Human fat cell adenylate cyclase. Enzyme characterization and guanine nucleotide effects on epinephrine responsiveness in cell membranes.

Human adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) has been studied in preparations of fat cell membranes ("ghosts"). As reported earlier, under ordinary assay conditions (1.0 mM ATP, 5 mM Mg2+, 30 degrees C, 10 min incubation) the enzyme was activated 6-fold by epinephrine in the presence of the GTP analog, 5'-guanylyl-imidodiphosphate [GMP-P(NH)P] (Cooper, B. et al. (1975) J. Clin. Invest. 56, 1350-1353). Basal activity was highest during the first 2 min of incubation then slowed and was linear for at least the next 18 min. Epinephrine, added alone, was often without effect. but sometimes maintained the initial high rate of basal activity. GMP-P(NH)P alone produced inhibition ("lag") of basal enzyme early in the incubation periods. Augmentation of epinephrine effect by GMP-P(NH)P, which also proceeded after a brief (2 min) lag period, was noted over a wide range of substrate (ATP) concentrations. GTP inhibited basal levels of the enzyme by about 50%. GTP also allowed expression of an epinephrine effect, but only in the sense that the hormone abolished the inhibition by GTP. Occasionally a slight stimulatory effect on epinephrine action was seen with GTP. At high Mg2+ concentration (greater than 10 mM) or elevated temperatures (greater than 30 degrees C) GMP-P(NH)P alone activated the enzyme. Maximal activity of human fat cell adenylate cyclase was seen at 50 mM Mg2+, 1.0 mM ATP, pH 8.2, and 37 degrees C in the presence of 10(-4) M GMP-P(NH)P; under these conditions addition of epinephrine did not further enhance activity. Human fat cell adenylate cyclase of adults was insensitive to ACTH and glucagon even in the presence of GMP-P(NH)P.     

Cholera toxin action on rabbit corpus luteum membranes: effects on adenylyl cyclase activity and adenosine diphospho-ribosylation of the stimulatory guanine nucleotide-binding regulatory component

Cholera toxin elicited 5- to 7-fold stimulation of adenylyl cyclase activity. Half-maximal activation was at 4.42 micrograms/ml cholera toxin. Cholera toxin-mediated activation was time dependent. At 0.1 mM ATP, both guanosine triphosphate (GTP) and nicotinamide adenine dinucleotide (NAD+) were required for cholera toxin activation of luteal adenylyl cyclase. The concentrations of GTP and NAD+ required for half-maximal activation were 1 and 200 microM, respectively. The GTP requirement could be eliminated by increasing the ATP concentration to 1.0 mM. Guanosine-5'-O-(2-thiodiphosphate) [GDP beta S] did not support cholera toxin activation of the luteal enzyme. Cholera toxin treatment increased GTP-stimulated activity, did not significantly alter guanyl-5'-yl imidodiphosphate [GMP-P(NH)P]-stimulated activity, and depressed NaF-stimulated activity. Furthermore, toxin treatment resulted in a 3.4-fold reduction in the Kact values for ovine luteinizing hormone (oLH) to activate adenylyl cyclase. A similar reduction in Kact values for oLH was obtained when concentration-effect curves performed in the presence of GMP-P(NH)P were compared to those performed in the presence of GTP. In addition, luteal membranes treated with cholera toxin and [32P]NAD+ were subjected to autoradiographic analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This treatment resulted in the [32P] adenosine diphospho (ADP)-ribosylation of a 45,000-dalton protein doublet, corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (Ns). As with activation of adenylyl cyclase activity, cholera toxin-specific [32P] ADP-ribosylation was time dependent and increased with increasing concentrations of cholera toxin. GTP, GMP-P(NH)P, and NaF, but not GDP beta S, were capable of supporting [32P] ADP-ribosylation of the protein doublet. oLH did not alter the ability of cholera toxin to ADP-ribosylate the protein activation of luteal adenylyl cyclase activity is due to the ADP-ribosylation of the alpha subunit of Ns and the concomitant inhibition of a GTPase associated with adenylyl cyclase.     

