 kDa on SDS/polyacrylamide-gel electrophoresis. T beta (36 kDa), after ADP-ribosylation, migrated as a 38 kDa peptide. With purified transducin subunits, it was observed that the GTPase activity of ADP-ribosylated T alpha, reconstituted with unmodified T beta gamma and photolysed rhodopsin, was decreased by 80%; conversely, reconstitution of T alpha with ADP-ribosyl-T beta gamma resulted in only a 19% inhibition of GTPase. Thus ADP-ribosylation of T alpha, the transducin subunit that contains the guanine nucleotide-binding site, has more dramatic effects on GTPase activity than does modification of the critical 'helper subunits' T beta gamma. To elucidate the mechanism of GTPase inhibition by transferase, we studied the effect of ADP-ribosylation on p[NH]pp[3H]G binding to transducin. It was shown previously that modification of transducin by choleragen, which like transferase ADP-ribosylates arginine residues, did not affect guanine nucleotide binding. ADP-ribosylation by the transferase, however, decreased p[NH]pp[3H]G binding, consistent with the hypothesis that choleragen and transferase inhibit GTPase by different mechanisms.     

Inhibition of ADP-ribosyltransferase activity of cholera toxin by MDL 12330A and chlorpromazine

ADP-ribosylation by cholera toxin of the guanine nucleotide binding regulatory protein (Gs) of rat liver membrane adenylate cyclase was inhibited by 0.1-1 mM MDL 12330A or 0.1-1 mM chlorpromazine. Basal as well as cholera toxin activated adenylate cyclase activity in liver membranes was also inhibited by the two drugs. NAD glycohydrolase activity and self-ADP-ribosylation of cholera toxin were also inhibited by MDL 12330A and chlorpromazine. These effects of MDL 12330A and chlorpromazine may be related to their effects on cholera toxin-induced fluid secretion in vivo.     

Cholera toxin action on rabbit corpus luteum membranes: effects on adenylyl cyclase activity and adenosine diphospho-ribosylation of the stimulatory guanine nucleotide-binding regulatory component

Cholera toxin elicited 5- to 7-fold stimulation of adenylyl cyclase activity. Half-maximal activation was at 4.42 micrograms/ml cholera toxin. Cholera toxin-mediated activation was time dependent. At 0.1 mM ATP, both guanosine triphosphate (GTP) and nicotinamide adenine dinucleotide (NAD+) were required for cholera toxin activation of luteal adenylyl cyclase. The concentrations of GTP and NAD+ required for half-maximal activation were 1 and 200 microM, respectively. The GTP requirement could be eliminated by increasing the ATP concentration to 1.0 mM. Guanosine-5'-O-(2-thiodiphosphate) [GDP beta S] did not support cholera toxin activation of the luteal enzyme. Cholera toxin treatment increased GTP-stimulated activity, did not significantly alter guanyl-5'-yl imidodiphosphate [GMP-P(NH)P]-stimulated activity, and depressed NaF-stimulated activity. Furthermore, toxin treatment resulted in a 3.4-fold reduction in the Kact values for ovine luteinizing hormone (oLH) to activate adenylyl cyclase. A similar reduction in Kact values for oLH was obtained when concentration-effect curves performed in the presence of GMP-P(NH)P were compared to those performed in the presence of GTP. In addition, luteal membranes treated with cholera toxin and [32P]NAD+ were subjected to autoradiographic analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This treatment resulted in the [32P] adenosine diphospho (ADP)-ribosylation of a 45,000-dalton protein doublet, corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (Ns). As with activation of adenylyl cyclase activity, cholera toxin-specific [32P] ADP-ribosylation was time dependent and increased with increasing concentrations of cholera toxin. GTP, GMP-P(NH)P, and NaF, but not GDP beta S, were capable of supporting [32P] ADP-ribosylation of the protein doublet. oLH did not alter the ability of cholera toxin to ADP-ribosylate the protein activation of luteal adenylyl cyclase activity is due to the ADP-ribosylation of the alpha subunit of Ns and the concomitant inhibition of a GTPase associated with adenylyl cyclase.     

Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin

A factor (ARF) that is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory, GTP-binding regulatory component (Gs) of adenylate cyclase has been purified about 2000-fold from cholate extracts of rabbit liver membranes. ARF is an intrinsic membrane protein with Mr = 21,000. The final product can be resolved into two polypeptides with very similar molecular weights; each of these has ARF activity. The ADP-ribosylation of Gs can now be studied with defined components. GTP and ARF are both necessary cofactors. The data imply that the substrates for the activated toxin are NAD and a GTP X Gs X ARF complex, and the reaction proceeds in a lipid environment. The apparent ability of ARF to bind to the alpha subunit of Gs suggests that it may play another, unknown role in the regulation of adenylate cyclase activity.     

