The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Properties and function of the purified protein

Treatment of membranes with islet activating protein (IAP), a toxin from Bordetella pertussis, results in abolition of GTP-dependent, receptor-mediated inhibition of adenylate cyclase. This appears to result from IAP-catalyzed ADP-ribosylation of a 41,000-Da membrane-bound protein. A protein with 41,000- and 35,000-Da subunits has been purified from rabbit liver membranes as the predominant substrate for IAP. This protein has now been shown to be capable of regulating membrane-bound adenylate cyclase activity of human platelets under various conditions. The characteristics of the actions of the IAP substrate are as follows. 1) Purified 41,000/35,000-Da dimer is capable of restoring the inhibitory effects of guanine nucleotides and the alpha 2-adrenergic agonist, epinephrine, on the adenylate cyclase activity of IAP-treated membranes. 2) The subunits of the dimer dissociate in the presence of guanine nucleotide analogs or A1(3+), Mg2+, and F-. The 41,000-Da subunit has a high affinity binding site for guanine nucleotides. 3) The resolved 35,000-Da subunit of the dimer mimics guanine nucleotide- and epinephrine-induced inhibition of adenylate cyclase. 4) The resolved (unliganded) 41,000-Da subunit stimulates adenylate cyclase activity and relieves guanine nucleotide- +/- epinephrine-induced inhibition of the enzyme. In contrast, the GTP gamma S-bound form of the 41,000-Da subunit inhibits adenylate cyclase activity, although with lower apparent affinity than does the 35,000-Da subunit. 5) The 35,000-Da subunit increases the rate of deactivation of Gs, the stimulatory regulatory protein of adenylate cyclase. In contrast, the 41,000-Da subunit can interact with Gs and inhibit its deactivation. These data strongly suggest that the IAP substrate is another dimeric, guanine nucleotide-binding regulatory protein and that it is responsible for inhibitory modulation of adenylate cyclase activity.     

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and the inhibition of adenylate cyclase in S49 lymphoma cyc- and wild type membranes

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta subunits are functionally indistinguishable. GTP-dependent hormonal inhibition of adenylate cyclase and that caused by guanine nucleotide analogs seem to result from dissociation of the subunits of Gi. Such inhibition can be explained by reduction of the concentration of the free alpha subunit of Gs as a result of its interaction with the beta subunit of Gi in normal Gs-containing membranes. However, inhibition in S49 lymphoma cyc- cell membranes presumably cannot be explained by the Gi-Gs interaction, since the activity of the alpha subunit of Gs is not detectable in this variant. Several characteristics of Gi-mediated inhibition of adenylate cyclase have been studied in both S49 cyc- and wild type membranes. There are several similarities between inhibition of forskolin-stimulated adenylate cyclase by guanine nucleotides and somatostatin in cyc- and wild type membranes. 1) Somatostatin-induced inhibition of the enzyme is dependent on GTP; nonhydrolyzable GTP analogs are also effective inhibitors. 2) The effect of guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) is essentially irreversible, and somatostatin accelerates GTP gamma S-induced inhibition. 3) Inhibition of adenylate cyclase by somatostatin or Gpp(NH)p is attenuated by treatment of cells with islet-activating protein (IAP). 4) Both cyc- and wild type membranes contain the substrate for IAP-catalyzed ADP-ribosylation (the alpha subunit of Gi). 5) beta Subunit activity in detergent extracts of membranes is liberated by exposure of the membranes to GTP gamma S. The alpha subunit of Gi in such extracts has a reduced ability to be ADP-ribosylated by IAP, which implies that this subunit is in the GTP gamma S-bound form. The resolved subunits of Gi have been tested as regulators of cyc- and wild type adenylate cyclase under a variety of conditions. The alpha subunit of Gi inhibits forskolin-stimulated adenylate cyclase activity in cyc-, while the beta subunit stimulates; these actions are opposite to those seen with wild type membranes. The inhibitory effects of GTP plus somatostatin (or GTP gamma S) and the alpha subunit of Gi are not additive in cyc- membranes. In wild type, the inhibitory effects of the hormone and GTP gamma S are not additive with those of the beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Amiloride interacts with guanine nucleotide regulatory proteins and attenuates the hormonal inhibition of adenylate cyclase