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

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

Effects of luteinizing hormone-releasing hormone agonist and calcium upon adenylyl cyclase activity of human corpus luteum membranes

We have investigated the ability of the agonist analog of luteinizing hormone-releasing hormone (LH-RH), D-Trp6-LH-RH (LH-RHa), and of CaCl2 to inhibit directly gonadotropin stimulation of adenylyl cyclase in a cell-free system prepared from human corpus luteum. In the presence of a submaximally effective concentration of hCG, addition of 10(-5)M final concentration of LH-RHa did not alter the gonadotropin-stimulated enzyme activity, nor did LH-RHa alone show any effect upon basal levels of the enzyme. The failure to inhibit adenylyl cyclase would indicate that the LH-RHa does not affect gonadotropin receptor binding or cAMP synthesis and/or degradation in this membrane system, suggesting that the luteolytic effects of LH-RH are unlikely to involve a direct antigonadotropic activity at the level of the human corpus luteum. In great contrast to LH-RHa, addition of CaCl2 resulted in a dose-dependent inhibition of hCG-stimulable adenylyl cyclase. Thus, in the presence of either a maximally or submaximally effective concentration of hCG, inhibition was significant at 0.5 mM CaCl2 added in excess of ATP (2 mM) and EDTA (1 mM), being about 90% upon addition of 2.5 mM CaCl2. We also found that calcium reduced enzyme stimulation by forskolin and the GTP analog, guanyl 5'-yl imidodiphosphate [GMP-P(NH)P] in a dose-related manner and that activation by NaF was less sensitive to inhibition by calcium. Accordingly, at 2.5 mM CaCl2, guanyl nucleotide and forskolin stimulations were inhibited 96% and 86%, respectively, while NaF stimulation was reduced by 40%. Because previous studies have shown that calcium does not impair gonadotropin binding activity, the calcium-dependent inhibition of gonadotropin responsiveness reported here would imply an alteration in the functional coupling of the components of the luteal adenylyl cyclase system. These data suggest that calcium may play a role in the regulation of gonadotropin action in the human corpus luteum.     

Hormone-sensitive adenylate cyclase of prolactin-producing rat pituitary adenoma (GH4C1) cells: molecular organization

Hormonal activation and inhibition of the GH4Cl1 cell adenylate cyclase complex is delineated. In the presence of the guanyl nucleotide GTP, enzyme activity was enhanced twofold by thyroliberin, sixfold by vasoactive intestinal peptide (VIP), twofold by prostaglandin E2 and twofold by isoproterenol. The diterpene, forskolin, increased, the activity 14-fold. In the presence of high GTP (400 microM) and NaCl (150 mM) concentrations, somatostatin inhibited (ED50 = 0.5 microM) the cyclase activity by 40%. In the presence of 10 microM somatostatin, the ED50 values (5 nM) for thyroliberin- and VIP-stimulated adenylate cyclase activities were shifted to 20 nM. Forskolin-elicited activation was, however, not affected by somatostatin. Cholera-toxin and pertussis-toxin pretreatment of the enzyme brought about some 20-fold and twofold activation, respectively. Inhibition by somatostatin was abolished upon pre-exposure to pertussis toxin. Mild alkylation by N-ethylmaleimide increased basal and hormone-activated adenylate cyclase while somatostatin again failed to express its inhibitory potential. Further alkylation caused a gradual decline and convergence of hormone-modulated cyclase activities towards zero. The N-ethylmaleimide-induced attenuation of thyroliberin-elicited activity was paralleled by a decrease in [3H]thyroliberin binding. Trifluoperazine and an anti-calmodulin serum reduced basal and net thyroliberin-, VIP- and forskolin-enhanced cyclase activities by some 30%, 100%, 70% and 80%, respectively. The Vmax of basal and thyroliberin-stimulated adenylate cyclase was diminished by 65%, leaving the apparent Km values (7.2 mM and 2.6 mM, respectively) for Mg2+ unaltered. Finally, the phorbol ester 12-O-tetra-decanoyl-phorbol 13-acetate (TPA) doubled the activity. This effect was counteracted by the protein kinase C inhibitor, polymyxin B, while thyroliberin-enhanced adenylate cyclase remained unaffected. In summary, we have described an adenylate cyclase with stimulatory (Rs) and inhibitory (Ri) receptors coupled to a calmodulin-sensitive holoenzyme through the Gs and Gi type of GTP-binding proteins. The ratio of the Gs to Gi is high. It appears that the GH4C1 cell adenylate cyclase is also activated by protein kinase C by interference with Gi. Apparently, thyroliberin activates the cyclase both directly through Gs and indirectly via protein kinase C stimulation.     

