Evidence for a rabbit luteal ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase.

1. An ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase was identified in a plasma membrane fraction from rabbit corpora lutea and partially characterized by comparing the properties of the luteal transferase with those of cholera toxin. 2. Incubation of luteal membranes in the presence of GTP and varying concentrations of NAD resulted in concentration-dependent increases in adenylyl cyclase activity. 3. Stimulation of adenylyl cyclase by NAD and cholera toxin plus NAD was observed in the presence of GTP but not in the presence of guanosine-5'-O-(2-thiodiphosphate) or guanyl-5'-yl imidodiphosphate. 4. NAD or cholera toxin plus NAD reduced the Kact values for luteinizing hormone to activate adenylyl cyclase 3- to 3.5-fold. 5. NAD or cholera toxin plus NAD increased the extent to which cholate extracts from luteal membranes were able to reconstitute adenylyl cyclase activity in S49 cyc- mouse lymphoma membranes. 6. It was necessary to add ADP-ribose and arginine to the incubation mixture in order to demonstrate cholera toxin-specific ADP-ribosylation of a protein corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (alpha Gs). 7. Treatment of luteal membranes with NAD prior to incubation in the presence of [32P]NAD plus cholera toxin resulted in reduced labeling of alpha Gs. 8. Endogenous ADP-ribosylation of alpha Gs was enhanced by Mg but was not altered by guanine nucleotide, NaF or luteinizing hormone and was inhibited by cAMP. 9. Incubation of luteal membranes in the presence of [32P]ADP-ribose in the absence and presence of cholera toxin did not result in the labeling of any membrane proteins.     

Interaction of the stimulatory and inhibitory regulatory proteins of the adenylyl cyclase system with the catalytic component of cyc-S49 cell membranes

The mechanism by which Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclases, regulate the activity of the catalytic component (C) of adenylyl cyclase was investigated using cyc-S49 cell membranes which contain a functional inhibitory regulatory protein (Ni) but not the active subunit of the stimulatory regulatory protein (Ns). To this end, purified Ns protein was preactivated (Ns) in solution with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and Mg2+, and then added to cyc- membranes under conditions where Ni was either unactivated or activated (Ni) by GTP gamma S and Mg2+. Activation of Ni in cyc- membranes resulted in a lowered expression of Ns activity under all conditions tested. Upon dilution of the reactants (Ns and cyc- membranes) the reconstituted activity declined in proportion to the dilution with an approximate t 1/2 of 30-45 min, being unaffected by activation of Ni. Postactivation of Ni after reconstitution of cyc- membranes with Ns resulted in a time-dependent decline in Ns activity to a level that was the same as that obtained when Ns was added to cyc- membranes with preactivated Ni. These data indicated that the effects of Ns on C are of a reversible type. The following indicated that Ns and Ni affect C activity in a noncompetitive manner: (a) the per cent reduction in Ns activity due to activation of Ni was constant and independent of the concentration of Ns, (b) double reciprocal plots of activities reconstituted in control and Ni-containing cyc- membranes versus Ns concentration were linear with an unaltered apparent Km for Ns, and (c) the onset of inhibition of C prereconstituted with Ns was much faster (approximate t 1/2 = 2-5 min) than expected if it were due to occupancy of a common site on C left vacant by Ns.(ABSTRACT TRUNCATED AT 400 WORDS)     

Treatment of oocyte membranes with the 2',3'-dialdehyde of guanosine triphosphate reduces progesterone inhibition of adenylyl cyclase

Treatment of Xenopus laevis membranes with the 2',3'-dialdehyde of GTP (dial GTP) drastically inhibits their adenylyl cyclase activity. Optimal inhibition is obtained by treatment with 1 mM dial GTP for 1h at 32 degrees C. Using guanyl-5'-yl imidodiphosphate, F-, forskolin and Mn2+ as activators of the enzyme it can be concluded that dial GTP preferentially reacts with the stimulatory subunit (Ns) and slightly with the catalytic subunit. Dial GTP treatment greatly reduces the inhibition of adenylyl cyclase by progesterone. Pure exogenous Ns stimulates the enzyme but does not restore progesterone inhibition. Treatment with dial [alpha-32P]GTP labels several membrane proteins some of which have similar Mr to Ns and Ni.     