ADP-ribosylation of membrane proteins and activation of adenylate cyclase by cholera toxin in fat cell ghosts from euthyroid and hypothyroid rats.

Incubation of fat cell ghosts with activated cholera toxin, nucleoside triphosphate, cytosol, and NAD results in increased adenylate cyclase activity and the transfer of ADP-ribose to membrane proteins. The major ADP-ribose protein comigrates on sodium dodecyl sulfate-polyacrylamide gels with the putative GTP-binding protein of pigeon erythrocyte membranes (Mr 42 000), which is also ADP-ribosylated by cholera toxin. The treatment with cholera toxin enhances the stimulation of the fat cell membrane adenylate cyclase by GTP, but the stimulation by guanyl-5'-yl imidodiphosphate is unaltered. Subsequent stimulation of fat cell adenylate cyclase by 10 micrometers epinephrine is not particularly affected. These changes were qualititatively the same for membranes isolated from fat cells of hypothyroid rats. Although the cyclase of these membranes has a reduced response to epinephrine, guanyl-5'-yl imidodiphosphate or GTP, as compared to euthyroid rat fat cell membranes, the defect is not rectified by toxin treatment and cannot be explained by a deficiency in the cholera toxin target.     

Thyroid membrane ADP ribosyltransferase activity. Stimulation by thyrotropin and activity in functioning and nonfunctioning rat thyroid cells in culture

Bovine thyroid membranes possess both ADP ribosyltransferase and NAD glycohydrolase activities with the same Km values for NAD and the same pH optima. In intact membranes, the ADP ribosyltransferase is limited in its extent by the amount of available membrane acceptor which can be ADP-ribosylated; in membranes solubilized with lithium diiodosalicylate, an artificial acceptor, L-arginine methyl ester, can be substituted to eliminate this limitation. The product of the ADP ribosyltransferase is a mono-ADP-ribosylated acceptor whether the intact or solubilized membrane provides the enzyme activity and whether membrane or exogenous acceptor, L-arginine methyl ester, is utilized. The intact membranes and the solubilized preparation also have an enzyme activity which can release AMP from the mono-ADP-ribosylated acceptor whether formed by the action of the membrane ADP ribosyltransferase or the A promoter of cholera toxin. The NAD glycohydrolase activity appears to represent the half-reaction of the ADP ribosyltransferase, i.e. an activity measurable substituting water for a membrane acceptor or L-arginine methyl ester. Membranes from functional rat thyroid cells in culture, i.e. cells chronically stimulated by thyrotropin and unresponsive to further additions of thyrotropin, have low ADP-ribosylation but high NAD glycohydrolase activities. In contrast, membranes from nonfunctional rat thyroid cells, i.e. cells unresponsive to thyrotropin, have high ADP-ribosylation and low NAD glycohydrolase activities. NAD hydrolysis by the NAD glycohydrolase activity cannot account for the low ADP-ribosylation activity in membranes from the functioning cells, and its low level of ADP-ribosylation can be eliminated by solubilizing the membranes and substituting an artificial acceptor, L-arginine methyl ester. The ADP ribosyltransferase activity of rat thyroid cell membrane preparations can be enhanced by thyrotropin in a dose-dependent manner but not by insulin, glucagon, hydrocortisone, adrenocorticotropin, or its glycoprotein hormone analog, human chorionic gonadotropin. It is thus suggested (i) that, in analogy to cholera toxin, thyrotropin-stimulated ADP-ribosylation may be important in the regulation of the adenylate cyclase response and (ii) that the level of membrane acceptor available for ADP-ribosylation may relate both to a stable "'activated" state of the adenylate cyclase system in cells chronically stimulated with thyrotropin and/or to a desensitized state with regard to a failure of more thyrotropin to elicit additional functional responses.     