Pertussis toxin-mediated ADP-ribosylation of rabbit luteal Gi uncouples enkephalin inhibition of adenylyl cyclase

1. Some of the actions of pertussis toxin on the rabbit luteal adenylyl cyclase system were analyzed. 2. Incubation of luteal membranes with pertussis toxin and [32P]NAD resulted in the [32P]ADP-ribosylation of a 40,000 Da protein that is distinct from the proteins ADP-ribosylated by cholera toxin. 3. Pertussis toxin specific [32P]ADP-ribosylation was time-dependent and dependent upon the concentration of pertussis toxin present during the incubation. 4. Pertussis toxin mediated [32P]ADP-ribosylation was enhanced by ATP, ADP, adenylyl imidodiphosphate, GTP, guanosine-5'-O-(2-thiodiphosphate), guanosine-5'-O-(3-thiotriphosphate), and NaF but not AMP or guanylyl imidodiphosphate [GMP-P(NH)P]. 5. Treatment of luteal membranes with NAD and pertussis toxin prevents GTP and enkephalin but not GMP-P(NH)P mediated inhibition of forskolin stimulated adenylyl cyclase, demonstrating the existence of a functional Gi in the rabbit corpus luteum.     

Coupling of glucagon receptor to adenylyl cyclase. Requirement of a receptor-related guanyl nucleotide binding site for coupling of receptor to the enzyme.

Effects of glucagon and guanyl nucleotides on the rat liver plasma membrane adenylyl cyclase were studied. It was established that: 1) glucagon stimulates the fully guanyl-5'-yl imidodiphosphate (GMP-P(NH)P)-activated enzyme between 20 and 70%, provided a guanyl nucleotide is present in the assay; 2) glucagon has no effect on adenylyl cyclase activity in membranes activated fully by GMP-P(NH)P and then washed free of nucleotides. It is concluded that occupancy of the guanyl nucleotide binding site that activates the catalytic moiety of the system is not sufficient to promote hormone-receptor coupling to adenylyl cyclase and that occupancy of a second site by guanyl nucleotides is essential to effect stimulation of adenylyl cyclase by the glucagon-receptor complex. The data presented raise the question whether the guanyl nucleotide site that promotes coupling is distinct from the guanyl nucleotide site that modulates binding of glucagon to receptor and whether the occupancy of the guanyl nucleotide site associated with the catalytic moiety is necessary for coupling.     

Association of binding sites for guanine nucleotides with adenylate cyclase activation in rat pancreatic plasma membranes. Interaction of gastrointestinal hormones

1. The activation of rat pancreatic adenylate cyclase by guanosine 5'-(beta-gamma-imido)triphosphate (p[NH]ppG) and GTP, and by the two gastrointestinal hormones pancreozymin (as C-terminal octapeptide) and secretin was correlated with the binding of [8-3H]guanosine 5'-(beta-gamma-imido)triphosphate to rat pancreatic plasma membranes. 2. The low basal adenylate cyclase activity was stimulated 17-fold by p[NH]ppG (after a 2 min lag period), 3,5-fold only by GTP, 21-fold by C-terminal octapeptide of pancreozymin, and 8-fold by secretin. GTP inhibited competitively the activation of adenylate cyclase by p[NH]ppG with a Ki,app almost identical with the Ka,app (0.3 micron). p[NH]ppG and GTP enhanced the stimulation by secretin more markedly than that by the C-terminal octapeptide of pancreozymin, leading to the same maximal activity. Both hormones suppressed the lag period of activation by p[NH]ppG. 3. The binding of [8-3H]p[NH]ppG was dependent on time, temperature and Mg2+ and it was also a saturable and reversible process. Scatchard plots with a concavity upward were linearized after co-addition of ATP, Mg2+ and an ATP-regenerating system that abolished low-affinity sites for p[NH]ppG without saturating higher affinity sites, GTP, ITP and UTP inhibited [8-3H]p[NH]ppG binding to the high-affinity sites in concentration ranges identical with those found for adenylate cyclase activation. Considerable binding of [8-3H]p[NH]ppG was still evident at 20 degrees C, but enzyme activation was not observed any more, except in the presence of hormones.     

