The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and guanine nucleotide-dependent hormonal inhibition

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 (35,000 Da) subunits are functionally indistinguishable. Gi and Gs both dissociate in the presence of guanine nucleotide analogs or Al3+, Mg2+, and F- in detergent-containing solutions. Several characteristics of Gi- and Gs-mediated regulation of adenylate cyclase activity have been studied in human platelet membranes. The nonhydrolyzable analog of GTP, guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) mimics GTP-dependent hormonal inhibition or stimulation of adenylate cyclase under appropriate conditions. This inhibition or stimulation follows a lag period. The combined addition of epinephrine or prostaglandin E1 with GTP gamma S results in the immediate onset of steady state inhibition or activation. The effects of the GTP analog are essentially irreversible. Fluoride is also an effective inhibitor of prostaglandin E1-stimulated adenylate cyclase, while it markedly stimulates the basal activity of the enzyme. The addition of the resolved 35,000-Da subunit of Gi to membranes results in inhibition of adenylate cyclase, and the resolved 41,000-Da subunit has a stimulatory effect on enzymatic activity. The inhibitory action of the 35,000-Da subunit is almost completely abolished in membranes that have been irreversibly inhibited by GTP gamma S plus epinephrine; this irreversible inhibition is almost completely relieved by the 41,000-Da subunit. Detergent extracts of membranes that have been treated with GTP gamma S plus epinephrine contain free 35,000-Da subunit. The 41,000-Da subunit of Gi contained in such extracts has a reduced ability to be ADP-ribosylated by islet-activating protein (IAP), which implies that this subunit is in the GTP gamma S-bound form. The irreversible inhibition of adenylate cyclase caused by GTP gamma S (plus epinephrine) in membranes is highly correlated with the liberation of free 35,000-Da subunit activity and is inversely related to the 41,000-Da IAP substrate activity in detergent extracts prepared therefrom. The increase in free 35,000-Da subunit activity in extracts and the inhibition of adenylate cyclase activity in GTP gamma S (plus epinephrine)-treated membranes are both markedly inhibited by treatment with IAP.(ABSTRACT TRUNCATED AT 400 WORDS)     

The subunits of the stimulatory regulatory component of adenylate cyclase. Resolution of the activated 45,000-dalton (alpha) subunit

Activation of the stimulatory guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase by guanine nucleotides or by Al3+, Mg2+, and F-stabilizes the protein to thermal denaturation or to inactivation by LiBr, guanidine HCl, or urea. Such activation allows the resolution of the active 45,000-Da alpha subunit from the 35,000-Da beta subunit by a high performance gel filtration procedure. Separation of the active alpha subunit has allowed definitive evaluation of the subunit dissociation model for the activation of G/F. The resolved alpha subunit is sufficient to reconstitute the adenylate cyclase activity of the cyc-S49 cell mutant. The alpha subunit alone is also sufficient to activate a preparation of the catalyst of adenylate cyclase that had been resolved from all other identified components of the enzyme system. The resolved alpha subunit displays hydrodynamic properties characteristic of activated G/F. The alpha subunit contains a high affinity guanine nucleotide-binding site. Activation of G/F by guanine nucleotides or by Al3+ + Mg2+ + F- allows resolution of the activated alpha subunit. Reversal of the activated state of the resolved alpha subunit occurs only slowly. Addition of beta subunit enhances the rate of deactivation. Deactivation of the activated alpha subunit by the beta subunit changes the S20,w for G/F activity from 2.0 to 4.0 (in Lubrol), consistent with a formation of the alpha X beta heterodimer. These data, taken in aggregate, constitute proof for the proposed mechanism of activation of G/F by non-hydrolyzable analogs of GTP and by Al3+, Mg2+, and F-. They are analogous to data obtained for transducin, the GTP-binding regulatory protein from vertebrate rod outer segment discs, and for the putative inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase (the substrate for islet-activating protein). The model provides several powerful tests for study of mechanisms of hormonal regulation of adenylate cyclase in membranes.     

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 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.     

