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.     

Mutations of GS alpha designed to alter the reactivity of the protein with bacterial toxins. Substitutions at ARG187 result in loss of GTPase activity

We have introduced two types of mutations into cDNAs that encode the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase. The arginine residue (Arg187) that is the presumed site of ADP-ribosylation of Gs alpha by cholera toxin has been changed to Ala, Glu, or Lys. The rate constant for hydrolysis of GTP by all of these mutants is reduced approximately 100-fold compared with the wild-type protein. As predicted from this change, these proteins activate adenylyl cyclase constitutively in the presence of GTP. Despite these substitutions, cholera toxin still catalyzes the incorporation of 0.2-0.3 mol of ADP-ribose/mol of mutant alpha subunit. The sequence near the carboxyl terminus of Gs alpha was altered to resemble those in Gi alpha polypeptides, which are substrates for pertussis toxin. Despite this change, the mutant protein is a poor substrate for pertussis toxin. Although this protein has unaltered rates of GDP dissociation and GTP hydrolysis, its ability to activate adenylyl cyclase in the presence of GTP is enhanced by 3-fold when compared with the wild-type protein but only when these assays are performed after reconstitution of Gs alpha into cyc- (Gs alpha-deficient) S49 cell membranes.     

Pertussis toxin-mediated ADP-ribosylation of rabbit luteal Gi uncouples enkephalin inhibition of adenylyl cyclase

1. Some of the actions of pertussis toxin on the rabbit luteal adenylyl cyclase system were analyzed. 2. Incubation of luteal membranes with pertussis toxin and [32P]NAD resulted in the [32P]ADP-ribosylation of a 40,000 Da protein that is distinct from the proteins ADP-ribosylated by cholera toxin. 3. Pertussis toxin specific [32P]ADP-ribosylation was time-dependent and dependent upon the concentration of pertussis toxin present during the incubation. 4. Pertussis toxin mediated [32P]ADP-ribosylation was enhanced by ATP, ADP, adenylyl imidodiphosphate, GTP, guanosine-5'-O-(2-thiodiphosphate), guanosine-5'-O-(3-thiotriphosphate), and NaF but not AMP or guanylyl imidodiphosphate [GMP-P(NH)P]. 5. Treatment of luteal membranes with NAD and pertussis toxin prevents GTP and enkephalin but not GMP-P(NH)P mediated inhibition of forskolin stimulated adenylyl cyclase, demonstrating the existence of a functional Gi in the rabbit corpus luteum.     

Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19 kDa protein

Cholera toxin causes the devastating diarrheal syndrome characteristic of cholera by catalyzing the ADP-ribosylation of Gs alpha, a GTP-binding regulatory protein, resulting in activation of adenylyl cyclase. ADP-ribosylation of Gs alpha is enhanced by 19 kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors or ARFs. We investigated the effects of agents known to alter toxin-catalyzed activation of adenylyl cyclase on the stimulation of toxin- and toxin subunit-catalyzed ADP-ribosylation of Gs alpha and other substrates by an ADP-ribosylation factor purified from a soluble fraction of bovine brain (sARF II). In the presence of GTP, sARF II enhanced activity of both the toxin catalytic unit and a reduced and alkylated fragment ('A1'), as a result of an increase in substrate affinity with no significant effects on Vmax. Activation of toxin was independent of Gs alpha and was stimulated 4-fold by sodium dodecyl sulfate, but abolished by Triton X-100. sARF II therefore serves as a direct allosteric activator of the A1 protein and may thus amplify the pathological effects of cholera toxin.     

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.     

GTP-activated GTP binding protein(Gs) in membranes achieved by hormone plus GDP does not serve as a substrate for ADP-ribosylation by cholera toxin

Adenylate cyclase in the presence of GTP became active by the addition of cholera toxin irrespective of the presence of glucagon, and under the same condition the Gs of these activated enzymes were good acceptor of an ADP-ribose moiety. On the other hand, the cyclase in the presence of GDP remained inactive with cholera toxin but became active by the further addition of glucagon. However, neither of these Gs served as a cholera toxin substrate. Glucagon reduced an inhibitory action of added GDP for cholera toxin plus GTP-stimulated adenylate cyclase activity but did not for toxin plus GTP-enhanced ADP-ribosylation of Gs. These results demonstrate that Gs-GTP complex formation alone is not sufficient for Gs to serve as a cholera toxin substrate, and suggest an additional GTP binding site responsible for ADP-ribosylation by the toxin. Hormone dependent preferential interaction between the GTP binding site on Gs coupled with adenylate cyclase regulation and membrane-associated nucleoside diphosphate kinase is discussed.     