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.     

Identification of a gamma subunit associated with the adenylyl cyclase regulatory proteins Ns and Ni

The subunit composition of the Ns and Ni, the human erythrocyte stimulatory and inhibitory regulatory proteins of adenylyl cyclase, respectively, were analyzed by a sodium dodecyl sulfate-containing discontinuous urea and polyacrylamide gradient gel electrophoresis system designed for the study of low molecular weight polypeptides. This system disclosed that these proteins, in addition to their known alpha and beta subunits, contain an additional small peptide of apparent molecular weight of 5,000 (5K). This "5K peptide" is also present in preparations of another protein which we termed "40K protein" on the basis of its hydrodynamic behavior and whose primary protein constituent is the Mr 35,000 beta subunit of the above regulatory proteins. Analyzing Ni, the 5K peptide was functionally related to the protein by showing that its apparent Stokes radius changes from 5.9 to 5.1 nm after treatment with guanyl-5'-yl imidodiphosphate and magnesium in parallel with the alpha and beta subunits. These data are interpreted as evidence for the existence of a third subunit associated with the regulatory proteins of adenylyl cyclase. We call this subunit gamma and propose a minimum subunit structure for these proteins of the alpha beta gamma type.     

Glucagon receptor-mediated activation of Gs is accompanied by subunit dissociation

The effect of the glucagon receptor on the activation of the stimulatory GTP-binding protein of adenylyl cyclase (Gs) in the native rat liver membrane environment was studied. The activated state of Gs was assessed by its ability to reconstitute the cyc- S49 cell membrane adenylyl cyclase. The Gs protein was activated by saturating concentrations of guanosine 5'-thiotriphosphate (GTP gamma S) or guanyl-5'-yl imidodiphosphate in a hormone-dependent manner at 0.4 mM Mg2+ in native membranes or in membranes that had been treated with 1 mM N-ethylmaleimide to eliminate the catalytic activity of adenylyl cyclase. At 50 mM Mg2+, Gs was fully activated by GTP gamma S in the absence of hormone. The unactivated Gs protein migrates around 4 S, whereas activated Gs migrates around 2 S on sucrose density gradients. When pure Gs is analyzed on sucrose density gradients, it is found that the unactivated protein migrates at 4.1 S. Gs was activated by saturating concentrations of GTP gamma S and Mg2+, and the alpha subunit of Gs was chromatographically purified. The resolved alpha subunit of Gs that is capable of stimulating the cyc- adenylyl cyclase migrates at 2.1 S. From these data, we conclude that activation of Gs results in the dissociation of this protein in the membrane environment and that the hormone-occupied receptor promotes this dissociation process under conditions where Mg2+ ions are limiting.     

GTP hydrolysis by pure Ni, the inhibitory regulatory component of adenylyl cyclases

The stimulatory and inhibitory regulatory components of adenylyl cyclase (Ns and Ni), purified to apparent homogeneity without the use of regulatory ligands such as Mg, NaF, and guanyl-5'-yl imidodiphosphate, were tested for GTPase activity by incubating them with [gamma-32P]GTP and measuring 32Pi liberation using a charcoal adsorption assay to separate hydrolyzed from nonhydrolyzed radioactivity. We found that Ni is capable of hydrolyzing GTP. The activity was shown to be due to Ni itself and not to presence of one of its minor contaminants by correlating activity with abundance of the 40,000 Da alpha i subunit throughout the last stages of purification and by showing co-migration on a sucrose density gradient of the GTP-hydrolyzing activity with the alpha i, beta, and gamma subunits of Ni and not with any one of three minor contaminants present in the preparation tested. Preparations of Ns, free of detectable Ni, exhibited less than 10% the capacity to hydrolyze GTP, as compared to Ni on an equal protein basis. The basic properties of the GTP-hydrolyzing activity of Ni were determined. The activity is dependent on Mg ion (apparent Km = 5 to 15 nM), and is rapidly lost upon incubation with Mg2+ in the absence of GTP. MgGTP and free GTP serve equally well as substrate (apparent Km about 40 nM). Isotopic dilution studies indicate that the GTP binding site has a relative affinity for guanine nucleotides in the order GTP = GTP gamma S greater than GDP = GMP-P(NH)P greater than GDP beta S with the highest difference (GTP versus GDP beta S) being about 10-fold. NaF inhibited GTP hydrolysis by Ni at concentrations at which it activates Ni in intact membranes.     

