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.     

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.     

Limulus ventral photoreceptors contain a homologue of the alpha-subunit of mammalian Ns

Membranes from ventral photoreceptors of Limulus were incubated with cholera toxin and [32P]NAD+. Cholera toxin catalyzes a specific ADP-ribosylation of a 43-kDa peptide from Limulus ventral photoreceptors. Possible homologies between the 43-kDa peptide of Limulus and the alpha-subunits of mammalian stimulatory, guanine nucleotide-binding regulatory component of adenylate cyclase (Ns) were investigated by comparing the electrophoretic patterns of proteolytic fragments derived from each of these peptides that are radiolabeled by [32P]NAD+ and cholera toxin. Evidence is provided for structural homology between this invertebrate peptide and mammalian Ns.     

Stimulation by cholera toxin of ADP-ribosylation of membrane proteins, adenylate cyclase and insulin release in pancreatic islets

In rat pancreatic islet membranes exposed to [alpha-32P]NAD, cholera toxin stimulated the labelling of three peptides with Mr close to 22 000, 42 000 and 48 000, respectively. In the islets, the toxin-stimulated ADP-ribosylation of the heavy form of the Ns alpha-subunit predominated over that of the light form, in mirror image of the situation found in the exocrine pancreas. When intact islets were preincubated with cholera toxin, the adenylate cyclase activity of a subcellular particulate fraction was increased. The responsiveness of adenylate cyclase to GTP was also augmented, but that to NaF was decreased. In intact islets, the production of cyclic AMP and the glucose-stimulated release of insulin were also enhanced after pretreatment with cholera toxin. These findings reveal the presence in pancreatic islets of the guanyl nucleotide regulatory protein of adenylate cyclase, with an unusual predominance of the heavy form of the Ns alpha-subunit.     

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.     

The activation of adenylate cyclase from small intestinal epithelium by cholera toxin

ADP-ribosylation of membrane proteins from rabbit small intestinal epithelium was investigated following incubation of membranes with [32P]NAD and cholera toxin. Cholera toxin catalyzes incorporation of 32P into three proteins of 40 kDA, 45 kDa and 47 kDa located in the brush-border membrane. In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and [32P]NAD. The modification of membrane proteins from brush border occurred in spite of the virtual absence in these membranes of adenylate cyclase activatable either by cholera toxin, vasoactive intestinal peptide (VIP) or fluoride. The three agents activated adenylate cyclase when crude plasma membrane were used. Cholera toxin activated fivefold at 10 micrograms/ml. Vasoactive intestinal peptide activated at concentrations from 10-300 nM, the maximal stimulation being sixfold. Fluoride activated 10-fold at 10 mM. When basal lateral membranes were assayed for adenylate cyclase it was found that, with respect to the crude membranes, the specific activity of fluoride-activated enzyme was 3.3-fold higher, VIP stimulated enzyme was maintained while cholera-toxin-stimulated enzyme showed half specific activity. Moreover, while fluoride stimulated ninefold and VIP stimulated fivefold, cholera toxin only stimulated twofold at the highest concentration. The results suggest that the activation by cholera toxin of adenylate cyclase located at the basal lateral membrane requires ADPribosylation of proteins in the brush border membrane.     

Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins

Cholera toxin catalyzes transfer of radiolabel from [32P]NAD+ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of Mr = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (Mr = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and [32P]NAD+ caused radiolabeling of purified microtubule and intermediate filament proteins.     

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.     

Thyroliberin receptor binding and adenylyl cyclase activation in cultured prolactin-producing rat pituitary tumor cells (GH cells)

A thyroliberin (TRH)-responsive particulate bound adenylyl cyclase is present in two rat anterior pituitary tumor cell strains (GH4C1 and GH3) which synthesize and secrete prolactin. At a given Mg2+ concentration, ATP and the guanyl nucleotides GTP and guanyl 5'-yl-imidodiphosphate (GMP-P(NH)P) caused a dose-dependent increase in adenylyl cyclase activity. The maximum response to thyroliberin occurred with ATP and GTP at concentrations above 0.30 mM and 2 microM, respectively. The maximal stimulatory effect of thyroliberin on adenylyl cyclase activity was 2-fold in the presence of GTP. GMP-P(NH)P increased the basal enzyme activity 4- to 10-fold over and above that of equimolar concentrations of GTP but supported poorly the TRH-induced response. Mg2+ caused a dose-dependent increase in the basal enzyme activity and reduced TRH and fluoride-induced responses. Also, Mn2+ and Co2+ stimulated the basal adenylyl cyclase activity while Zn2+, Ca2+, and Cu2+ inhibited the enzyme, and neither cations supported the TRH response. Half-maximal stimulation of the adenylyl cyclase by TRH and half-maximum binding of [3H]TRH to membranes at 35 degrees C were 102 and 56 nM, respectively. Pretreatment with TRH decreased the apparent Vmax of the enzyme and the maximal binding of [3H]TRH. Of 6 TRH analogs tested, only one was able to displace [3H]TRH from its receptor and to increase the adenylyl cyclase activity. We suggest that adenylyl cyclase activation is an early event in the stimulus secretion coupling between TRH and prolactin-producing GH cells.     