Mono-ADP-ribosylation of Gs by an eukaryotic arginine-specific ADP-ribosyltransferase stimulates the adenylate cyclase system

An arginine-specific ADP-ribosyltransferase, named ADP-ribosyltransferase A, was partially purified from human platelets using polyarginine as an ADP-ribose acceptor. When human platelet membranes were incubated with the transferase A in the presence of NAD+, Gs, a stimulatory guanine nucleotide-binding protein of the adenylate cyclase was specifically mono-ADP-ribosylated. ADP-ribose transfer to Gs by this enzyme was suppressed when membranes were pre-ADP-ribosylated by cholera toxin. Incubation of membranes with the transferase A resulted in activation of the adenylate cyclase system. This stimulatory effect of the transferase A on the adenylate cyclase system was inhibited by the presence of polyarginine. These results indicate a role of ADP-ribosyltransferase A in regulation of the adenylate cyclase system via endogenous mono-ADP-ribosylation of Gs.     

Cholera toxin induces cAMP-independent degradation of Gs

Cholera toxin stimulates adenylyl cyclase by catalyzing ADP-ribosylation of the alpha chain (alpha s) of Gs, a guanine nucleotide binding regulatory protein. In a rat pituitary cell line, GH3, the toxin-induced increase in GTP-dependent adenylyl cyclase activity is maximal at 1 h; adenylyl cyclase remains elevated for at least 32 h. Surprisingly, cholera toxin also induces a 74-95% decrease in the amount of immunoreactive alpha s in the same cells, as assessed on immunoblots probed with either of two antisera directed against separate alpha s peptide sequences. The decrease in immunoreactive alpha s, which begins after 1 h of toxin treatment and is complete by 8 h, is accompanied by a comparable decrease in the amount of biochemically active alpha s, as assessed by its ability to complement the biochemical defect of alpha s-deficient S49 cyc- membranes. Cholera toxin induces similar decreases in alpha s in wild type S49 lymphoma cells, in S49 kin- mutants, which lack cAMP-dependent protein kinase, and in S49 H21 a mutants, in which alpha s is unable to assume an active conformation upon binding GTP. The toxin-induced decrease in alpha s is somewhat temperature-dependent, but is not blocked by agents that increase lysosomal pH or by colchicine, which promotes breakdown of microtubules. alpha s in detergent-solubilized GH3 membranes is susceptible to proteolysis by an endogenous protease; this susceptibility is markedly increased in membranes from cells previously exposed to cholera toxin for 1 h. Taken together, these results suggest that cholera toxin-induced covalent modification of alpha s marks the protein for accelerated degradation. In addition, the persistence of elevated GTP-dependent adenylyl cyclase activity despite loss of a substantial fraction of alpha s suggests that the amount of alpha s membranes is greater than the amount necessary for maximal activation of cAMP synthesis by cholera toxin.     

The stimulatory guanine-nucleotide regulatory unit of adenylate cyclase from bovine cerebral cortex. ADP-ribosylation and purification.