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)     

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 effects of forskolin on adenylate cyclase in S49 wild type and cyc- cells

The effect of forskolin on adenylate cyclase in S49 wild type and cyc- cells was tested. Forskolin stimulated adenylate cyclase activity in cyc- membranes, particularly with Mn++ as cofactor. Forskolin stimulation of adenylate cyclase in wild type membranes was greater than in cyc- membranes, and the ability of forskolin to stimulate cyc- membranes was enhanced by Lubrol PX extracts of human erythrocyte membranes. Compared to its potent effect on intact wild type cells, forskolin was a poor stimulator of cAMP accumulation in cyc- cells. Cyc- cells proliferated in medium containing forskolin, while the growth of wild type cells in such medium was inhibited and the wild type cells ultimately died. Clones selected from a suspension of wild type cells on the basis of forskolin resistance showed the characteristics of cyc- cells. Thus, forskolin does not substantially activate adenylate cyclase activity in intact cyc- cells. Our data indicate that the guanine nucleotide regulatory protein (G/F) enhances forskolin activation of adenylate cyclase.     

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.     

A monoclonal antibody against the rod outer segment guanyl nucleotide-binding protein, transducin, blocks the stimulatory and inhibitory G proteins of adenylate cyclase

GTP-binding proteins have been implicated as transducers of a variety of biological signaling processes. These proteins share considerable structural as well as functional homology. Due to these similarities, it was thought that a monoclonal antibody that inhibits the light activation of the rod outer segment GTP-binding protein, tranducin (Gt), might exert some functional effect upon the G proteins that regulate the adenylate cyclase system. Antibody 4A, raised against the alpha subunit of Gt, cross-reacted (by hybridization on nitrocellulose) with purified alpha subunits of other G proteins (Gi and Gs, regulatory guanyl nucleotide-binding proteins that mediate inhibition and stimulation of adenylate cyclase, respectively) as long as they were not denatured. This antibody, which interferes with rod outer segment cGMP phosphodiesterase activation by blocking interaction between rhodopsin and Gt, also interfered with actions of both the stimulatory and inhibitory G proteins of adenylate cyclase from rat cerebral cortex membranes. Effects of monoclonal antibody (mAb) 4A were dose-dependent and not reversed by washing. mAb 4A also blocked the Gi-mediated inhibition of adenylate cyclase in the cyc- variant of S49 lymphoma and in doing so raised the level of adenylate cyclase activity in both the cyc- variant and the S49 wild type. There was no effect of mAb 4A on adenylate cyclase activity of the resolved catalytic subunit. These results suggest that the well known sequence homologies among the G proteins involved in cellular signal transduction may extend to the sites that interact with other members of signal-transducing cascades (receptors and effector molecules). Therefore, antibody 4A may serve as a useful tool to probe the similarities and differences among the various systems.     

Adenylate cyclase inhibition and GTPase stimulation by somatostatin in S49 lymphoma cyc- variants are prevented by islet-activating protein

cyc--Variants of S49 lymphoma cells are defective in the stimulatory guanine nucleotide site of the adenylate cyclase but contain an inhibitory site. Treatment of cyc- cells with islet-activating protein (IAP), which causes ADP-ribosylation of an Mr 40 000 polypeptide in cyc- membranes, abolishes adenylate cyclase inhibition by GTP and the peptide hormone, somatostatin, but not that induced by GTP gamma S. Furthermore, somatostatin-induced stimulation of GTP hydrolysis is lost. Thus, the data indicate that IAP interferes with the adenylate cyclase system by an action at the inhibitory guanine nucleotide site.     

Reconstitution of the Gs protein from B16 melanoma clones of high and low experimental metastatic potential into S49 cyc-membranes

The ability of a series of B16 melanoma clones to form experimental lung metastases in syngeneic mice has been shown to correlate positively with adenylate cyclase activity. (Sheppard et al, Int. J. Cancer 37 (1986) 713-722). To begin to identify the components of the adenylate cyclase complex that account for enhanced enzyme activity in highly metastatic tumor populations, cholate extracts containing the GTP-binding protein GS from B16 melanoma clones of different metastatic capacities were reconstituted with membranes prepared from S49 cyc-, a variant lymphoma cell line that lacks GS function. The results revealed that extracts from a highly metastatic B16 clone (F10-C23) reconstituted significantly greater adenylate cyclase activities in S49 cyc- membranes than parallel preparations from a B16 clone (F1-C29) of low metastatic capacity. The data suggest that aberrations in GS function may contribute to the heightened responsiveness of adenylate cyclase observed in B16 melanoma clones of increased metastatic potential.     

Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Withdrawal of GTP-dependent inhibition

12-O-Tetradecanoylphorbol-13-acetate (TPA) enhances the apparent maximal velocity of adenylate cyclase in S49 lymphoma cells, an effect that seems not to result from an increased rate of activation of the catalytic subunit by the stimulatory GTP-binding protein (Gs) (Bell, J. D., Buxton, I. L. O., and Brunton, L. L. (1985) J. Biol. Chem. 260, 2625-2628). In membranes from wild type S49 cells, this enhancing effect of TPA is largely GTP-dependent; TPA enhances forskolin-stimulated adenylate cyclase activity by 35% in the presence of guanine nucleotide but only slightly (approximately 10%) in its absence. TPA causes comparable results in membranes from the cyc- variant that lacks the GTP-binding subunit of Gs. Blockade of the activity of the inhibitory GTP-binding protein (Gi) by high concentrations of Mg2+ (100 mM) or Mn2+ (3 mM) abolishes the effect of TPA to enhance adenylate cyclase activity in wild type membranes. The potentiation by TPA of cAMP accumulation in intact cells is greater than and not additive with the similar effect of pertussis toxin (an agent known to abolish hormonal inhibition of adenylate cyclase). Kinetic experiments indicate that TPA decreases the rate of activation of Gi by guanine nucleotide. We conclude that the resultant withdrawal of tonic inhibition of adenylate cyclase is one mechanism by which phorbol esters enhance guanine nucleotide-dependent cAMP synthesis.     

Alterations in components of adenylate cyclase associated with transformation of chicken embryo fibroblasts by Rous sarcoma virus

Regulation of adenylate cyclase coincident with transformation of chicken embryo fibroblasts by Rous sarcoma virus is manifest as a 10-50% decrease in basal, Mg2+-, and forskolin-stimulated activities; activities elicited by fluoride and guanosine 5'-O-(3-thiotriphosphate) are unaltered. The level of the catalytic component of adenylate cyclase, assessed with activated stimulatory guanine nucleotide-binding protein (Gs), increases approximately 1.5-fold. The level of the beta subunit common to Gs and the inhibitory regulatory protein assessed by enzyme-linked immunotransfer blotting, increases 2.7-fold. The isoelectric behavior of the beta subunit is unaltered. The amount of radiolabel incorporated into the alpha subunit of Gs (Mr = 45,000) upon incubation of membranes with 32P-labeled NAD and cholera toxin increases 3-fold upon transformation. Detergent extracts prepared from membranes of untransformed and transformed fibroblasts nevertheless exhibit equivalent abilities to reconstitute fluoride-stimulated activities to membranes of the cyc-variant of mouse S49 lymphoma cells. Islet-activating protein catalyzes incorporation of radiolabel from 32P-labeled NAD into 39,000- and 41,000-dalton proteins; the extent of radiolabel incorporation does not change upon transformation. Modest alterations in the isoelectric behaviors of substrates for cholera toxin and islet-activating protein occur.     

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.     

Prevention of the agonist binding to gamma-aminobutyric acid B receptors by guanine nucleotides and islet-activating protein, pertussis toxin, in bovine cerebral cortex. Possible coupling of the toxin-sensitive GTP-binding proteins to receptors

Using the membranes treated with Triton X-100, we studied the interaction between gamma-aminobutyric acid (GABA)B receptors and the GTP-binding proteins which are the substrates for ADP-ribosylation by the islet-activating protein (IAP), pertussis toxin. The addition of guanine nucleotides to the membranes markedly decreased the binding of GABA to GABAB receptors. Preincubation of the membranes with IAP plus NAD caused ADP-ribosylation of the 41,000- and 39,000-Da proteins selectively and decreased GABA binding to GABAB receptors in a time- and dose-dependent manner. This decrease of binding appeared to be due to the reduction of receptor affinity for agonist. The GTP-binding proteins which are ADP-ribosylated by IAP were purified from the membrane fraction of bovine cerebral cortex. The addition of the purified GTP-binding proteins to IAP-treated membranes restored the high affinity binding of GABA to GABAB receptor. The two GTP-binding proteins which were resolved by octyl-Sepharose column chromatography showed similar efficacy in restoring GABA binding. Thus, GABAB receptors are coupled to GTP-binding proteins, IAP-specific substrates, in the brain membranes.     

Alteration in the protein components of catecholamine-sensitive adenylate cyclase during maturation of rat reticulocytes

The maturing rat reticulocyte was used as a model system in which to study developmental changes in the protein components of hormone-sensitive adenylate cyclase. Plasma membranes from rat erythrocytes display 10 to 20% of the adenylate cyclase activity and 30 to 50% of the beta-adrenergic receptors which are measured in membranes from rat reticulocytes, as noted by others. Reticulocyte membranes also display equal activities in response to (-)-isoproterenol in the presence of either GTP or GTP gamma S, whereas erythrocyte membrane adenylate cyclase is twice as active in the presence of isoproterenol plus GTP gamma S as in the presence of isoproterenol plus GTP. We have studied this system in greater detail by developing or applying independent assays for the catalytic protein (C) and the guanine nucleotide-binding regulatory protein (G/F) of adenylate cyclase. C was assayed in membranes by its intrinsic Mn2+-stimulated activity. It was also measured by reconstituting membranes with saturating amounts of GTP gamma S-activated G/F, yielding an operationally defined Vmax for the catalyst. By either assay, reticulocytes display about 3-fold greater C activity than do erythrocytes. G/F was assayed by its ability to confer GTP gamma S-stimulated activity upon C (which was supplied by membranes of cyc- S49 lymphoma cells). This assay indicates that reticulocyte membranes contain about 3 times as much G/F as do erythrocyte membranes. Cholera toxin and [32P]NAD were used to [32P]ADP-ribosylate the 45,000- and 52,000-dalton subunits of G/F. Total incorporation of 32P into these subunits decreased 3- to 4-fold with reticulocyte maturation. The ratio of label in the 52,000-dalton peptide to that in the 45,000-dalton peptide decreased from 0.29 in reticulocyte membranes to 0.14 in erythrocyte membranes. The apparently coordinate decrease in the amounts of C, G/F, and beta-adrenergic receptors suggest that the stoichiometry between these components is maintained during maturation, and may account for the decrease in adenylate cyclase in the membranes. However, the qualitative changes in responsiveness to hormones in the presence of GTP or GTP gamma S may be related to loss or proteolysis of the 52,000-dalton subunit of G/F.     

Reconstitution of resolved muscarinic cholinergic receptors with purified GTP-binding proteins

The association of agonists with muscarinic receptors in membranes from bovine brain was affected only slightly by guanine nucleotides. However, solubilization of these membranes with deoxycholate and subsequent removal of detergent resulted in a preparation of receptors with increased affinity for agonists and a large increase in response to guanine nucleotides. Chromatography of deoxycholate extracts of membranes on DEAE-Sephacel resulted in the separation of receptors from 95% of the guanine nucleotide-binding activity. Guanine nucleotides had no effect on the binding of agonists to these resolved receptors. The effect of guanine nucleotides was restored after the addition of either of two purified guanine nucleotide-binding proteins from bovine brain. One of these proteins, presumably brain GI, is composed of subunits with the same molecular weights (alpha, 41,000; beta, 35,000; gamma, 11,000) and functions as the inhibitory guanine nucleotide-binding protein isolated from liver. The other protein, termed Go, is a novel guanine nucleotide-binding protein that possesses a similar subunit composition (alpha, 39,000; beta, 35,000; gamma, 11,000) but whose function is not yet known. Addition of either protein to the resolved receptor preparation increased agonist affinity by at least 10-20-fold, and low concentrations of guanine nucleotides specifically reversed this effect. Reconstitution of receptors with the resolved subunits of Go demonstrates that the beta subunit alone had no effect on agonist binding, but that this subunit does appear to enhance the effects observed with the alpha subunit alone.     

The regulatory component of adenylate cyclase. Purification and properties of the turkey erythrocyte protein

The regulatory component (G/F) of adenylate cyclase has been purified from turkey erythrocyte plasma membranes by adaptation of procedures developed for purification of the rabbit liver protein. The major modifications entail inclusion of high concentrations of NaCl to facilitate extraction and reconstitution of the protein. A typical preparation yields 200 micrograms of protein with a reconstitutive specific activity of 3-4 mumol . min-1 mg-1. Turkey erythrocyte G/F contains two putative subunits of 35,000 and 45,000 daltons. The 52,000-dalton polypeptide that appears to be a component of rabbit liver G/F is lacking. In solution, G/F behaves as a particle with Mr = 81,000. This value is reduced to 50,000 in the presence of activating ligands, suggesting dissociation of subunits. Activation of G/F by guanine nucleotide analogs is markedly accelerated in the presence of high concentrations of Mg2+. Reconstitutive and physical properties of the protein are also affected by fluoride. Cyc- S49 lymphoma membranes reconstituted with turkey erythrocyte G/F acquire properties that are characteristic of the turkey adenylate cyclase system; at least certain differing characteristics of adenylate cyclase systems are thus dictated by the nature of their G/F.     

Prostaglandin E-induced heterologous desensitization of hepatic adenylate cyclase. Consequences on the guanyl nucleotide regulatory complex

Prostaglandin E (PGE) receptor density in hepatic plasma membranes can be down-regulated by in vivo exposure to the 16,16-dimethyl analog of PGE2, and this is associated with desensitization of PGE-sensitive adenylate cyclase. These studies examined adenylate cyclase response to other agonists in membranes whose PGE receptor density was 51% decreased and whose maximal PGE-stimulated adenylate cyclase activity was 31% decreased. Down-regulated membranes had a 37% decrease in their maximal response to glucagon, indicating that treatment with the PGE analog had induced both homologous and heterologous desensitization. To determine whether adenylate cyclase had been affected, stimulation with NaF, guanyl 5'-yl imidodiphosphate (GppNHp), and forskolin was examined in both intact and solubilized membranes. Intact membranes had decreased adenylate cyclase responses to all three stimulators (NaF, -41%; GppNHp, -25%; forskolin, -41%) as did solubilized membranes (NaF, -51%; GppNHp, -50%; forskolin, -50%), suggesting alterations in adenylate cyclase rather than indirect membrane effects. Cholera toxin activation and labeling were examined to more directly assess whether the guanine nucleotide (G/F) regulatory component of adenylate cyclase had been affected. Cholera toxin activation was 42% less in down-regulated membranes, and these membranes incorporated less label when the incubation was performed in the presence of [32]NAD. Solubilized G/F subunit activity from down-regulated membranes was less effective in reconstitution of adenylate cyclase activity from cyc- cell membranes than G/F activity from control membranes. These data indicate that in vivo exposure to the PGE analog causes both homologous and heterologous desensitization of adenylate cyclase as well as an apparent quantitative decrease in G/F.     

Protein kinase C stimulates adenylate cyclase activity in prolactin-secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP-binding protein Gi

The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4C1 pituicytes in culture. Both TPA and thyroliberin activated the adenylate cyclase in broken cell membranes. When combined, the secretagogues displayed additive effects. TPA did not alter the time course (time lag) of adenylate cyclase activation by hormones, guanosine 5'-[beta,gamma-imino]triphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberin-enhanced, 2.2 mM) for free Mg2+. The TPA-mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED50 (dose yielding half-maximal activation) was 60 microM. Access to free Ca2+ was necessary to express TPA activation of the enzyme, however, the presence of calmodulin was not mandatory. TPA-stimulated adenylate cyclase activity was abolished by the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberin-sensitive adenylate cyclase remained unaffected. Experimental conditions known to translocate protein kinase C to the plasma membrane and without inducing adenylate cyclase desensitization, increased both basal and thyroliberin-stimulated enzyme activities, while absolute TPA-enhanced adenylate cyclase was maintained. Association of extracted GTP-binding inhibitory protein, Gi, from S49 cyc- murine lymphoma cells with GH4C1 cell membranes yielded a reduction of basal and hormone-stimulated adenylate cyclase activities, while net inhibition of the cyclase of somatostatin was dramatically enhanced. However, TPA restored completely basal and hormone-elicited adenylate cyclase activities in the Gi-enriched membranes. Finally, TPA completely abolished the somatostatin-induced inhibition of adenylate cyclase in both hybrid and non-hybrid membranes. These data suggest that, in GH4C1 cells, protein kinase C stimulation by phorbol esters completely inactivates the n alpha i subunit of the inhibitory GTP-binding protein, leaving the n beta subunit functionally intact. It can also be inferred that thyroliberin conveys its main effect on the adenylate cyclase through activation of the stimulatory GTP-binding protein, Gs.