Guanosine 5'-O-(3-thiotriphosphate) reduces ADP-ribosylation of the inhibitory guanine nucleotide-binding regulatory protein of adenylyl cyclase (Ni) by pertussis toxin without causing dissociation of the subunits of Ni. Evidence of existence of heterotrimeric pt+ and pt- conformations of Ni

The effect of the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), the GTP analog which activates the inhibitory guanine nucleotide-binding regulatory protein of adenylyl cyclase (Ni), on the pertussis toxin-mediated ADP-ribosylation reaction was studied in detail. Two effects were discerned: a stimulation of the ADP-ribosyltransferase activity of the toxin, akin to what was described for ATP and GDP in a previous report (Mattera, R., Codina, J., Sekura, R., and Birnbaumer, L. (1986) J. Biol. Chem. 261, 11173-11179), and a decrease in the ability of Ni to be a substrate for the activated toxin. Both effects were time-dependent with activation of the toxin being somewhat faster than inactivation of Ni. The effect of the addition of GTP gamma S on Ni was readily reversed by excess GDP and attenuated by increasing EDTA in the medium from 0.35 to 10 mM, suggesting dependence on trace concentrations of a divalent cation. It is suggested that this cation is Mg2+ on the basis that low (5-10 nM) concentrations of Mg2+ are needed for the endogenous GTPase activity of Ni (Sunyer, T., Codina, J., and Birnbaumer, L. (1984) J. Biol. Chem. 259, 15447-15451). Sucrose density gradient analysis of the Ni X GTP gamma S complexes with decreased susceptibility to ADP-ribosylation by pertussis toxin showed the same sedimentation parameters as Ni or Ni X GDP complexes, indicating that the molecule of Ni with GTP gamma S bound is heterotrimetric as opposed to dissociated into alpha i X GTP gamma S plus beta X gamma. Thus, these experiments define two conformations of heterotrimeric Ni: one -pt+, ADP-ribosylated by pertussis toxin, and the other pt-, poorly or not ADP-ribosylated by pertussis toxin. This latter, hitherto unrecognized conformation, is stabilized by the addition of strongly activating guanine nucleotides such as GTP gamma S and guanyl-5'-yl imidodiphosphate and should be important in the train of events that lead from an inactive heterotrimeric Ni to a fully active and dissociated Ni.     

Relationship Among Calmodulin-, Forskolin-, and Guanine Nucleotide-Dependent Adenylate Cyclase Activities in Cerebellar Membranes: Studies by Limited Proteolysis