The effect of amiloride on the hormonal regulation of adenylate cyclase was studied in the rat anterior pituitary. The diuretic did not alter basal adenylate cyclase but augmented the enzyme activity in an irreversible manner in the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S) stimulated adenylate cyclase at lower concentrations and inhibited at higher concentrations. Amiloride treatment enhanced the stimulatory and abolished the inhibitory phase of GTP gamma S action. In addition, amiloride also attenuated the inhibitory effects of atrial natriuretic factor (ANF 99-126) and angiotensin II on cAMP levels and adenylate cyclase activity. On the other hand, amiloride showed an additive effect on the stimulation exerted by corticotropin-releasing factor and vasoactive intestinal peptide on adenylate cyclase in anterior pituitary and on isoproterenol-stimulated cAMP levels in cultured vascular smooth muscle cells. Pertussis toxin, in the presence of [alpha-32 P]NAD, catalyzed the ADP-ribosylation of two protein bands of Mr 41,000 and 39,000, referred to as Gi and Go, respectively, in the anterior pituitary, and 40,000-Da protein in the aorta, referred to as Gi. Amiloride treatment inhibited the labeling of all these bands in a concentration- and time-dependent manner. Similarly, the pertussis toxin-catalyzed ADP-ribosylation of purified Gi from bovine brain was also inhibited by amiloride treatment. However, amiloride had no significant effect on the cholera toxin-catalyzed ADP-ribosylation of Gs. These data suggest that amiloride interacts with the guanine nucleotide regulatory proteins Gi and Go. Modification of Gi results in the attenuation of hormone-induced adenylate cyclase and cAMP inhibition. However, the interaction between amiloride and Go and the consequent Ca2+ mobilization and phosphatidylinositol turnover have to be investigated.     

The subunits of the stimulatory regulatory component of adenylate cyclase. Resolution, activity, and properties of the 35,000-dalton (beta) subunit

The stimulatory guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase is activated by exposure to guanine nucleotide analogs or to Al3+ + F-. Activated G/F can reconstitute adenylate cyclase activity when mixed with the catalytic moiety of the enzyme system in the absence of an effective free concentration of stimulatory ligand. Activation is explained by dissociation of the alpha (45,000-Da) and beta (35,000-Da) subunits of G/F. The beta subunit of G/F facilitates reversal of the activated state of the regulatory protein. This phenomenon, which has been exploited as an assay for the resolved beta subunit, has the following properties. 1) Addition of the resolved beta subunit to fluoride-activated G/F increases the initial rate of deactivation from a t 1/2 of 10 min to less than 0.5 min. 2) The enhancement of the rate of deactivation is a saturable process with a K 1/2 value of 60 ng/ml (approximately 2 nM). 3) G/F does not display beta subunit activity unless the alpha subunit has been inactivated or the subunits have been resolved. beta Subunit activity is measurable in detergent extracts of rabbit liver membranes or plasma membranes from S49 cell clones. The activity in such extracts is similar to that found with purified G/F, in that incubation at 30 degrees C in the presence of Mg2+ is required for its expression. However, cyc-, UNC, and H21a (S49 cell mutants with deficient or altered G/F activity) have amounts of beta subunit activity similar to that found in wild type S49 cells. Furthermore, the amount of beta subunit activity exceeds by 5- to 10-fold the amount expected based on the quantity of G/F in wild type extracts. All of the beta subunit activity in detergent extracts of liver membranes can be purified as a 35,000-Da polypeptide that is indistinguishable from the beta subunit of G/F. The beta subunit activity in extracts of cyc- membranes is expressed after incubation with guanine nucleotide analogs, implying association of the beta subunit with a GTP-binding protein. By analysis of the chromatographic behavior of G/F and the recently identified 41,000/35,000-Da heterodimeric substrate for the islet-activating protein from Bordetella pertussis, we have identified the 41,000-Da subunit of the substrate for islet-activating protein as the GTP-binding protein with which the majority of the beta subunit activity associates. These data have direct bearing on the mechanisms of hormonal activation and inhibition of adenylate cyclase.     

