Rhodopsin-enhanced GTPase activity of the inhibitory GTP-binding protein of adenylate cyclase

Work in several laboratories has shown that Gi, the inhibitory guanyl nucleotide-binding protein of the adenylate cyclase system, is similar in many ways to transducin, the guanyl nucleotide-binding protein of the retinal light-activated cGMP phosphodiesterase system. Separated subunits of purified transducin, T alpha (approximately 39 kDa) and T beta gamma (approximately 35 and approximately 10 kDa), do not exhibit GTPase activity; GTPase activity is observed when the subunits are combined in the presence of rhodopsin ( Fung , B. K.-K. (1983) J. Biol. Chem. 258, 10495-10502). Subunits of Gi, Gi alpha (approximately 41 kDa), and Gi beta gamma (approximately 35 and approximately 10 kDa) were prepared from rabbit liver membranes. It was found that Gi beta gamma could replace T beta gamma in reconstituting the rhodopsin-stimulated GTPase activity of T alpha. Gi alpha exhibited rhodopsin-stimulated GTPase activity when reconstituted with Gi beta gamma or T beta gamma. GTPase activity was a function of Gi alpha concentration when Gi beta gamma or T beta gamma was constant, and the GTPase activity of a given amount of Gi alpha was dependent on Gi beta gamma concentration. These studies demonstrate that the GTPase activity of Gi resides in Gi alpha and further establish that Gi alpha and Gi beta gamma are functionally analogous to T alpha and T beta gamma, respectively.     

Sweet tastants stimulate adenylate cyclase coupled to GTP-binding protein in rat tongue membranes.

Sucrose and other saccharides, which produce an appealing taste in rats, were found to significantly stimulate the activity of adenylate cyclase in membranes derived from the anterior-dorsal region of rat tongue. In control membranes derived from either tongue muscle or tongue non-sensory epithelium, the effect of sugars on adenylate cyclase activity was either much smaller or absent. Sucrose enhanced adenylate cyclase activity in a dose-related manner, and this activation was dependent on the presence of guanine nucleotides, suggesting the involvement of a GTP-binding protein ('G-protein'). The activation of adenylate cyclase by various mono- and di-saccharides correlated with their electrophysiological potency. Among non-sugar sweeteners, sodium saccharin activated the enzyme, whereas aspartame and neohesperidin dihydrochalcone did not, in correlation with their sweet-taste effectiveness in the rat. Sucrose activation of the enzyme was partly inhibited by Cu2+ and Zn2+, in agreement with their effect on electrophysiological sweet-taste responses. Our results are consistent with a sweet-taste transduction mechanism involving specific receptors, a guanine-nucleotide-binding protein and the cyclic AMP-generating enzyme adenylate cyclase.     

N-Chloroacetylhydrazone of oxo-GTP irreversibly inhibits the activating function of GTP-binding protein coupled with adenylate cyclase

Guanine nucleotides are successfully used in the studies of regulatory N-proteins coupled with adenylate cyclase. In the present work N-chloroacetylhydrazones of oxo-GTP and oxo-GDP are described. After 4 hr preincubation of these nucleotides with plasma membranes from bovine brain caudate nucleus, the ability of adenylate cyclase to be activated by guanylyl-5'-methylene-diphosphonate is blocked. The degree of inhibition depends on preincubation time and increases in the presence of Mg2+. Guanylyl-5'-methylenediphosphonate protects adenylate cyclase from the action of N-chloroacetylhydrazone of oxo-GTP. These findings suggest that adenylate cyclase activation is diminished as a result of covalent modification of the Ns. N-chloroacetyl-hydrazone of oxo-GDP also causes a loss of the adenylate cyclase sensitivity to the fluoride ion and cholera toxin.     

Reconstitution of catecholamine-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) to the stimulatory GTP-binding protein of adenylate cyclase

The stimulatory GTP-binding protein (Gs) of adenylate cyclase, purified from rabbit liver, and beta-adrenergic receptors, partially purified 1000-4000-fold from turkey erythrocyte plasma membranes, were coreconstituted into unilamellar phospholipid vesicles. The molar ratio of Gs to receptors in the vesicles varied from 3 to 10 in different preparations, as measured by guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) binding to Gs and [125I]iodocyanopindolol binding to receptors. Activation of reconstituted Gs by GTP gamma S was stimulated up to 10-fold by the addition of the beta-adrenergic agonist (-)-isoproterenol. Activation was assayed functionally by reconstitution with the catalytic unit of adenylate cyclase. Because of the relative purity of this preparation, the quasi-irreversible binding of [35S]GTP gamma S could also be measured in the vesicles and was shown to parallel the functional activation of Gs under all conditions. Most of the assayable Gs in the vesicles could interact with the receptors and undergo agonist-stimulated activation. Agonist-stimulated activation and [35S]GTP gamma S binding were complete in less than 3 min, even under suboptimal conditions, and could go to completion in less than 20 s under maximal stimulation. Agonist-stimulated binding did not require appreciable free Mg2+ (less than 0.1 mM). Activation in the absence of agonist was stimulated by free Mg2+, but maximal activation took up to 10 min in the presence of 50 mM MgCl2. Reconstitution increased the stability of Gs to thermal denaturation. The addition of beta-adrenergic agonist further stabilized Gs, presumably by the formation of a stable agonist-receptor-Gs complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of bovine cerebellum GTP-binding protein G39 and its effect on the adenylate cyclase system

The primary structure of bovine cerebellum GTP-binding protein alpha-subunit, protein G39, was determined by parallel analysis of the protein amino acid sequence and the corresponding cDNA nucleotide sequence. The protein consists of 354 amino acid residues and has a molecular mass of 40064 Da. High homology between G39 and other G-proteins, especially rat brain G0, was shown. An assumption is made that certain brain adenylate cyclase system properties are determined by the presence of G39.     

Stimulation of Escherichia coli adenylate cyclase activity by elongation factor Tu, a GTP-binding protein essential for protein synthesis

A unique feature of eucaryotic adenylate cyclases is their interaction with GTP-binding proteins that mediate hormonal responses. Until now, there has been no evidence for regulation of Escherichia coli adenylate cyclase by a GTP-binding protein. We describe here that the most abundant protein in E. coli, the GTP-binding protein EF-Tu, which is important as an elongation factor in protein synthesis, also serves as a stimulator of adenylate cyclase activity. Homogeneous EF-Tu specifically increased the activity of purified adenylate cyclase as much as 70%; other E. coli GTP-binding proteins had no effect on enzyme activity. A study of the guanine nucleotide specificity for EF-Tu-mediated stimulation of adenylate cyclase activity suggested that the preferred activator is EF-Tu X GDP. To account for the GTP-specific stimulation of adenylate cyclase activity observed in intact cells, we propose that the nucleotide specificity for EF-Tu-dependent activation of adenylate cyclase is governed by other factors in the cell.     

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.     

Age-related decline in the activites of erythrocyte membrane adenylate cyclase and protein kinase.

The circular dichroism and thermal denaturation properties of chromatin isolated from duck erythrocytes have been carefully examined as has the chromatography of sonicated erythrocyte chromatin on ECTHAM-cellulose. The circular dichroism spectrum and thermal denaturation profile resemble much more closely those of chromatin from liver than has been previously reported by other workers. The chromatography of erythrocyte chromatin on ECTHAM-cellulose gave results that differ dramatically from those obtained from chromatography of f1-containing chromatins on this weak anion exchanger, in that no variations in histone content, circular dichroism spectra or thermal denaturation profiles were observed in the eluted material. Coupled with our earlier finding of no variation in relative content of individual histones (Reeck, G. R. et al. (1974) Eur. J. Biochem. 49, 407–414), we interpret the results of ECTHAM-cellulose chromatography of erythrocyte chromatin to indicate that f2c-depleted regions analogous to the f1-depleted regions found in f1-containing chromatins do not exist in duck erythrocyte chromatin.     

GTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediates

The GTPase activity of the stimulatory guanine nucleotide-binding regulatory protein (Gs) of hormone-sensitive adenylate cyclase was investigated using purified rabbit hepatic Gs and either [alpha-32P]- or [gamma-32P] GTP as substrate. The binding of [35S]guanosine 5'-O-(thiotriphosphate) (GTP gamma S) was used to quantitate the total concentration of Gs. 1) GTPase activity was a saturable function of the concentration of GTP, with Km = 0.3 microM. MgCl2 monotonically increased the activity. The maximum observed turnover number was about 1.5 min-1. 2) During steady-state hydrolysis, 20-40% of total Gs could be trapped as a Gs-GDP complex and 1-2% could be trapped as Gs-GTP. The hydrolysis of Gs-GTP to Gs-GDP occurred with t 1/2 less than or equal to 5 s at 30 degrees C and t 1/2 approximately 1 min at 0 degrees C. Hydrolysis of Gs-GTP was inhibited by 1.0 mM EDTA in the absence of added Mg2+. 3) The rate of formation of Gs-GDP and the initial GTPase rate varied in parallel as functions of the concentrations of either GTP or MgCl2 (above 0.1 mM Mg2+). The ratio of the rate of accumulation of Gs-GDP to the GTPase rate was constant at 0.3-0.4. 4) The rate of dissociation of assayable Gs-GDP was biphasic. The initial phase accounted for 60-80% of total assayable Gs-GDP and was characterized by a t 1/2 of about 1 min. 5) Lubrol 12A9 potently inhibited the GTPase reaction and the dissociation of Gs-GDP in parallel, and inhibition of product release may account for the inhibition of steady-state hydrolysis. 6) The beta and gamma subunits of Gs markedly inhibited the dissociation of GDP from Gs in contrast to their ability to stimulate the dissociation of GTP gamma S. 7) GDP, GTP gamma S, and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) competitively inhibited the accumulation of Gs-GDP. GTP gamma S and Gpp(NH)p inhibited the GTPase reaction noncompetitively, GDP displayed mixed inhibition, and Pi did not inhibit. These data are interpretable in terms of the coexistence of two specific mechanistic pathways for the overall GTPase reaction.     

A novel mechanism for the inhibition of adenylate cyclase via inhibitory GTP-binding proteins. Calmodulin-dependent inhibition of the cyclase catalyst by the beta gamma-subunits of GTP-binding proteins

The adenylate cyclase catalytic protein partially purified from rat brain membranes was activated by the stimulatory GTP-binding protein (Gs), forskolin, and Ca2+-calmodulin. The Ca2+-calmodulin-stimulated activity was markedly, but the Gs- or forskolin-stimulated activity was essentially not, inhibited by low concentrations of the beta gamma-subunits of the inhibitory GTP-binding protein (Gi). The inhibition appeared to be competitive with calmodulin. On the other hand, the association of increasing amounts of beta gamma with the alpha of Gi, which was measured based on the ADP-ribosylation by islet-activating protein, pertussis toxin, was apparently competed by Ca2+-calmodulin. Furthermore, beta gamma bound to calmodulin-Sepharose in the presence of Ca2+, but not in its absence. Thus, the direct interaction of beta gamma with calmodulin is a likely mechanism involved in beta gamma-induced inhibition of the calmodulin-stimulated adenylate cyclase.     

Effects of pertussis toxin-catalyzed ADP-ribosylation on interactions of transducin and the inhibitory GTP-binding protein of adenylate cyclase with guanyl nucleotides

Release of bound [3H]Gpp(NH)p from NG108-15 cell membranes was induced by carbamylcholine, enkephalinamide, and norepinephrine, all of which inhibit adenylate cyclase. Release was blocked by antagonist, was greater with multiple agonists than with one, and required guanyl nucleotides. With membranes from pertussis toxin-treated cells, both total [3H] Gpp(NH)p binding and agonist-induced [3H]Gpp(NH)p release was decreased. ADP-ribosylation by toxin of transducin, the retinal GTP-binding protein which is similar in structure and function to that in cyclase, decreased [3H]Gpp(NH)p binding. Thus, the inability to demonstrate agonist-induced [3H]Gpp(NH)p release from toxin-treated NG108-15 membranes may result in part from absence of bound [3H]Gpp(NH)p.     

A proper transmembrane Ca2+ gradient is essential for the stimulation of adenylate cyclase activity by stimulatory GTP-binding protein

Stimulatory GTP-binding Protein (Gs) and adenylate cyclase prepared from bovine brain cortices were co-reconstituted into asolectin vesicles with or without 1000-fold transmembrane Ca²⁺ gradient. The results showed that both basal activity and Gs-stimulated activity of adenylate cyclase were highest in proteoliposomes with a transmembrane Ca²⁺ gradient similar to physiological condition (1 M Ca²⁺ outside and 1 mM Ca²⁺ inside) and lowest when the transmembrane Ca²⁺ gradient was in the inverse direction. Such a difference could be diminished following dissipation of the transmembrane Ca²⁺ gradient by A23187. Comparable conformational changes of Gs in proteoliposomes were also observed when Gs was labeled with the fluorescence probe, acrylodan. These results may indicate that a proper transmembrane Ca²⁺ gradient is essential not only for higher adenylate cyclase activity but also for its stimulation by Gs.     

Signal transduction pathway for IL-1. Involvement of a pertussis toxin-sensitive GTP-binding protein in the activation of adenylate cyclase

Human Il-1 alpha induces the synthesis of kappa Ig L chains by the pre-B cell line 7OZ/3, IL-2R alpha by the human NK cell line YT, and PGE2 by human rheumatoid synovial cells. Pertussis toxin (PT) markedly inhibited all three IL-1-induced activation events. The inhibition by PT was associated with a decrease in IL-1-mediated cAMP production. PT also inhibited IL-1-stimulated cAMP production in crude membrane fractions from 7OZ/3, YT, and 3T3 fibroblasts. In addition, IL-1 stimulated GTPase activity present in the membranes IL-1-responsive cells. Furthermore, the IL-1-induced GTPase activity was sensitive to PT. PT induced the ADP-ribosylation of a 46-kDa substrate in membrane preparations from IL-1-responsive cells. Cholera toxin also induced the ADP-ribosylation of a 46-kDa substrate in the same membrane preparations. The present findings indicate that the IL-1R is linked to a PT-sensitive G protein that stimulates the activity of adenylate cyclase.     

Identification of a prostacyclin receptor coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mouse mastocytoma P-815 cells.

A stable analogue of prostacyclin, iloprost, specifically bound to 30,000 x g pellet (the membrane fraction) prepared from mouse mastocytoma P-815 cells. The binding was dependent on time, temperature and pH, and absolutely required a divalent cation. The equilibrium dissociation constant and the maximal concentration of the binding site as determined by Scatchard plot analysis were 10.4 nM and 1.12 pmol/mg of protein, respectively. The Hill coefficient was 1.0, indicating a single entity of binding site and no cooperativity. The binding site was highly specific for iloprost among PGs tested (iloprost much greater than PGE1 greater than carbacyclin greater than PGE2). In contrast, the membrane fraction had the binding site specific for PGE2 and PGE1, which was distinct from the prostacyclin receptor. The dissociation of bound [3H]iloprost from the membrane fraction was specifically enhanced by guanine nucleotides. Furthermore, iloprost dose-dependently enhanced the activity of adenylate cyclase in a GTP-dependent manner. These results indicate that a specific prostacyclin receptor is coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mastocytoma cells.     

Interleukin 2 modulation of adenylate cyclase. Potential role of protein kinase C

Interleukin 2 (IL 2) stimulated DNA synthesis of murine T lymphocytes (CT6) in a concentration-dependent manner, over a range of 1-1000 units/ml. This proliferative effect of IL 2 was attenuated by simultaneous exposure to prostaglandin E2 (PGE)2. In intact cells, IL 2 inhibited both basal and PGE2-stimulated cAMP production; the amount of cAMP generated was dependent upon the relative concentrations of IL 2 and PGE2. The effect of IL 2 on CT6 cell proliferation and cAMP production was mimicked by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which, like IL 2, causes a translocation and activation of protein kinase C. While PGE2 stimulated adenylate cyclase activity in membrane preparations, neither IL 2 nor TPA inhibited either basal or stimulated membrane adenylate cyclase activity. However, when CT6 cells were pretreated with IL 2 or TPA and membranes incubated with calcium and ATP, both basal and PGE2-and NaF-stimulated membrane adenylate cyclase activity was inhibited. This inhibition of adenylate cyclase activity was also observed if membranes from untreated cells were incubated with protein kinase C purified from CT6 lymphocytes in the presence of calcium and ATP. The data suggest that the decreased cAMP production which accompanies CT6 cell proliferation results from an inhibition of adenylate cyclase activity mediated by protein kinase C and that these two distinct protein phosphorylating systems interact to modulate the physiological response to IL 2.