Adenylate cyclase activity in bovine cerebellar membranes is regulated by calmodulin, forskolin, and both stimulatory (Ns) and inhibitory (Ni) guanine nucleotide-binding components. The susceptibility of the enzyme to chymotrypsin proteolysis was used as a probe of structure-function relationships for these different regulatory pathways. Pretreatment of membranes with low concentrations of chymotrypsin (1-2 micrograms/ml) caused a three- to fourfold increase in basal adenylate cyclase activity and abolished the Ca2+-dependent activation of the enzyme by calmodulin. In contrast, the stimulation of the enzyme by GTP plus isoproterenol was strongly potentiated after protease treatment, an effect that mimics the synergistic activation of adenylate cyclase by Ns and calmodulin in unproteolyzed membranes. Limited proteolysis revealed low- and high-affinity components in the activation of adenylate cyclase by forskolin. The low-affinity component was readily lost on proteolysis, together with calmodulin stimulation of the enzyme. The activation via the high-affinity component was resistant to proteolysis and nonadditive with the Ns-mediated activation of the enzyme, suggesting that both effectors utilize a common pathway. The inhibitory effect of low concentrations (10(-7) M) of guanyl-5'-yl imidodiphosphate [Gpp(NH)p] on forskolin-activated adenylate cyclase was retained after limited proteolysis of the membranes, indicating that the proteolytic activation does not result from an impairment of the Ni subunit. Moreover, in the rat cerebellum, proteolysis as well as calmodulin was found to enhance strongly the inhibitory effect of Gpp(NH)p on basal adenylate cyclase activity. Our results suggest that calmodulin and Ns/Ni interact with two structurally distinct but allosterically linked domains of the enzyme. Both domains appear to be involved in the mode of action of forskolin.     

N-ethylmaleimide uncouples the glucagon receptor from the regulatory component of adenylyl cyclase

125I-Glucagon binding to rat liver plasma membranes was composed of high- and low-affinity components. N-Ethylmaleimide (NEM) and several other alkylating agents induced a dose-dependent loss of high-affinity sites. This diminished the apparent affinity of glucagon receptors for hormone without decreasing the binding capacity of membranes. Solubilized hormone-receptor complexes were fractionated as high molecular weight (Kav = 0.16) and low molecular weight (Kav = 0.46) species by gel filtration chromatography; NEM or guanosine 5'-triphosphate (GTP) diminished the fraction of high molecular weight complexes, suggesting that NEM uncouples glucagon receptor-N-protein complexes. Exposure of intact hepatocytes to the impermeable alkylating reagent p-(chloromercuri)benzenesulfonic acid failed to diminish the affinity of glucagon receptors on subsequently isolated plasma membranes, indicating that the thiol that affects receptor affinity is on the cytoplasmic side of the membrane. Hormone binding to plasma membranes was altered by NEM even after receptors were uncoupled from N proteins by GTP. These data suggest that a sensitive thiol group that affects hormone binding resides in the glucagon receptor, which may be a transmembrane protein. Alkylated membranes were fused with wild-type or cyc- S49 lymphoma cells to determine how alkylation affects the various components of the glucagon-adenylyl cyclase system. Stimulation of adenylyl cyclase with fluoride, guanylyl 5'-imidodiphosphate, glucagon, or isoproterenol was observed after fusion of cyc- S49 cells [which lack the stimulatory, guanine nucleotide binding, regulatory protein of adenylyl cyclase (Ns)] with liver membranes alkylated with 1.5 mM NEM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of a hormone-sensitive adenylate cyclase with membrane extracts from Neurospora and avian erythrocytes

Adenylate cyclase activity associated to wild type Neurospora membranes is highly dependent on Mn2+ and insensitive to fluoride, guanyl nucleotides, and cholera toxin. These membranes are able to interact with components of detergent extracts from turkey erythrocyte ghosts. The reconstituted cyclase system is catalytically active in the presence of Mg2+ and it is activated by guanyl-5'-yl imidodiphosphate plus isoproterenol and fluoride. When detergent extracts were prepared from avian erythrocyte membranes treated with cholera toxin, the reconstituted system was stimulated by guanyl-5'-yl imidodiphosphate in the absence of isoproterenol and cyclase activities were higher than those observed with extracts from membranes not treated with the toxin. Dose-response curves for isoproterenol and fluoride in the reconstituted system were similar to those reported for avian erythrocyte and liver membranes, respectively.     