The guanine nucleotide-binding regulatory component of adenylate cyclase in human erythrocytes

The guanine nucleotide-binding regulatory component of adenylate cyclase (G/F) has been purified from human erythrocyte membranes. It is composed of two major polypeptides with molecular weights of 35,000 and 45,000. When cyc- S49 lymphoma cell plasma membranes are reconstituted with purified human erythrocyte G/F, stimulation of adenylate cyclase by beta-adrenergic agonists, guanine nucleotides, and fluoride is restored. Binding of GTP gamma S to human erythrocyte G/F and GTP gamma S-mediated activation of the protein are closely correlated. The agreement between the apparent dissociation constants for these two reactions suggests that the measured binding site is identical to the site responsible for activation. A 41,000-dalton protein has been identified as a contaminant of preparations of G/F that have been purified by four successive chromatographic steps. This protein serves as a specific substrate for ADP-ribosylation and labeling by islet activating protein (IAP) and [32P]NAD, and it appears to contribute an additional high-affinity guanine nucleotide binding site to such preparations.     

The regulatory components of adenylate cyclase and transducin. A family of structurally homologous guanine nucleotide-binding proteins

G/F and transducin are guanine nucleotide-binding regulatory proteins that mediate activation of adenylate cyclase and of a rod outer segment cyclic GMP-specific phosphodiesterase, respectively. The substrate for islet-activating protein is a third guanine nucleotide-binding protein that is postulated to mediate inhibition of adenylate cyclase. The extent of structural homology among subunits of all three proteins was examined by analyses of amino acid compositions and electrophoretic patterns of proteolytic peptides. The lower molecular weight subunits (beta subunits; Mr = 35,000) of these proteins have identical amino acid compositions and yield similar peptides upon proteolysis with Staphylococcus aureus V8 protease and elastase. The higher molecular weight subunits (alpha subunits; Mr = 39,000, 41,000, and 45,000) are also similar to each other in these respects. Similarity between the subunits of transducin and the islet-activating protein (IAP) substrate is especially evident. Substantial differences do, however, exist between the lower and higher molecular weight subunits within each protein. In addition, evidence was obtained that the 41,000-Da subunit of the IAP substrate is not derived from the 45,000-Da subunit of G/F. It is concluded that transducin, the IAP substrate, and G/F represent a family of structurally homologous guanine nucleotide-binding regulatory proteins.     

Guanine nucleotide activation and inhibition of adenylate cyclase as modified by islet-activating protein, pertussis toxin, in mouse 3T3 fibroblasts

Guanine nucleotide regulation of membrane adenylate cyclase activity was uniquely modified after exposure of 3T3 mouse fibroblasts to low concentrations of islet-activating protein (IAP), pertussis toxin. The action of IAP, which occurred after a lag time, was durable and irreversible, and was associated with ADP-ribosylation of a membrane Mr = 41,000 protein. GTP, but not Gpp(NH)p, was more efficient and persistent in activating adenylate cyclase in membranes from IAP-treated cells than membranes from control cells. GTP and Gpp(NH)p caused marked inhibition of adenylate cyclase when the enzyme system was converted to its highly activated state by cholera toxin treatment or fluoride addition, presumably as a result of their interaction with the specific binding protein which is responsible for inhibition of adenylate cyclase. This inhibition was totally abolished by IAP treatment of cells, making it very likely that IAP preferentially modulates GTP inhibitory responses, thereby increasing GTP-dependent activation and negating GTP-mediated inhibition of adenylate cyclase.     