Regulation of glucagon receptor binding. Lack of effect of Mg and preferential role for GDP

The effects of Mg2+ and guanine nucleotides on glucagon binding to its receptor were studied using [125I-Tyr10]monoiodoglucagon. Contrary to findings with beta-adrenergic receptors, high affinity binding of the stimulatory hormone was not dependent on Mg2+ and low affinity binding could be obtained on nucleotide addition regardless of presence of Mg2+. GDP, guanyl-5'-yl thiophosphate (GDP beta S), GTP, and guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) were all able to induce low affinity hormone binding. Since the Ns component of adenylyl cyclase, with which the receptor interacts, is inactive in stimulating the catalytic component C of adenylyl cyclase in the absence of Mg2+, both before and after GDP addition, it is suggested that Ns has at least two domains that change conformation independently of each other: a r domain, that interacts with the receptor and confers to it high affinity binding, and a c domain, that interacts with the catalyst C and stimulates it. It is suggested further that Ns is r+c- when stabilizing the receptor in its conformation with high affinity for hormone, and r-c- when under the influence of GDP which results in the receptor adopting the conformation that exhibits low affinity for the hormone. Comparison of potencies of the four nucleotides to induce low affinity binding showed that GDP and GDP beta S were equipotent and 10 times more potent than GTP and 100 times more potent than GMP-P(NH)P. Under the conditions used it was impossible to substantiate that the effects of GTP or GMP-P(NH)P were not due to formation of GDP from GTP or presence of GDP-like material in GMP-P(NH)P. It is suggested that, contrary to widely held opinions, GDP and GDP-like compounds, and not GTP or its analogs, are responsible for the lowering of the affinity of adenylyl cyclase stimulating receptors for their hormones or agonists. Furthermore, the experiments suggest that the c+ conformation of the c domain of Ns co-exists with the r+ and not the r- conformation of its r domain.     

Physical and functional association of follitropin receptors with cholera toxin-sensitive guanine nucleotide-binding protein

We have previously reported detergent (Triton X-100) solubilization of a follitropin (FSH) receptor-rich fraction from light membranes of bovine testis that responded to exogenous FSH by activation of adenylate cyclase (Dattatreyamurty, B., Schneyer, A., and Reichert, L. E., Jr. (1986) J. Biol. Chem. 261, 13104-13113). Upon gel filtration of the detergent-extract through Sepharose-6B, two fractions were separated. Each specifically bound [3H]guanosine 5'-imidotriphosphate (Gpp(NH)p) and had guaninetriphosphatase (GTPase) activity. Of these, one fraction (6B-Fraction-1) also bound radioiodinated human follitropin (hFSH), indicating a coelution of the nucleotide-binding protein with receptor. The other fraction (6B-Fraction-2) did not contain detectable FSH receptor activity. Several lines of evidence suggest that 6B-Fraction-1 is a complex consisting of FSH receptor and a guanine nucleotide regulatory protein, probably Ns. 1) The GTP-binding and FSH-binding activities of 6B-Fraction-1 were retained by a GTP-affinity column, and their retention by the affinity matrix could be prevented by simultaneous addition of free Gpp(NH)p. 2) When exogenous GTP was added to 6B-Fraction-1, binding of 125I-hFSH was reduced compared to controls lacking exogenous GTP. This effect of GTP was highly specific and noncompetitive, indicating that GTP did not bind to receptor. In addition, the affinity of receptor for FSH was decreased, and the rate and degree of dissociation of bound labeled FSH from receptor were increased in the presence of exogenous GTP, each in concentration-dependent manner. 3) Exposure of 6B-Fraction-1 to higher concentration of Triton X-100 reduced significantly the receptor-associated GTP-binding activity and also rendered the hormone-binding activity insensitive to GTP. 4) Treatment of highly purified testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminated the ability of GTP to modulate the 125I-hFSH binding to receptor. 5) After cholera toxin-induced [32P]ADP-ribosylation of testis membranes, a major peak of radioactivity (presumably Ns) was coeluted with FSH receptor activity from the Sepharose-6B column. These results and the observation that the effect of GTP is noncompetitive at FSH receptor level suggest that FSH binding inhibition and the increased rate of hormone dissociation from receptor were the result of GTP interaction with a guanine nucleotide regulatory protein, probably Ns, which itself was functionally associated with the FSH receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determination of G-protein levels, ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db) mice

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of Gi alpha-2, Gi alpha-3 and G-protein beta-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of Gs alpha-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of Gs alpha-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 microM), GTP (100 microM), p[NH]ppG (100 microM), NaF (10 mM) and glucagon (10 microM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 microM) was lower by about 22%. Lower concentrations (EC50-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC50-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional Gi activity. The lower levels of expression of G-protein beta-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

Fasting increases fat cell adenylate cyclase sensitivity to stimulatory agonists through enhanced ability of the stimulatory regulatory component Ns to dissociate

In rat fat cell membranes, a 72-hour fasting fails to alter the adenylate cyclase stimulatory responses to Mn2+, forskolin and cholera toxin and the cholera toxin catalyzed [alpha-32P] ADP ribose incorporation into the Mr = 42,000 and 46,000/48,000 alpha s peptides of Ns. In contrast, dose-response curves for GTP-stimulation of basal and isoproterenol-stimulated adenylate cyclase display higher maximal responses in fasted rats under conditions restraining (2 mM Mg2+) but not promoting (10 mM Mg2+) the dissociation of Ns. Moreover, at 10 mM Mg2+, the sensitivity of isoproterenol-stimulated adenylate cyclase to GTP is clearly increased in fasted rats. Finally, fasting reduces by 40% the lag-phase of adenylate cyclase activation by Gpp(NH)p. Taken together, these results are consistent with the hypothesis that the permissive effect of fasting on the fat cell adenylate cyclase response to stimulatory agonists is related to increased ability of Ns and the ternary H.R.Ns. complex to dissociate which is likely due to enhanced Ns affinity for guanine nucleotides.     

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 mM) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34-54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5'-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the beta-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25-800 mM) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li+ greater than Na+ greater than K+. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (Ns and Ni) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%. Ni did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 mM) increased both the Kd for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the Kact for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (-)-[125I]iodopindolol binding sites or the Kact for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, Ns from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of Ni from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.     

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.     

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.     

Hepatic adenylate cyclase. Development-dependent coupling to the beta-adrenergic receptor in the neonate

Guanine nucleotide-dependent modulation of agonist binding to the beta-receptor reflects coupling of the receptor to the nucleotide regulatory protein. Similarly, guanine nucleotide-dependent stimulation of adenylate cyclase can be used as an index of coupling between the regulatory protein and the catalytic unit of the cyclase. Using both approaches we have studied coupling in the beta-adrenergic receptor-adenylate cyclase system in rabbit liver during neonatal development. With [3H]dihydroalprenolol as ligand, the Bmax was relatively unchanged (200-300 fmol/mg of protein) between birth and end of day 1 and was similar to adult values. Guanyl-5'-yl imidodiphosphate-dependent shift in agonist (l-isoproterenol) competition curves was biphasic, decreasing from 10-fold in membranes isolated from animals at term to about 6-fold in membranes from 6-h-old neonates, and increasing progressively in older animals to a maximal measurable value of 42-fold in the adult. The ability of guanyl-5'-yl imidodiphosphate, GTP, GTP plus isoproterenol, NaF, or forskolin to activate adenylate cyclase was also biphasic and age-dependent. With Mn2+ the measured activity was not at any time greater than the activity at term. Pretreatment of membranes with cholera toxin resulted in differential levels of enhancement of adenylate cyclase activity wherein much lower enhancement was observed in membranes from neonatal animals. With [32P]NAD as substrate, cholera toxin-catalyzed ADP-ribosylation of membranes indicated development-dependent accumulation of Ns peptides. From these results we suggest that there is a decreased efficiency in the coupling of the beta-adrenergic receptor to hepatic adenylate cyclase in early neonatal life. The molecular basis for the biphasic nature of the coupling is presently unclear.     

Determination of G-protein levels,ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db)

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of G_i α-2, G_i α-3 and G-protein β-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of G_s α-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of G_s α-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 μM), GTP (100 μM), p[NH]ppG (100 μM), NaF (10 mM) and glucagon (10 μM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 μM) was lower by about 22%. Lower concentrations (EC₅₀-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC₅₀-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional G_i activity. The lower levels of expression of G-protein β-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

Treatment of oocyte membranes with the 2',3'-dialdehyde of guanosine triphosphate reduces progesterone inhibition of adenylyl cyclase

Treatment of Xenopus laevis membranes with the 2',3'-dialdehyde of GTP (dial GTP) drastically inhibits their adenylyl cyclase activity. Optimal inhibition is obtained by treatment with 1 mM dial GTP for 1h at 32 degrees C. Using guanyl-5'-yl imidodiphosphate, F-, forskolin and Mn2+ as activators of the enzyme it can be concluded that dial GTP preferentially reacts with the stimulatory subunit (Ns) and slightly with the catalytic subunit. Dial GTP treatment greatly reduces the inhibition of adenylyl cyclase by progesterone. Pure exogenous Ns stimulates the enzyme but does not restore progesterone inhibition. Treatment with dial [alpha-32P]GTP labels several membrane proteins some of which have similar Mr to Ns and Ni.     

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.