1,25-Dihydroxycholecalciferol and glucocorticosteroid regulation of adenylate cyclase in an osteoblast-like cell line

To study regulation of the parathyroid hormone (PTH)-responsive adenylate cyclase of osteoblast-like cells by 1,25-dihydroxyvitamin D (1,25(OH)2D), cAMP levels and adenylate cyclase activity were assayed in the hormone-responsive ROS 17/2.8 rat osteosarcoma cell line. Treatment of cells with 1,25(OH)2D3: alone markedly attenuated the cAMP response to subsequent PTH; decreased adenylate cyclase stimulated by PTH; and completely antagonized the positive regulatory effects of cell treatment with glucocorticosteroid (GC) on these responses to PTH. Sterol receptor mediation was indicated by specificity for the 1,25(OH)2D metabolite and high sensitivity (half-maximal attenuation at 7 X 10(-11) M). The effects of 1,25(OH)2D and GC were primarily on the maximal activity of adenylate cyclase and not on sensitivity to Mg2+, guanine nucleotide, or PTH. GC augmentation of ROS 17/2.8 cell cAMP accumulation was also seen with another receptor agonist (beta-adrenergic), cholera toxin or forskolin; 1,25(OH)2D antagonized all these GC effects. Opposing effects of GC and 1,25(OH)2D were seen as well on activation of the guanine nucleotide-binding regulatory protein (Ns) by guanyl-5'-yl imidodiphosphate and F- and on activation of the catalyst (C) by Mn2+. In contrast, with the activators other than PTH, cell treatment with 1,25(OH)2D in the absence of GC produced only minor attenuation of cAMP accumulation and no effect on adenylate cyclase activities. The data suggest that GC acts strongly on or near the PTH receptor-Ns complex in ROS 17/2.8 and to a lesser degree on the Ns-C interaction. Direct GC enhancement of C could not be concluded because of the influence of Ns on forskolin action and present data that Mn2+ does not uncouple Ns from C in this system. A GC effect on membrane structure or composition, as seen in other cell types, could explain these changes in adenylate cyclase function without the need to postulate multiple mechanisms. The data dissociate two 1,25(OH)2D effects, direct attenuation of activation of Ns via the PTH receptor and interference with the as yet undefined mechanism(s) of GC augmentation. These may represent dissimilar pathways of 1,25(OH)2D action on osteoblasts.     

Regulation of hormone-receptor coupling to adenylyl cyclase. Effects of GTP and GDP

GDP and GTP regulation of receptor-mediated stimulation of adenylyl cyclases in membranes of S49 murine lymphoma cells (S49), NS-20 murine neuroblastoma cells (NS-20), rabbit corpora lutea (CL), and turkey erythrocytes were studied under assay conditions which minimized conversion of added GTP to GDP and of added GDP to GTP. Hormonal stimulation in all systems required guanine nucleotide addition. In the presence of GTP, adenylyl cyclase activity in S49, NS-20, and CL was stimulated respectively by isoproterenol and prostaglandin E1 (PGE1), by PGE1 and the adenosine analog, phenylisopropyladenosine, and by PGE1 and isoproterenol, with the first of the listed stimulants eliciting higher activities than the second. Activity in turkey erythrocyte membranes was stimulated by isoproterenol. GDP was partially effective in promoting hormonal stimulation, being able to sustain stimulation by isoproterenol and PGE1 in S49 cell membranes and by PGE1 in CL membranes. In NS-20 membranes, both GDP and guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) were inhibitory on basal activity, yet promoted limited but significant stimulation by PGE1. In turkey erythrocytes, stimulation by isoproterenol could not be elicited with GDP or GDP beta S. Thus, although less effective than GTP in promoting hormonal stimulation of several adenylyl cyclase systems, GDP was clearly not inactive. Concentration effect curves for active hormone in the presence of GDP had higher apparent Ka values than in the presence of GTP. In spite of differences between the effects of GTP and GDP on hormonal stimulation of adenylyl cyclase activities, GTP and GDP affected equally well isoproterenol binding, regardless of whether or not its receptor could be shown to stimulate adenylyl cyclase in the presence of GDP. Determination of transphosphorylation of GDP to GTP showed that at saturating concentrations, the proportion of GDP converted to GTP is negligible and unaffected by hormonal stimulation. Concentrations giving 50% inhibition were determined for GTP- and GDP-mediated inhibition of guanyl-5'-yl imidodiphosphate stimulation in the absence and presence of stimulatory hormones. In all four systems studied, GTP and GDP interacted with about equal potency and hormonal stimulation was not accompanied by a selective decrease in affinity for GDP. One way to explain all of the results obtained is to view hormonally sensitive adenylyl cyclase systems as two-state enzymes whose activities are regulated by GTP and GDP through an allosteric site related to the catalytic moiety, and receptors as entities that are inactive and hence unable to couple unless occupied by hormones and activated by any guanine nucleotide through a distinct receptor-related process.     