Carbamylcholine inhibits beta-adrenergic receptor-coupled Gs protein function proximal to adenylate cyclase

The specific mechanism by which the inhibitory guanine nucleotide binding protein (Gi) mediates the inhibition of adenylate cyclase activity is still unclear. The subunit dissociation model, based on studies in purified or reconstituted systems, suggests that the beta gamma subunit, which is dissociated with activation of Gi, inhibits the function of the stimulatory guanine nucleotide binding protein (Gs) by reducing the concentration of the free alpha s subunit. In the present study, Gs protein function is determined by measuring cholera toxin-blockable, isoproterenol-induced increases in guanosine triphosphate (GTP) binding capacity to rat cardiac ventricle membrane preparations. Carbamylcholine totally inhibited this beta-adrenergic receptor-coupled Gs protein function. Pretreatment of the cardiac ventricle membrane with pertussis toxin prevented this muscarinic agonist effect. These results confirm the possibility of an inhibitory agonist-receptor coupled effect through Gi on Gs protein function proximal to the catalytic unit of adenylate cyclase in an intact membrane preparation.     

Inhibition of hormonally regulated adenylate cyclase by the beta gamma subunit of transducin.

Transducin (T), the GTP-binding protein of the retina activates the cGMP phosphodiesterase system, and presents analogies with the proteins GS and Gi which respectively mediate adenylate cyclase activation and inhibition by hormone receptors. These proteins are all comprised of an alpha subunit carrying the GTP-binding site and a beta gamma subunit made of two peptides. The beta peptide (35 kd) appears similar in the three proteins. We demonstrate here that purified T beta gamma inhibits adenylate cyclase from human platelet membranes. This inhibition was observed when adenylate cyclase was stimulated by GTP, prostaglandin E1 (PGE1), NaF and forskolin, but not when stimulated by GTP(gamma)S. In the presence of GTP and forskolin, the T beta gamma-induced maximal inhibition was not additive with the alpha 2-receptor-induced adenylate cyclase inhibition mediated by Gi. Both inhibitions were suppressed at high Mg2+ concentrations, which as also known to dissociate T beta gamma from T alpha-GDP. This suggests that these adenylate cyclase inhibitions are due to the formation of inactive complexes of GS alpha-GDP with T beta gamma or Gi beta gamma. T beta gamma-induced inhibition did not require detergent and could be suppressed by simple washing. T beta gamma effects are dependent on its concentration rather than on its total amount. This suggests that T beta gamma can operate in solution with no integration into the membrane. Similar inhibitory effects of T beta gamma are observed on adenylate cyclase from anterior pituitary and lymphoma S49 cell lines.     

Activation of the inhibitory GTP-binding protein of adenylate cyclase, Gi, by beta-adrenergic receptors in reconstituted phospholipid vesicles

beta-Adrenergic receptors and the inhibitory GTP-binding protein, Gi of the adenylate cyclase system were reconstituted into phospholipid vesicles by the method described previously for reconstituting receptors and the stimulatory GTP-binding protein, Gs (Brandt, D. R., Asano, T., Pedersen, S. E., and Ross, E. M. (1983) Biochemistry 22, 4357-4362). In the receptor-Gi vesicles, beta-adrenergic agonists stimulated both the high-affinity binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to Gi and GTPase activity to an extent similar to that observed in vesicles containing beta-adrenergic receptors and Gs. Stimulation required receptors and displayed appropriate beta-adrenergic specificity. The prior treatment of receptor-Gi vesicles with islet-activating protein (pertussis toxin) plus NAD markedly inhibited both the isoproterenol-stimulated binding of GTP gamma S and the isoproterenol-stimulated GTPase activity. No contamination of Gi by Gs was apparent. These data suggest that receptors that typically stimulate adenylate cyclase activity may also activate the inhibitory system, perhaps as one mechanism of desensitization.     