Stimulatory and inhibitory effects of guanyl-5'-yl imidodiphosphate on adenylate cyclase activity of cardiac sarcolemma.

A nucleotide phosphohydrolase-resistant analog of GTP, guanyl-5′-yl imidodiphosphate [GMP-P(NH)P], caused stimulation of basal adenylate cyclase activity of cardiac sarcolemma when ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid (EGTA) was absent in the assay mixture, whereas the nucleotide, in the presence of EGTA, inhibited basal cyclase activity. GTP, GDP, GMP, and guanosine failed to show such an inhibition of basal enzyme activity. The degree of both stimulatory and inhibitory effects of GMP-P(NH)P depended on the concentration of magnesium ions. The apparent affinities toward magnesium ions of the metal binding site and toward MgATP^2? of the catalytic site of control and ?GMP-P(NH)P-inhibited” enzyme were similar. Isoproterenol reversed the inhibitory effect, whereas calcium ions failed to revert it. Both in the presence and absence of EGTA, GMP-P(NH)P plus isoproterenol caused a synergistic stimulation of the enzyme activity, the degree of stimulation being lower with EGTA present. Exposure of sarcolemma to GMP-P(NH)P (with and without isoproterenol and in the absence and presence of EGTA) caused an activation of adenylate cyclase, the degree of activation being higher with isoproterenol present. The activated enzyme displayed increased affinity toward Mg²⁺ at the metal binding site. When activated enzyme preparations were assayed in the presence of EGTA, reversal of the activated state was observed in the case of the GMP-P(NH)P-activated enzyme but not in the case of the GMP-P(NH)P + isoproterenol-activated enzyme.     

Reversible inactivation of guanylate cyclase by mixed disulfide formation

Highly purified preparations of guanylate cyclase from rat lung were inactivated by several disulfide compounds in a time- and dose-dependent manner. Cystamine and cystine were the most potent disulfides tested, but other compounds which contained the cysteamine moiety (NH2CH2CH2S-), including pantethine and oxidized coenzyme A, were also able to partially inactivate the enzyme. In addition to the decrease in basal activity (measured with either Mg2+-GTP or Mn2+-GTP), disulfide-inhibited enzyme was activated to a lesser extent by nitric oxide. Treatment with dithiothreitol or other reducing agents restored basal activity and increased the level of cGMP production following nitric oxide activation. Control enzyme samples exhibited a single GTP Km of 25 microM or 150 microM with Mn2+ or Mg2+, respectively. However, cystamine-treated enzyme showed these same Km values as well as an additional GTP Km of 2 to 3 microM using either metal ion as cofactor. When [35S]cystine was incubated with purified enzyme, radioactivity was incorporated into the trichloroacetic acid-precipitable protein, and the counts were released following dithiothreitol treatment. In addition, [35S]cystine-labeled enzyme co-migrated with native guanylate cyclase on nondenaturing polyacrylamide gels. These data indicate that mixed disulfides can be formed between guanylate cyclase and certain naturally occurring compounds, and that disulfide formation leads to a reversible loss of enzyme activity.     

Challenge of hepatocytes by glucagon triggers a rapid modulation of adenylate cyclase activity in isolated membranes.

Membrane fractions obtained from hepatocytes treated with glucagon exhibited a decreased glucagon (with or without GTP)-stimulated adenylate cyclase activity. A maximum effect was seen in around 5 min. No change in the rate of cyclic AMP production was observed for the basal, NaF-, p[NH]ppG (guanosine 5'-[beta, gamma-imido]-triphosphate)- and GTP-stimulated states of the enzyme. The lag observed in the p[NH]ppG-stimulated adenylate cyclase activity of native membranes was abolished when membranes from glucagon-pretreated cells were used. When Mn2+ replaced Mg2+ in the assays, the magnitude of the apparent desensitization was decreased. Mn2+ abolished the lag of onset of p[NH]ppG-stimulated activity in native membranes. The desensitization process was dose-dependent on glucagon, which exhibited a Ka of 4 X 10(-10) M. Depletion of intracellular ATP did not affect this process. It is suggested that this desensitization occurs at the level of the guanine nucleotide-regulatory protein.     