Fat cell beta-adrenergic receptor in the hypothyroid rat. Impaired interaction with the stimulatory regulatory component of adenylate cyclase

Fat cells from the hypothyroid rat fail to synthesize cyclic AMP in response to beta-adrenergic agonists, although possessing normal amounts of beta-adrenergic receptors (R) and catalytic adenylate cyclase activity. Membranes of hypothyroid rat fat cells contain Mr = 42,000 (major form), 46,0000, and 48,000 (minor forms) peptides of the stimulatory guanine nucleotide-binding regulatory component (Ns) radiolabeled in the presence of cholera toxin and [32P]NAD+. Maps of fragments generated by partial proteolysis of these radiolabeled peptides are virtually identical in hypothyroid and euthyroid preparations. Two-dimensional gel electrophoresis showed that the size and charge of the Mr = 42,000, 46,000, and 48,000 radiolabeled peptides are similar in euthyroid and hypothyroid rat fat cell membranes. Extracts of hypothyroid rat fat cell membranes express normal amounts of Ns activity as measured by their ability to reconstitute the adenylate cyclase of membranes of S49 mouse lymphoma cyc- mutant cells which lack functional Ns activity. Hybridization of hypothyroid rat fat cells with donor membranes of normal rat fat cells, rat hepatocytes, or S49 cyc- cells restores the beta-adrenergic response of these fat cells. Pretreating the donor membranes with a beta-adrenergic antagonist covalent label blocks the ability of these membranes to restore the response of the cells. Rat hepatocytes pretreated with a beta-adrenergic antagonist covalent label do not accumulate cyclic AMP in response to isoproterenol. Hybridization of these receptor-deficient hepatocytes with fat cell ghosts of euthyroid rats restores beta-adrenergic stimulation of cyclic AMP accumulation, whereas hybridization with fat cell ghosts of hypothyroid rat does not restore this response. Ns of pigeon erythrocyte membranes radiolabeled with cholera toxin and [32P]NAD+, extracted in cholate, and reconstituted with fat cell membranes interacts with fat cell R. The ability of R to interact with Ns of pigeon erythrocyte membranes is impaired when the reconstitution is performed with membranes from the hypothyroid rat fat cell. Hypothyroidism appears to affect the ability of R to interact productively with Ns, without affecting either R number or Ns structure and function.     

Properties of beta-adrenergic receptors of cultured mammalian cells. Interaction of receptors with a guanine nucleotide-binding protein in membranes prepared from L6 myoblasts and from wild-type and cyc- S49 lymphoma cells

The radiolabeled agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) and antagonist [125I]iodopindolol ([125I]IPIN) were used to investigate the properties of beta-adrenergic receptors on membranes prepared from L6 myoblasts and S49 lymphoma cells. The high affinity binding of (-)-[3H]HBI to membranes prepared from L6 myoblasts was stereoselectively inhibited by the active isomers of isoproterenol and propranolol. The density of receptors determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. The binding of (-)-[3H]HBI was inhibited by guanine nucleotides, suggesting an agonist-mediated association of the receptor with a guanine nucleotide-binding protein, presumably the stimulatory guanine nucleotide-binding protein (Ns) of adenylate cyclase. Results obtained in studies with membranes prepared from wild-type S49 lymphoma cells and the adenylate cyclase-deficient variant (cyc-) were similar to those obtained in experiments carried out with membranes prepared from L6 myoblasts. Thus, the high affinity binding of (-)-[3H]HBI to membranes prepared from wild-type and cyc- S49 lymphoma cells was stereoselectively inhibited by the active isomers of isoproterenol and propranolol, and was inhibited by GTP. Moreover, the density of sites determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. These results suggest either that cyc- cells contain a partially functional Ns, or alternatively, that the inhibitory guanine nucleotide-binding protein (Ni) is capable of interacting with beta-adrenergic receptors.     