Synergistic inhibition of human platelet adenylate cyclase by stable GTP analogs and epinephrine

Adenylate cyclase inhibition by stable GTP analogs and their interaction with epinephrine were studied in human platelet membranes. Whereas basal enzyme activity was increased by these nucleotides, the stable GTP analogs decreased the adenylate cyclase activity stimulated by fluoride or forskolin by maximally 60 to 70%, with the potency order, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) greater than guanyl-5'-ylimidodiphosphate greater than guanyl-5'-ylmethylenediphosphate. The inhibition of the forskolin-stimulated enzyme by GTP gamma S was half-maximal at about 4 nM, occurred after a time lag period, which was inversely related to the GTP gamma S concentration, and was resistant to washing of the membranes. Prostaglandin E1-stimulated activity exhibited a biphasic response towards GTP gamma S, with activation occurring at low (1 nM) and inhibition at higher GTP gamma S concentrations. The inhibitory effect of GTP gamma S was competitively antagonized by GTP. This antagonism was prevented by epinephrine, which inhibited the stimulated platelet adenylate cyclase in the presence of GTP to the same degree as observed with GTP gamma S alone. In the absence of GTP, epinephrine largely diminished the time lag required for the inhibitory action of GTP gamma S. Furthermore, the decrease in final activity induced by GTP gamma S was amplified by epinephrine. Whereas the acceleration of the inhibitory action of GTP gamma S was observed at low and high GTP gamma S concentrations, the amplification by epinephrine was observed only at submaximally effective concentrations of GTP gamma S.     

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.     

Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system

The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O-(thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neither alpha T . GTP gamma S nor beta gamma T having any direct effect on the intrinsic activity of the catalytic moiety. Increasing the MgCl2 concentration in the assay incubations significantly decreases the inhibitions by both alpha T . GTP gamma S and beta gamma T. Similarly, when the pure hamster lung beta-adrenergic receptor is included in the lipid vesicles with Ns and C, the levels of inhibition of the GppNHp-stimulated activity by both alpha T . GTP gamma S and beta gamma T are reduced compared to those obtained in vesicles containing just Ns and C (but not stimulatory receptor). These inhibitions are reduced still further under conditions where the agonist stimulation of adenylate cyclase activity is maximal, i.e. when stimulating with isoproterenol plus GTP. In these cases the alpha T . GTP gamma S inhibitory effects are completely eliminated and the inhibitions observed with holotransducin can be fully accounted for by the beta gamma T complex. The ability of the beta-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to Ns (or alpha s) during the activation of the regulatory protein and the stimulation of adenylate cyclase. These results also suggest that under physiological conditions the beta gamma subunit complex is primarily responsible for mediating the inhibition of adenylate cyclase activity.     

Cholera toxin ADP-ribosylates the islet-activating protein substrate in adipocyte membranes and alters its function

In adipocyte membranes, cholera toxin may ADP-ribosylate the islet-activating protein (IAP) substrate, under certain conditions. Covalent modification is maximal in the absence of a guanosine triphosphate; in the presence of 5'-guanylylimidodiphosphate, incorporation of [32P]ADP-ribose is markedly reduced. ADP-ribosylation by cholera toxin has similar functional consequences as does IAP-mediated modification, i.e. the biphasic response of isoproterenol-stimulated adenylate cyclase to GTP and the inhibition by N6-phenylisopropyladenosine is abolished, and only the stimulatory phase remains. In contrast, membranes treated with cholera toxin in the presence of GTP display both the stimulatory and inhibitory responses to GTP. The binding of the adenosine analog [3H]N6-phenylisopropyladenosine is increased in the presence of GTP. Treatment of the membranes with IAP, but not with cholera toxin in the absence of GTP, reverses this GTP effect on [3H]N6-phenylisopropyladenosine binding. However, [3H]N6-phenylisopropyladenosine binding is still sensitive to GTP in membranes treated with cholera toxin in the presence of GTP. In adipocyte and cerebral cortical membranes, the IAP substrate appears as a 39,000/41,000-Da doublet which does not appear to reflect protease activity. On two-dimensional polyacrylamide gels, these two proteins migrate with approximate pI values 6.0 and 5.6, respectively. Although both behave similarly under all conditions explored in this study, it is unknown whether both, or only one, are involved in inhibition of adenylate cyclase activity. These results extend the already striking homology between the adenylate cyclase complex and the visual system. Ni, as well as transducin, may be ADP-ribosylated by cholera toxin and by IAP, and, in all cases, there are functional consequences.     