Guanine nucleotide regulation of adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae

Adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae was examined. Among various permeabilization procedures, including organic solvents, detergents and other reagents, dimethylsulfoxide (DMSO) and digitonin treatments resulted in the highest recovery of adenylate cyclase activity. Incubation of cells at 30 degrees C with digitonin at 0.01% to 0.1%, or DMSO at 20% to 40% for 15 to 30 min gave optimal adenylate cyclase activity. The enzyme activity in digitonin-permeabilized cells could be supported only by Mn2+, whereas Mg2+ with or without guanine nucleotides did not support cyclase activity. DMSO-permeabilized cells exhibit efficient Mn2+- and Mg2+/Gpp[NH]p-dependent stimulation. Furthermore, digitonin added to yeast membranes at a 1:50 detergent to protein ratio (w/w) abolishes guanyl nucleotide regulation without significantly affecting the Mn2+-supported cyclase activity. The superiority of DMSO is further supported by the fact that recovery of adenylate cyclase activity is better in the DMSO-treated cells than in the digitonin-treated cells. DMSO most probably causes less disturbance of the fabric of the native cell. We conclude that digitonin, but not DMSO, uncouples the catalytic unit of adenylate cyclase from the regulatory GTP binding (ras) proteins.     

A transplantable rat Leydig cell tumor--3. Isoproterenol and prostaglandin E1 (PGE1) responsive adenylyl cyclase (AC). Regulatory effects of nucleotides and magnesium

The activity of the membrane bound adenylyl cyclase (AC), the effects of nucleotides, Mg2+-cations and its responsiveness to isoproterenol and prostaglandin E1 (PGE1) were examined in a transplantable rat Leydig cell tumor (H-540). Both isoproterenol and PGE1 caused activation of the AC in Leydig cell tumors. The degree of activation by PGE1 (4-5-fold) was approximately twice that of isoproterenol (2-3-fold). The addition of both AC agonists simultaneously was not additive indicating that they activate AC of the same cell. Increasing concentrations of ATP (0.025-2.0 X 10 mM) caused a concentration dependent increase in both the basal and hormone stimulated AC activity, and the activation by isoproterenol and PGE1 (relative response) revealed a slight but significant increase with increasing ATP concentrations. Lineweaver-Burke analysis of these data indicated an apparent Km for ATP (Mg X ATP) of 0.16 mM. Free magnesium did not influence the apparent Km of the AC for ATP. Increasing concentrations of free Mg2+ (0.24-13.2 mM) also caused a concentration dependent increasing activation of AC activity up to a concentration of approx 6 mM in excess of Mg2+-binding ingredients. Higher concentrations of free Mg2+ (13.1 mM) caused a small but significant decrease in both basal and agonist stimulated AC activity. In contrast to other reports, activation by isoproterenol and PGE1 was in general not influenced by the concentration of Mg2+. Both GTP and GMP-P(NH)P stimulated basal and hormone stimulated AC activity (Kact 1 microM), but with different kinetics. In the presence of GTP, AC activity was almost constant for 90 min. In the presence of GMP-P(NH)P, AC activity was much higher, but constant AC activity occurred after a certain lag time (7-10 min), which was reduced by PGE1 and isoproterenol. In conclusion, cAMP production in Leydig cell tumors is stimulated by both PGE1 and isoproterenol. The AC activity and activation by these agonists are regulated by Mg2+ and nucleotides in a slightly different manner from most other cells. The association between AC activation and stimulation of steroid production by Leydig cell tumors remains to be investigated.     

Effect of GTP analogues on purified soluble guanylate cyclase

In our studies with purified soluble guanylate cyclase from rat lung, we have tested a number of guanosine 5'-triphosphate (GTP) analogues as substrates and inhibitors, 5'-Guanylylimidodiphosphate (GMP-P(NH)P), guanylyl (beta, gamma-methylene) diphosphate (GMP-P(CH2)P), and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) were found to be substrates for guanylate cyclase. GTP gamma S supported cyclic GMP formation at 20 or 75% of the rate seen with Mn2+-GTP and Mg2+-GTP, respectively. GMP-P(NH)P and GMP P(CH2)P supported cyclic GMP formation at 10-20% of the GTP rate with either cation cofactor. These analogues were found to have multiple Km values; one Km value was similar to GTP (150 microM with Mg2+, 20-70 microM with Mn2+), but an additional high affinity catalytic site (3 microM) was also observed. Guanosine tetraphosphate (Ki = 10 microM), adenosine triphosphate (Ki = 9 microM) and the 2'3'-dialdehyde derivative of GTP (dial GTP) (Ki = 1 microM) were not good substrates for the enzyme; however, they were potent competitive inhibitors. These GTP analogues will be useful tools for the study of GTP binding sites on guanylate cyclase and they may also help elucidate the effects of free radicals and other agents on guanylate cyclase regulation.     