Hormonal stimulation of adenylate cyclase from bovine cerebral cortex is mediated by a guanine-nucleotide regulatory protein (Gs). This protein contains at least three polypeptides: a guanine nucleotide-binding alpha s component and a beta X gamma component, which modulates the function of alpha s. The alpha s component from many tissues can be ADP-ribosylated with cholera toxin, but has been unusually difficult to modify in brain. We have improved incorporation of ADP-ribose by including isonicotinic acid hydrazide to inhibit the potent NAD glycohydrolase activity of brain. ADP-ribosylation is further improved by addition of detergent to render the substrates accessible and 20 mM-EDTA to chelate metal ions. Although Mg2+ is absolutely required for activation of adenylate cyclase by the GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), it is not obligatory for p[NH]ppG-stimulated ADP-ribosylation by cholera toxin. Under these conditions, the ADP-ribosylation of brain membranes is not enhanced by a cytosolic protein. We find that there are two major sizes of brain alpha s, which we have named 'alpha sL', with an apparent Mr of 42,000-45,000, and 'alpha sH' with an apparent Mr of 46,000-51,000 depending on the gel-electrophoretic system used. The alpha sL and alpha sH components can incorporate different amounts of ADP-ribose depending on the reaction conditions, so that one or the other may appear to predominate. Thus we show that incomplete ADP-ribosylation by cholera toxin is not a good indication of the relative amounts of alpha s units. Functionally, however, both forms of alpha s appear to be similar. Both forms associate with the catalytic unit of adenylate cyclase, but neither of them does so preferentially. There is an excess of each of them over the amount associated with catalytic unit. We have now substantially purified Gs from brain by a modification of the method of Sternweis et al. [(1981) J. Biol. Chem. 256, 11517-11526] as well as by a new, simplified, procedure. On SDS/polyacrylamide-gel electrophoresis, the purified brain Gs contains both the 45 and 51 kDa alpha s polypeptides revealed by ADP-ribosylation and a beta X gamma component. Activation of purified alpha s by guanine nucleotides or fluoride can be reversed by addition of purified beta X gamma component. The activated form of purified brain Gs has an Mr of 49,000 as determined by hydrodynamic measurements, which is consistent with the idea that the active form of brain Gs is the dissociated one.     

Polyphosphoinositide labeling in rat liver plasma membranes is reduced by preincubation with cholera toxin

Incubation of a crude rat liver plasma membrane preparation with [gamma-32P]ATP resulted in a rapid Mg2+-dependent incorporation of 32P into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Preincubation of the membranes with cholera toxin under ADP-ribosylating conditions reduced the labeling of the polyphosphoinositides. This action of cholera toxin required NAD+ and guanine nucleotides, was dose-dependent with respect to cholera toxin, and could not be mimicked by cAMP. It therefore appears that ADP-ribosylation of the stimulatory guanine nucleotide-binding regulatory protein of adenylate cyclase, or another G-protein, in rat liver plasma membranes affects the activity of enzymes in the polyphosphoinositide pathway.     

Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs.

Coupling of the three alpha 2-adrenergic receptor (alpha 2AR) subtypes to Gi and Gs was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the alpha 2AR agonist UK-14304, alpha 2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, alpha 2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of alpha 2C10 to both Gi and Gs. To isolate functional Gs and Gi coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only alpha 2C10-mediated inhibition (approximately 50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only alpha 2C10-mediated stimulation (approximately 60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl-terminal portion of Gs alpha blocked this functional alpha 2AR.Gs coupling to the same extent as that found for beta 2AR.Gs coupling. In addition to functional Gs coupling, we also verified direct, agonist-dependent, physical coupling of alpha 2AR to Gs alpha. In agonist-treated membranes, an agonist-receptor-Gs alpha complex was immunoprecipitated with a specific alpha 2C10 antibody, and the Gs component identified by both western blots using Gs alpha antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the alpha 2AR subtypes, we investigated whether G-protein coupling was subtype-selective, using UK-14304 and cells with the same alpha 2AR expression levels (approximately 5 pmol/mg). Coupling to Gi was equivalent for alpha 2C10, alpha 2C4, and alpha 2C2: 53.4 +/- 8.8% versus 54.9 +/- 1.0% versus 47.6 +/- 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to Gs were found between the three alpha 2AR subtypes: stimulation of adenylyl cyclase was 57.9 +/- 6.3% versus 30.7 +/- 1.1% versus 21.8 +/- 1.7% for alpha 2C10, alpha 2C4, and alpha 2C2, respectively. Thus, alpha 2AR have the potential to couple physically and functionally to both Gi and Gs; for Gi coupling we found a rank order of alpha 2C10 = alpha 2C4 = alpha 2C2, while for Gs coupling, alpha 2C10 greater than alpha 2C4 greater than alpha 2C2.     

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.     

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.     