Identification of the predominant substrate for ADP-ribosylation by islet activating protein

Islet activating protein (IAP), a toxin isolated from Bordetella pertussis, blocks the ability of inhibitory hormones to attenuate adenylate cyclase activity and enhances the ability of stimulatory hormones to activate the enzyme. The toxin appears to act by catalyzing the transfer of ADP ribose from NAD to a 41,000-dalton protein in target cell membranes. A protein purified from rabbit liver membranes, apparently composed of 41,000- and 35,000-dalton subunits, is shown to be a specific substrate for IAP. Cholera toxin does not ADP-ribosylate this protein. In contrast, the purified guanine nucleotide-binding regulatory component of adenylate cyclase (G/F), which is ADP-ribosylated by cholera toxin, is not covalently modified by IAP. Equilibrium binding studies and photoaffinity labeling experiments demonstrate that the 41,000-dalton subunit of the IAP substrate has a specific binding site for guanine nucleotides.     

Specific epidermal growth factor receptors on porcine thyroid cell membranes

The influence of islet-activating protein (IAP), a Bordetella pertussis toxin, was studied on adenylate cyclase and GTPase activities in rat adipocyte membranes. Pretreatment of rats or intact rat adipocytes with IAP did not affect adenylate cyclase inhibition by the stable GTP analog, GTP gamma S, whereas inhibition by GTP was abolished. Concomitantly, activation of the adipocyte enzyme by sodium and its inhibition by nicotinic acid were prevented. Furthermore, IAP treatment of adipocyte membranes prevented nicotinic acid-induced stimulation of a high affinity GTPase. The data suggest that a GTP-hydrolyzing system involved in the inhibitory regulation of adenylate cyclase is the target of IAP's action.     

Evidence for receptor-regulated phosphotransfer reactions involved in activation of the adenylate cyclase inhibitory G protein in human platelet membranes

The activity of the adenylate cyclase inhibitory guanine-nucleotide-binding regulatory protein (Gi), measured as inhibition of forskolin-stimulated cyclic AMP formation, and its regulation by various nucleotides and the inhibitory alpha 2-adrenoreceptor agonist epinephrine was studied in membranes of human platelets. When adenylate cyclase activity was measured with ATP as substrate and in the absence of a nucleoside-triphosphate-regenerating system, GTP (0.1-10 microM) by itself potently and efficiently inhibited the enzyme. GDP was almost as potent and as effective as GTP. In the additional presence of epinephrine, the potencies of both GTP and GDP were increased about threefold, while maximal inhibition by these nucleotides was only slightly increased by the receptor agonist. In contrast to GTP and GDP, the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate, had only a very small effect, suggesting that GDP but not its stable analog is converted to the active GTP. Addition of UDP (1 mM), used to block the GDP to GTP conversion reaction, completely suppressed the inhibitory effect of GDP, while that caused by GTP was not affected. Most important, the inhibitory receptor agonist epinephrine counteracted the suppressive effect of UDP on GDP's action, suggesting that, while UDP inhibits the formation of GTP from GDP, the activated receptor stimulates this conversion reaction. In the presence of a complete nucleoside-triphosphate-regenerating system, which by itself had no influence on control forskolin-stimulated adenylate cyclase activity, GTP alone, at concentrations up to 10 microM, did not decrease enzyme activity, but required the presence of an inhibitory receptor agonist (epinephrine) to activate the Gi protein. Addition of the regenerating system creatine phosphate plus creatine kinase not only abolished adenylate cyclase inhibition by GTP alone, but also largely reduced both the potency and efficiency of epinephrine to activate the Gi protein in the presence of GTP. Furthermore, the nucleoside-triphosphate-regenerating system also largely delayed the onset of adenylate cyclase inhibition by the GTP analog, guanosine-5'-[beta-thio]triphosphate (10 nM), which was accelerated by epinephrine, and it also decreased the final enzyme inhibition caused by this GTP analog.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of lithium ion on the inhibitory GTP-binding protein and its coupling response.

Addition of lithium ion to the inhibitory GTP-binding (Gi) protein resulted in a decrease of its ADP-ribosylation by islet-activating protein (pertussis toxin, IAP). The possibility that this decrease was due to dissociation of the Gi protein trimer was examined. Results showed that lithium ions had no appreciable effect on either the Gi protein trimer or its dissociation into its three subunits induced by Mg2+ and GTP gamma S. Next, the effect of lithium ions on Gi protein-mediated adenylate cyclase inhibition and alpha 2-adrenoceptor in human platelet membranes was examined. Lithium ion was found to impair adenylate cyclase inhibition of alpha 2-adrenoceptor stimulation of forskolin-stimulated enzyme activities. The monovalent ion also abolished guanine nucleotide modulation (GTP shift) of agonist binding, while it had no remarkable effects on antagonist binding in alpha 2-adrenoceptor of human platelet membranes. These results suggested that lithium ion caused functional change of the Gi protein without remarkable change of its dissociation, causing modulation in a coupling between alpha 2-adrenoceptor and Gi protein.     

Pertussis toxin treatment modifies opiate action in the rat brain striatum

In this report we present evidence that a guanine nucleotide regulatory protein, Gi, mediates opiate action in the rat brain striatum. Opiates inhibit basal adenylate cyclase activity in rat brain striatum. This effect on adenylate cyclase is dose-dependently attenuated by pretreatment of membranes with pertussis toxin, which ADP-ribosylates a protein with a molecular mass of 41,000 daltons. This protein co-migrates with the GTP-binding subunit of Gi, which mediates inhibition of adenylate cyclase. Several brain regions were compared for the extent of radiolabeling and effects on adenylate cyclase activity. Although Gi was found in each region examined, opiate inhibition of adenylate cyclase is clearly seen only in the striatum.     

Modification of Gs-Stimulated Adenylate Cyclase in Brain Membranes by Low pH Pretreatment: Correlation with Altered Guanine Nucleotide Exchange

Pretreatment of rat brain membranes at pH 4.5 before assay at pH 7.4 modifies the function of GTP-binding proteins (G-proteins) by eliminating Gs-stimulated adenylate cyclase activity while increasing opiate-inhibited adenylate cyclase activity. To help characterize the molecular nature of the low pH effect, we labeled Gs and Gi alpha-subunits in both control and low pH-pretreated membranes with the GTP photoaffinity analog [32P]P3 (4-azidoanilido)-P1-5'-GTP ([32P]AAGTP). When membranes were preincubated with unlabeled AAGTP, a persistent inhibitory state of adenylate cyclase was produced, which was overcome in untreated membranes with high (greater than 1 microM) concentrations of guanylyl-5'-imidodiphosphate [Gpp(NH)p]. In low pH-pretreated membranes, this inhibition could not be overcome, and stimulation by Gpp(NH)p was eliminated. Maximal inhibition of adenylate cyclase achieved by incubation with AAGTP was not altered by low pH pretreatment. Incorporation of [32P]AAGTP into Gs (42 kilodaltons) or Gi/o (40 kilodaltons) was unaffected by low pH pretreatment; however, transfer of 32P from Gi/o to Gs, which occurs with low (10 nM) concentrations of Gpp(NH)p in untreated membranes, was severely retarded in low pH-pretreated membranes. Both the potency and efficacy of Gpp(NH)p in producing exchange of [32P]AAGTP from Gi/o to Gs were markedly reduced by low pH pretreatment. These results correlate the loss of Gs-stimulated adenylate cyclase with a loss of transfer of nucleotide from Gi/o to Gs alpha-subunits and suggest that the nucleotide exchange participates in the modulation of neuronal adenylate cyclase.