Solubilization of receptors for thyrotropin-releasing hormone from GH4C1 rat pituitary cells: demonstration of guanyl nucleotide sensitivity

TRH receptors have been solubilized from GH4C1 cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH [( 3H]MeTRH) attained equilibrium within 2-3 h at 4 C, and it was reversible, dissociating with a t1/2 of 7 h. Analysis of [3H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (Kd) of 3.8 nM and a total binding capacity (Bmax) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [3H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC50 of 11 microM. When [3H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [3H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [3H]MeTRH dissociation by 10 microM GTP gamma S occurred within about 45 min at 22 C. GTP gamma S, GTP, GDP beta S, and GDP were all effectors of [3H]MeTRH dissociation, exhibiting EC50s in the range of 14-450 nM. The rank order of potency of the tested nucleotides was GTP gamma S greater than GTP congruent to GDP beta S greater than GDP much greater than ATP gamma S greater than GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [3H]MeTRH to GH4C1 cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein.     

Adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is not regulated by guanyl nucleotides

The adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is localized to the plasma membrane of the cell. The enzyme utilizes Mn2+/ATP as substrate and free Mn2+ ions as an effector. Unlike the baker yeast Saccharomyces cerevisiae, S. pombe adenylyl cyclase does not utilize Mg2+/ATP as substrate and the activity is not stimulated by guanyl nucleotides. The optimal pH for the S. pombe adenylyl cyclase activity is 6.0. The activity dependence on ATP is cooperative with a Hill coefficient of 1.68 +/- 0.14.     

Thyrotropin-releasing hormone and GTP activate inositol trisphosphate formation in membranes isolated from rat pituitary cells

Stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by a phospholipase C to produce inositol trisphosphate (InsP3) and 1,2-diacylglycerol appears to be the initial step in signal transduction for a number of cell-surface interacting stimuli, including thyrotropin-releasing hormone (TRH). In suspensions of membranes isolated from rat pituitary (GH3) cells that were prelabeled to isotopic steady state with [3H]inositol and incubated with ATP, [3H] PtdIns(4,5)P2, and [3H]phosphatidylinositol 4-phosphate, the polyphosphoinositides, and [3H]InsP3 and [3H]inositol bisphosphate, the inositol polyphosphates, accumulated. TRH and GTP stimulated the accumulation of [3H]inositol polyphosphates in time- and concentration-dependent manners; half-maximal effects occurred with 10-30 nM TRH and with 3 microM GTP. A nonhydrolyzable analog of GTP also stimulated [3H] inositol polyphosphate accumulation. Moreover, when TRH and GTP were added together their effects were more than additive. Fixing the free Ca2+ concentration in the incubation buffer at 20 nM, a value below that present in the cytoplasm in vivo did not inhibit stimulation by TRH and GTP of [3H]inositol polyphosphate accumulation. ATP was necessary for basal and stimulated accumulation of [3H]inositol polyphosphates, and a nonhydrolyzable analog of ATP could not substitute for ATP. These data demonstrate that TRH and GTP act synergistically to stimulate the accumulation of InsP3 in suspensions of pituitary membranes and that ATP, most likely acting as substrate for polyphosphoinositide synthesis, was necessary for this effect. These findings suggest that a guanine nucleotide-binding regulatory protein is involved in coupling the TRH receptor to a phospholipase C that hydrolyzes PtdIns(4,5)P2.     

Photolysis of water coupled to nitrate reduction by Nostoc muscorum subcellular particles.