Membrane-bound form of ADP-ribosyl cyclase in rat cortical astrocytes in culture

ADP-ribosyl cyclase activities in cultured rat astrocytes were examined by using TLC for separation of enzymatic products. A relatively high rate of [3H]cyclic ADP-ribose production converted from [3H]NAD+ by ADP-ribosyl cyclase (2.015+/-0.554 nmol/min/mg of protein) was detected in the crude membrane fraction of astrocytes, which contained approximately 50% of the total cyclase activity in astrocytes. The formation rate of [3H]ADP-ribose from cyclic ADP-ribose by cyclic ADP-ribose hydrolase and/or from NAD+ by NAD glycohydrolase was low and enriched in the cytosolic fraction. Although NAD+ in the extracellular medium was metabolized to cyclic ADP-ribose by incubating cultures of intact astrocytes, the presence of Triton X-100 in the medium for permeabilizing cells increased cyclic ADP-ribose production three times as much. Isoproterenol and GTP increased [3H]cyclic ADP-ribose formation in crude membrane-associated cyclase activity. This isoproterenol-induced stimulation of membrane-associated ADP-ribosyl cyclase activity was confirmed by cyclic GDP-ribose formation fluorometrically. This stimulatory action was blocked by prior treatment of cells with cholera toxin but not with pertussis toxin. These results suggest that ADP-ribosyl cyclase in astrocytes has both extracellular and intracellular actions and that signals of beta-adrenergic stimulation are transduced to membrane-bound ADP-ribosyl cyclase via G proteins within cell surface membranes of astrocytes.     

Pertussis toxin inhibits enkephalin stimulation of GTPase of NG108-15 cells

In neuroblastoma-glioma (NG108-15) hybrid cells, opiates inhibit adenylate cyclase and stimulate a low Km GTPase. It has been postulated that the stimulation of GTPase plays a role in opiate inhibition of adenylate cyclase (Koski, G., and Klee, W. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4185-4189). Treatment of NG108-15 cells with pertussis toxin attenuates receptor-mediated inhibition of adenylate cyclase. The toxin acts by catalyzing the ADP-ribosylation of a 41,000-dalton substrate believed to be a part of the receptor-adenylate cyclase complex. We have found that toxin treatment of NG108-15 results in inhibition of the opiate-stimulated GTPase. The concentration of toxin required for inhibition of this GTPase was similar to that needed for both attenuation of opiate inhibition of adenylate cyclase and ADP ribosylation of the 41,000-dalton substrate. Inhibition of the opiate-induced GTPase by pertussis toxin in isolated membranes required NAD, consistent with the hypothesis that this effect of the toxin resulted from ADP ribosylation of a protein component of the system. Since the opiate-stimulated GTPase is believed to play a role in the receptor-mediated decrease in adenylate cyclase activity, inhibition of this GTPase may be an important part of the mechanism by which the toxin interferes with opiate action on adenylate cyclase.     

Effects of GTP on choleragen-catalyzed ADP ribosylation of membrane and soluble proteins

Choleragen-dependent ADP ribosylation of soluble proteins from bovine thymus, using [32P]NAD as substrate, was increased 3- to 4-fold by GTP. The effect was specific for nucleoside triphosphate, with GTP approximately equal to ITP greater than CTP greater than ATP greater than UTP. Half-maximal enhancement was observed with 0.5 mM GTP. The magnitude of the GTP effect decreased with increasing NAD concentration; GTP had no effect on hydrolysis of NAD at low NAD concentrations. Digestion of ADP-ribosylated proteins with snake venom phosphodiesterase yielded primarily 5'-AMP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of soluble proteins from thymus after incubation with choleragen and [32P]NAD separated numerous ADP-ribosylated proteins; radioactivity in all bands was increased by nucleoside triphosphate. Choleragen catalyzed the ADP ribosylation of several purified proteins; depending on the protein, GTP either increased, decreased, or had no effect on the extent of ADP ribosylation. Choleragen-dependent ADP ribosylation of a wide variety of proteins is consistent with the possibility that intoxication results in covalent modification of more than one cellular protein and perhaps alters the activity of other enzymes in addition to adenylate cyclase.     

The sites on the regulatory component of adenylate cyclase which are ADP-ribosylated by cholera toxin

In rat liver membranes cholera toxin ADP-ribosylated two polypeptides (M_r 42000 and 47000) in the regulatory component of adenylate cyclase. L-arginine methyl ester specifically inhibited both the activation of adenylate cyclase and ADP-ribosylation by cholera toxin, suggesting that cholera toxin modified arginine, or arginine-like, residues. A hydrolysis-resistant analogue of GTP (β, γ-imidoguanosine 5′-triphosphate, p(NH)ppG) bound to the regulatory protein in an essentially irreversible manner. Pretreatment with the analogue failed to inhibit the labelling of polypeptides by cholera toxin showing that the sites for ADP-ribosylation were different from those at which guanyl nucleotides were bound.