Effects of guanine nucleotides on cholera toxin catalyzed ADP-ribosylation in rat adipocyte plasma membranes

ADP-ribosylation of rat adipocyte plasma membrane proteins was investigated following incubation of membranes with [alpha-32P]NAD and cholera toxin in the presence and absence of various guanine nucleotides. In membranes incubated without guanine nucleotides, cholera toxin induced incorporation of 32P into three discrete proteins of 48, 45, and 41 kDa. In membranes containing 100 microM GTP or GDP, toxin-catalyzed incorporation of 32P into the 41-kDa protein was inhibited. GMP and Gpp(NH)p (100 microM) allowed moderate incorporation of 32P into the 41-kDa protein. Toxin-catalyzed labeling of all proteins was rapid, reaching maximal levels between 5 and 10 min. Toxin-catalyzed ADP-ribosylation of the 48- and 45-kDa proteins was stimulated by GTP, reaching maximal levels at 10(-5) M GTP. Inhibition of toxin-dependent labeling of the 41-kDa protein required GTP concentrations above 10(-7) M with complete inhibition occurring between 10(-5) and 10(-4) M GTP. Cholera toxin catalyzed ADP-ribosylation was increased up to 2-fold in membranes supplemented with adipocyte cytosol. These results indicate that cholera toxin catalyzes ADP-ribosylation of three distinct adipocyte plasma membrane proteins, each of which is regulated by the amount and type of added guanine nucleotides.     

ADP-ribosylation of Gs promotes the dissociation of its alpha and beta subunits

We have utilized purified reactants and cofactors to examine the form of the stimulatory guanine nucleotide-binding regulatory component (Gs) of adenylate cyclase that serves as a substrate for ADP-ribosylation by cholera toxin; we have also investigated some of the consequences of that covalent modification. Activation of Gs with nonhydrolyzable analogs of GTP, which causes dissociation of its subunits, completely inhibits the toxin-catalyzed covalent modification. However, this effect cannot be explained by subunit dissociation, since activation of Gs by fluoride is not inhibitory and ADP ribosylation of the alpha (45,000-Da) subunit of Gs proceeds equally well in the presence and absence of the beta (35,000-Da) subunit. ADP-ribosylation of the alpha subunit of Gs decreases its apparent affinity for the beta subunit; however, the affinity of alpha and ADP-ribosyl-alpha for GTP appear to be approximately the same. ADP-ribosylation of Gs thus promotes the dissociation of its alpha and beta subunits. This effect may account for or contribute to the activation of adenylate cyclase by cholera toxin.     

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.     

Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human alpha 2-C10 adrenergic receptor into rat 1 fibroblasts. Evidence for the direct interaction of a single receptor with two pertussis toxin-sensitive G-proteins, Gi2 and Gi3.

A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of [3H]yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study. When cholera toxin-catalyzed ADP-ribosylation was performed with [32P]NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha. Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells. Dose response curves for U.K.14304 enhancement of cholera toxin-labeling of the 40-kDa polypeptide(s) and stimulation of high affinity GTPase activity were identical. By contrast, U.K.14304 was ineffective in either assay in membranes from the 4D cells, demonstrating this effect to be dependent upon receptor activation. Furthermore, the alpha 2 receptor antagonist yohimbine blocked all effects of U.K.14304. The agonist promotion of cholera toxin-catalyzed ADP-ribosylation of Gi was completely blocked by guanine nucleotides. Whether GDP or GDP + fluoroaluminate (as a mimic of GTP) was used, blockade of the agonist effect was complete and indeed both conditions prevented agonist-independent labeling by cholera toxin of the 40-kDa polypeptide(s). Mg2+ produced an agonist-independent cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s) but even in the presence of [Mg2+], agonist-stimulation of cholera toxin-labeling of the 40-kDa polypeptide(s) was observed and was additive with the effect of [Mg2+]. Agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi was completely attenuated by pretreatment of the cells with pertussis toxin, which prevents contact between receptors and G-proteins which are substrates for this toxin. By contrast, pretreatment of the cells with concentrations of cholera toxin able to "down-regulate" essentially all of the membrane-associated Gs alpha did not prevent agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi.(ABSTRACT TRUNCATED AT 400 WORDS)     

Microinjection of a 19-kDa guanine nucleotide-binding protein inhibits maturation of Xenopus oocytes

ADP-ribosylation factors (ARFs) are 19-21-kDa proteins purified from bovine brain that bind guanosine 5'-triphosphate (GTP). They exhibit GTP-dependent activity as activators of cholera toxin-catalyzed ADP-ribosylation of the alpha-subunit of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system (Gs alpha). ARF, which interacts directly with the catalytic subunit of cholera toxin, has no known physiologic role. Intracellular microinjection of ARF was employed to investigate the effect of ARF on progesterone- and insulin-stimulated maturation of Xenopus oocytes. Maturation was inhibited by injection of ARF 3-8 h before exposure of oocytes to progesterone or insulin. ARF inhibition was dependent on progesterone concentration but not on insulin concentration. Inhibition was enhanced by concomitant injection of GTP and to a greater extent by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) which, in the absence of ARF, inhibited somewhat at early time points. The demonstration of this effect of ARF on both progesterone- and insulin-stimulated oocyte maturation may provide a clue to the physiologic role of this guanine nucleotide-binding protein.     

Adenylyl cyclase and G-proteins in Phytomonas

Phytomonas sp. membranes have an adenylyl cyclase activity which is greater in the presence of Mn2+ than with Mg2+. The Mg2+ and Mn2+ activity ratio varies from one membrane preparation to another, suggesting that the adenylyl cyclase has a variable activation state. A[35S]GTP-gamma-S-binding activity with a Kd of 171 nM was detected in Phytomonas membranes. Incubation of these membranes with activated cholera or pertussis toxin and [adenylate 23P]NAD+ led to incorporation of radioactivity into bands of about 40-44 kDa. Crude membranes were electrophoresed on SDS-polyacrylamide gels and analyzed, by Western blotting, with the 9188 anti-alpha[s] antibody and the AS/7 antibody (anti-alpha[i], anti-alpha[i1], and anti-alpha[i2]. These procedures resulted in the identification of polypeptides of approximately 40-44 kDa. Phytomonas adenylyl cyclase could be activated by treatment of membrane preparations with cholera toxin, in the presence of NAD+, while similar treatment with pertussis toxin did not affect this enzyme activity. These studies indicate that in Phytomonas, adenylyl cyclase activity is coupled to an unknown receptor entity through G alpha[s] proteins.     

Functional modification by cholera-toxin-catalyzed ADP-ribosylation of a guanine-nucleotide-binding regulatory protein serving as the substrate of pertussis toxin.

The alpha subunits of Gi (Gi alpha) and Gs (guanine-nucleotide-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in differentiated HL-60 cell membranes upon stimulation of chemotactic receptors by fMLF (fM, N-formylmethionine). The ADP-ribosylation site of Gi alpha modified by cholera toxin appeared to be different from that modified by pertussis toxin [Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M. & Katada, T. (1989) J. Biol. Chem. 264, 21,394-21,400]. This allowed us to investigate how the two types of ADP-ribosylation influence the function of the signal-coupling protein. The major findings observed in HL-60 cell membranes, where the same Gi alpha molecule was ADP-ribosylated by treatment of the membranes with either toxin, are summarized as follows. (a) More fMLF bound with a high affinity to cholera-toxin-treated membranes than to the control membranes. The high-affinity binding was, however, not observed in pertussis-toxin-treated membranes. (b) Although fMLF stimulated guanine nucleotide binding and GTPase activity in control membranes, stimulation was almost completely abolished in pertussis-toxin-treated membranes. In contrast, fMLF-dependent stimulation of GTPase activity, but not that of guanine nucleotide binding was attenuated in cholera-toxin-treated membranes. (c) Gi alpha, once modified by cholera toxin, still served as a substrate of pertussis-toxin-catalyzed ADP-ribosylation; however, the ADP-ribosylation rate of modified Gi was much lower than that of intact Gi. These results suggested that Gi ADP-ribosylated by cholera toxin was effectively capable of coupling with fMLF receptors, resulting in formation of high-affinity fMLF receptors, and that hydrolysis of GTP bound to the alpha subunit was selectively impaired by its ADP-ribosylation by cholera toxin. Thus, unlike the ADP-ribosylation of Gi by pertussis toxin, cholera-toxin-induced modification would be of great advantage to the interaction of Gi with receptors and effectors that are regulated by the signal-coupling protein. This type of modification might also be a candidate for unidentified G proteins which were less sensitive to pertussis toxin and appeared to be involved in some signal-transduction systems.