The binding of tritiated guanylylimidodiphosphate ([³H]GMP-P(NH)P) to turkey erythrocyte ghosts was studied in parallel with the activation by GMP-P(NH)P of adenylate cyclase. The high affinity binding capacity for GMP-P(NH)P, 50 pmoles per mg protein, exceeds the estimated quantity of adenylate cyclase of 1 pmole per mg of protein. The rate of nucleotide binding is not affected by isoproterenol. Further, in the presence of the hormone the rate of binding is much slower than the rate of activation. Although the rate of dissociation of bound [³H]GMP-P(NH)P is negligible at 37°, it is increased dramatically by unlabeled GMP-P(NH)P, GTP, EDTA, ATP, AMP-P(CH₂)P, or p-aminophenylmercuric acetate. In contrast, the rate of decay of the GMP-P(NH)P-simulated state is not altered by these agents. Thus, the major fraction of GMP-P(NH)P binding to membranes is not relevant to cyclase activation.     

Thyrotropin-releasing hormone activates a Ca2+-dependent polyphosphoinositide phosphodiesterase in permeable GH3 cells. GTP gamma S potentiation by a cholera and pertussis toxin-insensitive mechanism

Numerous hormones are known to rapidly activate polyphosphoinositide turnover in target cells by promoting phosphodiesteratic cleavage of the phospholipids; however, little is known about the enzymology of receptor-mediated phosphoinositide breakdown. In the present study, thyrotropin-releasing hormone (TRH) stimulation of polyphosphoinositide turnover has been characterized in electrically permeabilized, [3H]myoinositol-labeled GH3 cells. The permeable cells allow the influence of small molecular weight (Mr less than or equal to 1000) cofactors to be determined. We present evidence for the following: 1) TRH stimulates inositol phosphate generation in permeable cells; 2) optimal hormone-stimulated inositol phosphate generation requires Mg2+, ATP, and Ca2+; 3) Mg2+ and ATP requirements reflect polyphosphoinositide kinase reactions; 4) in the absence of MgATP, TRH stimulates the phosphodiesteratic breakdown of pre-existing polyphosphoinositides in a reaction which requires only low Ca2+ (10(-7) M); 5) hormone activation is potentiated in the presence of the stable guanine nucleotide, GTP gamma S; neither TRH-stimulated nor GTP gamma S-potentiated hydrolysis is inhibited by cholera or pertussis toxin treatment. These results demonstrate that hormone-induced phospholipid hydrolysis involves activation of a phosphoinositide phosphodiesterase; activation results in lowering the Ca2+ requirement of the phosphodiesterase such that maximal activity is observed at Ca2+ levels characteristic of a resting cell (10(-7) M). Furthermore, TRH regulation of polyphosphoinositide hydrolysis is modulated by guanine nucleotides; however, nucleotide regulation appears to involve a GTP-binding factor (Np) other than Ns or Ni.     

Constitutively active mu-opioid receptors inhibit adenylyl cyclase activity in intact cells and activate G-proteins differently than the agonist [D-Ala2,N-MePhe4,Gly-ol5]enkephalin

The most convincing evidence demonstrating constitutive activation of mu-opioid receptors is the observation that putative inverse agonists decrease basal G-protein activity in membrane preparations. However, it is not clear whether constitutively active receptors in isolated membranes have any physiological relevance in intact cells. GH3 cells expressing mu-opioid receptors (GH3MOR) exhibit higher basal G-protein activity and lower basal cAMP levels than wild-type GH3 cells, indicative of constitutively active receptors. This study determined whether alkylation of mu-opioid receptors by the irreversible antagonist beta-funaltrexamine would decrease spontaneous receptor activity in intact cells, revealing constitutive activity. GH3MOR cells were pretreated with increasing concentrations of beta-funaltrexamine followed by functional testing after removal of unbound drug. beta-Funaltrexamine pretreatment produced a concentration-dependent decrease in mu-opioid receptor binding with an IC50 of 0.98 nm and an Emax of 77%. Similar concentrations of beta-funaltrexamine pretreatment produced a half-maximal reduction in basal [35S]GTPgammaS binding, a decrease in basal photolabeling of G-proteins with azidoanilido-[alpha-32P]GTP, and an increase in basal adenylyl cyclase activity in intact cells. Therefore, mu-opioid receptors are constitutively active in intact cells, producing stimulation of G-proteins and inhibition of adenylyl cyclase. Importantly, photolabeling of Galpha-subunits with azidoanilido-[alpha-32P]GTP demonstrated that constitutively active mu-opioid receptors activate individual G-proteins differently than the agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin.