Regulation of calmodulin-sensitive adenylate cyclase by the stimulatory G-protein, Gs

Studies in bovine and rat brain membranes have suggested that calmodulin can potentiate neurotransmitter- and GTP-stimulated adenylate cyclase activities. To examine whether calmodulin and the stimulatory G-protein, Gs, are potentiative at a calmodulin-sensitive adenylate cyclase, Gs was purified from rabbit liver and reconstituted with a partially purified calmodulin-sensitive adenylate cyclase from bovine brain. Activated Gs (G*s) stimulated basal adenylate cyclase activity and enhanced the stimulation by calmodulin. The potentiation of the calmodulin-stimulated adenylate cyclase activity was dose-dependent with respect to G*s concentration. At the highest concentration of G*s tested (3 nM), a 2-fold enhancement of the calmodulin-stimulated adenylate cyclase activity was observed at all concentrations of calmodulin. The synergistic activation of adenylate cyclase by calmodulin and Gs was dependent on the presence of Ca2+ and occurred at physiologically relevant Ca2+ concentrations. The potentiation was not observed when either a nonactivated Gs or a mixture of activated Gi/Go was used. G*s was not able to stimulate or potentiate a calmodulin-stimulated adenylate cyclase purified from membranes pretreated with the nonhydrolyzable GTP analog, guanyl-5'-yl beta,gamma-imidodiphosphate. Photochemical cross-linking of 125I-calmodulin-diazopyruvamide to proteins having an Mr corresponding to the known Mr of adenylate cyclase was not enhanced by G*s. The results demonstrate that the guanyl nucleotide-dependent enhancement of calmodulin-stimulated adenylate cyclase activity is mediated by G*s and suggest that G*s modulates the enzymatic turnover of the calmodulin-stimulated activity.     

Prostacyclin stimulates the adenylate cyclase system of human thyroid tissue

Since Prostacyclin (PGI₂) is a major product of arachidonic acid metabolism in the human thyroid, we have studied the effects of PGI₂ on cAMP accumulation in human thyroid slices and cultured thyrocytes. In both systems, PGI₂ caused a dose- and time-dependent increase of cAMP accumulation with higher potency and efficacy than PGE₂. Two optically active isomers of 5,6-dihydro-PGI₂, i.e. stable synthetic analogs of PGI₂, had qualitatively similar effects to PGI₂. The relative potency ratio between the α- and β- isomer as well as their potency compared to PGI₂ were substantially similar to their potency in inhibiting human platelet aggregation. In thyroid slices, PGI₂ and its stable analogs had a greater than TSH in causing cAMP accumulation; however, in contrast to TSH, this effect was not associated with increased iodothyronine release except at maximal PGI₂ concentrations. TSH had no detectable effect on thyroidal PGI₂ synthesis and release. In cultured thyrocytes the effects of PGI₂ and its stable analogs were considerably less than those obtained with TSH and required higher concentrations. Such a discrepancy was not found in the case of PGE₂. These findings suggest the existence of a specific PGI₂-responsive adenylate cyclase system in human significance.     

Prostacyclin stimulates the adenylate cyclase system of human thyroid tissue

Since Prostacyclin (PGI2) is a major product of arachidonic acid metabolism in the human thyroid, we have studied the effects of PGI2 on cAMP accumulation in human thyroid slices and cultured thyrocytes. In both systems, PGI2 caused a dose- and time-dependent increase of cAMP accumulation with higher potency and efficacy than PGE2. Two optically active isomers of 5,6-dihydro-PGI2, i.e. stable synthetic analogs of PGI2, had qualitatively similar effects to PGI2. The relative potency ratio between the alpha- and beta- isomer as well as their potency compared to PGI2 were substantially similar to their potency in inhibiting human platelet aggregation. In thyroid slices, PGI2 and its stable analogs had a greater effect than TSH in causing cAMP accumulation; however, in contrast to TSH, this effect was not associated with increased iodothyronine release except at maximal PGI2 concentrations. TSH had no detectable effect on thyroidal PGI2 synthesis and release. In cultured thyrocytes the effects of PGI2 and its stable analogs were considerably less than those obtained with TSH and required higher concentrations. Such a discrepancy was not found in the case of PGE2. These findings suggest the existence of a specific PGI2-responsive adenylate cyclase system in human thyroid cells other than thyrocytes, of possible physiologic significance.     

L-arginine stimulates an endogenous ADP-ribosyltransferase

An ubiquitous biochemical pathway known to synthesize nitric oxide (NO) from L-arginine has been identified in many cell types. Recent studies indicate that besides activating soluble guanylate cyclase NO is likely to have effects unrelated to the known signal transduction pathway. Activation of the soluble NO synthase stimulates an endogenous ADP-ribosylation of a predominant 39 kDa protein, known to be activated by NO releasing agents. This is demonstrated using the cytosolic fraction of rat cerebellum and HL-60 cells. The ADP-ribosylation is suppressed by the known NO synthase inhibitors N-nitro-L-arginine and N-methyl-L-arginine. These observations indicate that NO derived from its physiological precursor L-arginine activates an endogenous ADP-ribosyltransferase.     

Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gs alpha

An antibody (RM) raised against the carboxyl-terminal decapeptide of the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Gs alpha) has been used to study the interaction of Gs alpha with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. RM antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-gamma-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-gamma-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gs alpha protein in the partially purified preparations was determined by immunoblotting with RM antibody. There was 3-fold more Gs alpha detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with RM antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gs alpha protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Go alpha, Gi alpha, and G beta. The G beta protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of G beta was about the same in adenylate cyclase from preactivated membranes and from control membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

ADP-ribosylation of actins by arginine-specific ADP-ribosyltransferase purified from chicken heterophils.

We reported the purification and characterization of an arginine-specific ADP-ribosyltransferase and acceptor protein p33 in granules of chicken peripheral polymorphonuclear leukocytes (heterophils) [Mishima, K., Terashima, M., Obara, S., Yamada, K., Imai, K. & Shimoyama, M. (1991) J. Biochem. (Tokyo) 110, 388-394]. In the present study, we obtained evidence that chicken non-muscle beta/gamma-actin, skeletal muscle alpha-actin and smooth-muscle gamma-actin were ADP ribosylated by the heterophil ADP-ribosyltransferase. The stoichiometry of ADP-ribose incorporation into these actins was 1.2 mol, 1.0 mol and 2.0 mol ADP-ribose/mol of beta/gamma-actin, alpha-actin and gamma-actin, respectively. The optimal pH for the ADP ribosylation was at pH 8.5, with the respective actin. Km values for NAD were calculated to be 30 microM with beta/gamma-actin, 35 microM with alpha-actin and 20 microM with gamma-actin. The Km values for the actin isoforms were 15 microM for beta/gamma-actin, 2.5 microM for alpha-actin and 10 microM for gamma-actin. ADP ribosylation of actin inhibited its capacity to polymerize, as determined by the increase in fluorescence intensity with N-(1-pyrenyl)iodoacetamide-labelled actin. Filamentous actin (F-actin) polymerized with the respective actin isoform was also ADP ribosylated, although the extent of the modification of F-actin was lower than that of globular actin (G-actin). In situ ADP ribosylation of beta/gamma-actin was evidenced with chicken peripheral heterophils permeabilized with saponin. Thus, the endogenous ADP ribosylation of actin in the heterophils may be involved in the cellular processes such as phagocytosis, secretion and migration.     

Gs alpha mediates epidermal growth factor-elicited stimulation of rat cardiac adenylate cyclase

In an earlier study we demonstrated that epidermal growth factor (EGF) increases the cellular accumulation of cAMP in perfused rat hearts by stimulating the cardiac adenylate cyclase via a stimulatory GTP-binding protein (Nair, B. G., Rashed, H. M., and Patel, T. B. (1989) Biochem. J. 264, 563-571). Employing antiserum, CS1, generated against a synthetic decapeptide RMHLRQYELL representing the carboxyl terminus of Gs alpha, the involvement of Gs in mediating the effects of EGF on cardiac adenylate cyclase was further investigated. The CS1 antiserum specifically recognized two forms, (52 and 40 kDa) of Gs alpha in rat cardiac membranes; the 52 kDa being the predominant species. In functional assays of adenylate cyclase activity, the CS1 antiserum did not alter either aluminum fluoride- or forskolin-stimulated adenylate cyclase activity. Similarly, basal adenylate cyclase activity in the absence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) was also not altered by the CS1 antiserum. However, as compared with controls performed in the presence of non-immune serum, preincubation of cardiac membranes with the CS1 antiserum resulted in a concentration-dependent inhibition of Gpp(NH)p-, isoproterenol-, and EGF-stimulated activities. In experiments which monitored Gi function as the ability of different G(pp)NHp, (-)N6-(R-phenylisopropyl)adenosine and carbachol to inhibit forskolin-stimulated adenylate cyclase, CS1 antiserum by inhibiting Gs, increased the apparent activity of Gi. Overall, our data demonstrate that the CS1 antiserum can specifically inhibit Gs function and therefore the stimulation of adenylate cyclase by agonists whose actions are mediated by Gs. In this respect, the data presented here demonstrate that Gs is the G-protein involved in mediating EGF-elicited stimulation of cardiac adenylate cyclase. Additionally, the finding that CS1 antiserum can overcome the effects of Gpp(NH)p on Gs, but not Gi, suggests that the carboxyl-terminal region of Gs alpha is important in the interactions with GTP or its analogs.     

Forskolin stimulates adenylate cyclase and iodine metabolism in thyroid

Forskolin is a potent activator of the cyclic AMP-generating system in many tissues. In dog thyroid slices, the enhancement of cyclic AMP level was rapid, sustained in the presence of forskolin, but easily reversible after its withdrawal. Contrary to TSH, forskolin induced little apparent desensitization. Forskolin potentiated the effects of TSH, PGE1 and cholera toxin. However, the forskolin-induced cyclic AMP accumulation was still sensitive to inhibitors of dog thyroid adenylate cyclase such as iodide, norepinephrine and adenosine. As fluoride, but contrary to TSH and PGE1, forskolin stimulated adenylate cyclase in a medium where Mg2+ was replaced by Mn2+. This suggests that in thyroid, as in other tissues, forskolin acts beyond the receptor level but, as it potentiates hormone action and does not impair modulation by inhibitors, it may interact with the nucleotide-binding regulatory proteins. Forskolin mimicked the effect of TSH on iodide organification and secretion.     

Parathyroid hormone stimulates adenylate cyclase in rat cerebral microvessels

We have studied the effect of parathyroid hormone (PTH) on adenylate cyclase of microvessels isolated from rat cerebral cortex. Native bovine (b) PTH-(1–84), the synthetic amino-terminal fragment bPTH-(1–34) and the synthetic analog [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH- (1–34) amide stimulated adenylate cyclase in a dose-dependent manner with apparent ED₅₀ values of 16 nM, 6.3 nM and 15 nM respectively. The stimulation by bPTH was greatly enhanced by guanosine triphosphate. The PTH antagonist, [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH-(3–34) amide inhibited the action of bPTH-(1–84) and bPTH-(1–34). In summary, PTH stimulated adenylate cyclase in rat cerebral microvessels in a very similar manner to its stimulation in the renal cortex.     

Corticotropin-releasing factor stimulates adenylate cyclase activity in the anterior pituitary gland

Ovine corticotropin-releasing factor (CRF) stimulates adenylate cyclase activity in rat anterior pituitary homogenate at an ED₅₀ value of 70 nM. GTP increases the stimulatory effect of CRF on [³²P] cyclic AMP formation in a rat adenohypophysial particulate fraction and in bovine anterior pituitary plasma membranes. The present data show that CRF stimulates adenylate cyclase activity in the anterior pituitary gland at least partly through a guanyl nucleotide-dependent mechanism.     

Angiotensin stimulates adenylate cyclase activity via prostaglandins in the adrenal glomerulosa membrane

The effects of angiotensin II (A II) on adenylate cyclase activities in membranes of the zona glomerulosa (the capsular fraction) and the zona fasciculata (the decapsulated fraction) from rat adrenocortical glands were investigated. A time- and GTP-dependent stimulation by A II of adenylate cyclase activity was observed in the capsular fraction but not in the decapsulated fraction. The activation of adenylate cyclase by ACTH and A II was additive. Stimulation by A II was inhibited by an angiotensin antagonist, DD-3487 (DD). Moreover, the addition of a prostaglandin antagonist, a mixture of polyesters of polyphloretin phosphate (PPP) and an inhibitor of prostaglandin synthesis, indomethacin, was effective in inhibiting A II-induced stimulation of the capsular adenylate cyclase activity, suggesting that the activation of A II receptors located on the capsular membrane leads to the release of prostaglandins, which in turn stimulates the adenylate cyclase.     

Phospholipase C digestion of rat liver plasma membrane stimulates adenylate cyclase

Brief treatment of rat liver plasma membranes with phospholipase C of Clostridium welchii increased both the ratio of saturated to unsaturated fatty acids and the ratio of cholesterol to phospholipids. Using 5-doxylstearic acid spin probes two breaks at 29 and 19.6 °C could be observed in the order parameter, S_A, vs temperature curve for untreated membranes. Upon phospholipase C digestion the lower phase transition temperature was shifted to 23 °C, while the higher phase transition temperature could not be detected up to 40 °C. The order parameter, S_A, was consistently higher at all temperatures in the phospholipase C-treated membranes. As phospholipase C is known to attack the outer lamella, these results can be interpreted as indicating an increase in ordering (i.e., decrease in fluidity) of the outer membrane lamella. On the other hand, an increase in basal activity of adenylate cyclase of the treated membranes was observed with an apparent reduction of the activation energies both below and above the break (at 20 °C) in the Arrhenius plot of enzyme activity. Phospholipase C treatment did not affect the temperature of the break in Arrhenius kinetics of the enzyme. The results are discussed in terms of the role of the ordering state of membrane lipids in adenylate cyclase activity.     

Growth hormone-releasing factor stimulates adenylate cyclase activity in the anterior pituitary gland

Synthetic human pancreatic growth hormone-releasing factor (hpGRF) (1–40)-NH₂ stimulates adenylate cyclase activity in rat anterior pituitary particulate fraction at an ED₅₀ value of approximately 150 nM. GTP more than doubles the stimulatory effect of hpGRF aand PGE₂ on [³²p] cyclic AMP formation. The present data show that hpGRF as well as PGE₂, another potent stimulus of GH secretion, act at least partly, through GTP-dependent mechanisms in their coupling with adenylate cyclase.     

Molecular basis for two forms of the G protein that stimulates adenylate cyclase

Most cells contain two forms of the alpha subunit of the G protein (Gs) that stimulates adenylate cyclase; their apparent molecular weights are 45,000 and 52,000. Two cDNAs that correspond to distinct mRNAs for the alpha subunit of Gs have been cloned from a bovine adrenal library and sequenced. The sequences of the two cDNAs, designated pGs-l and pGs-S, are identical except for a single stretch of 46 nucleotides in the coding region, where four are altered and 42 are deleted in pGs-S. Expression of pGs-S and pGs-l in COS-m6 cells yields protein products with apparent molecular weights of 45,000 and 52,000, respectively, based on their mobility in sodium dodecyl sulfate-polyacrylamide gels. We conclude that pGs-S and pGs-l encode the 45- and 52-kDa forms of Gs alpha, respectively, and propose that the mRNAs encoding these proteins arise from a single gene by internal alternative RNA splicing.     

Purification and partial characterization of arginine-specific ADP-ribosyltransferase from skeletal muscle microsomal membranes

Integral membrane-associated arginine-specific mono-ADP-ribosyltransferase was purified from rabbit skeletal muscle microsomes. The ADP-ribosyltransferase was solubilized from the 100,000 x g pellet with 0.3% sodium deoxycholate and purified to greater than or equal to 95% homogeneity by successive DE52, concanavalin A-agarose, 3-aminobenzamide-agarose, and size-exclusion high-performance liquid chromatography (HPLC) steps in the presence of detergents. Two molecular weight forms of the enzyme were isolated and partially characterized. The apparent Mr of the alpha-form of the enzyme purified to greater than or equal to 95% homogeneity was approximately 39,000 +/- 500 as estimated by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr of the beta-form purified to greater than or equal to 80% homogeneity was 38,500 +/- 500. The rapid procedure resulted in a 200-fold purification for the alpha-form and a 645-fold purification for the beta-form, relative to the microsomal fraction. Positive identification of the enzyme was confirmed by utilizing a zymographic in situ gel assay and by HPLC assay of polyacrylamide gel slice incubations with an NAD and guanylhydrazone substrate. The specificity of the mono-ADP-ribosyltransferase zymographic assay was characterized by time course incubations, hydroxylamine sensitivity, 3-aminobenzamide inhibition, and histone dependence. The ADP-ribosyltransferase is inactivated by reducing agents.     

Inhibition of thyrotropin-induced DNA synthesis in thyroid follicular cells by microinjection of an antibody to the stimulatory G protein of adenylate cyclase, Gs.

Thyrotropin (TSH) is an important regulator of thyroid follicular cells. While its role in the maintenance of differentiated functions is undisputed, its role as a mitogen is less clear. TSH induces DNA synthesis and cell proliferation in some cells, while in others, TSH is mitogenic only in the presence of additional growth factors such as insulin-like growth factor-1. TSH causes elevations in intracellular cAMP and is thought to utilize this second messenger system in its mitogenic action. We studied TSH as a mitogen in Wistar rat thyroid cells (WRT) (Brandi, M. L., Rotella, C. M., Mavilia, C., Franceschelli, F., Tanini, A., and Toccafondi, R. (1987) Mol. Cell. Endocrinol. 54, 91-103) and examined the role of the guanine nucleotide binding protein, Gs, in its mitogenic action. WRT cells synthesized DNA in response to TSH and elevations in cAMP. In addition, TSH caused a rapid stimulation of an indicator gene whose expression is regulated by cAMP response elements. Following microinjection of an inhibitory polyclonal antibody raised against the Gs protein, both TSH-induced changes in gene expression and DNA synthesis were significantly reduced. These results demonstrate that virtually all of the mitogenic action of TSH is transduced through the Gs protein in WRT cells, presumably through the regulation of adenylate cyclase. Whether all or only part of TSH action is mediated by cAMP and the cAMP-dependent protein kinase remains to be determined.     

Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture.

Primary cultures of embryonic chick skeletal myogenic cells were used as an experimental model to examine the possible role of mono(ADP-ribosyl)ation reactions in myogenic differentiation. Initial studies demonstrated arginine-specific mono(ADP-ribosyl)transferase activity in the myogenic cell cultures. We then examined the effect of a novel inhibitor of cellular arginine-specific mono(ADP-ribosyl)transferases, meta-iodobenzylguanidine (MIBG), on differentiation of cultured embryonic chick skeletal myoblasts. MIBG reversibly inhibited both proliferation and differentiation of embryonic chick myoblasts grown in culture. Micromolar (15-60 microM) concentrations of MIBG blocked myoblast fusion, the differentiation-specific increase in creatine phosphokinase activity, and both DNA and protein accumulation in myogenic cell cultures. Meta-iodobenzylamine, an analog of MIBG missing the guanidine group, had no effect. Low concentrations of methylglyoxal bis-guanylhydrazone, a substrate for cholera toxin with a higher Km than MIBG, also had no effect, but higher concentrations reversibly inhibited fusion. These findings suggest a possible role for mono(ADP-ribosyl)ation reactions in myogenesis. In addition, the total arginine-specific mono(ADP-ribosyl)transferase activity increased with differentiation in the myogenic cell cultures, and this increase was also blocked by MIBG treatment. Because high levels of activity were found in the membrane fraction derived from later, myotube cultures, the membrane fraction from 96-h cultures was incubated with [32P]NAD+ and subjected to electrophoresis and autoradiography. Three proteins, migrating at 21, 20, and 17 kDa, that were ADP-ribosylated in the absence, but not the presence, of MIBG were identified. These proteins may be endogenous substrates for this enzyme.