Absence of the inhibitory effect of guanine nucleotides on adenylate cyclase activity in white adipocyte membranes of the ob/ob mouse. Effect of the ob gene

In mice homozygous for the ob gene (ob/ob), the response of adipose tissue adenylate cyclase to stimulation by lipolytic hormones is abnormally low in comparison to that in lean mice (+/+). Studies on the kinetics of adenylate cyclase activation in white adipocyte membranes under a variety of conditions show the following differences between +/+ and ob/ob mice. 1) The inhibitory effects of GTP and guanyl-5'-yl imidodiphosphate, which were clearly seen in +/+ membranes, were absent in the ob/ob membranes. 2) Half-maximal activation by GTP (in the presence of isoproterenol) required at least 10 times more GTP in ob/ob than in +/+ membranes. 3) Increasing the magnesium concentration (up to 10 mM) of the assay medium facilitated the activation of cyclase by modulatory ligands proportionately more in ob/ob than in +/+ membranes; in the +/+ membranes, 10 mM Mg2+ abolished the inhibitory effects of GTP. 4) Treatment with pertussis toxin attenuated the inhibitory effects of guanine nucleotides in +/+ membranes; no effect of the treatment was seen in the ob/ob membranes. 5) Pretreatment of membranes with cholera toxin facilitated cyclase activation proportionately more in ob/ob than in +/+ membranes; in addition, this treatment led to a shift to the left of the GTP dose-response curve in the ob/ob membranes. Cholera and pertussis toxins catalyzed the incorporation of ADP-ribose into their respective substrates in both the +/+ and the ob/ob membranes, showing that the alpha subunits of the stimulatory and inhibitory proteins of the regulatory component Ns and Ni, respectively are present in both types of membranes. Taken together, the results are consistent with the hypothesis that an excess of beta subunit (either primary or secondary to an altered interaction between beta and Ni alpha or Ns alpha) is responsible for the altered sensitivity to activating ligands of the adipocyte adenylate cyclase of the ob/ob mouse. In addition to these findings, we report an effect of the ob gene on the expression of adenylate cyclase activity, since adipose tissue cyclase from heterozygous lean mice (+/ob) showed characteristics which were intermediate between those of +/+ and ob/ob membranes.     

Inhibition of Xenopus oocyte adenylate cyclase by progesterone and 2',5'-dideoxyadenosine is associated with slowing of guanine nucleotide exchange

Adenylate cyclase activity in Xenopus oocyte membranes measured in the presence of guanyl-5'-yl imidodiphosphate and 1.5 mM Mn2+ was maximally inhibited to 57% of control by progesterone and to 89% by the P site agonists, 2',5'-dideoxyadenosine and 9-beta-d-arabinofuranosyladenine. Inhibition by saturating concentrations of 2',5'-dideoxyadenosine and progesterone was not additive, suggesting that inhibition of oocyte adenylate cyclase by progesterone may share a common mechanism with P site inhibition. Kinetic analysis of the effect of progesterone and 2',5'-dideoxyadenosine on the hysteretic activation of adenylate cyclase by guanyl-5'-yl imidodiphosphate indicates that both hormones exert their effects, at least in part, by lengthening the lag in cAMP formation, and this hysteretic effect is inversely proportional to the concentration of guanine nucleotide in the incubation mixture. Direct measurement of [3H] guanine nucleotide release from oocyte membranes preloaded with [3H] GTP demonstrated that treatment with either progesterone or 2',5'-dideoxyadenosine slows the rate of nucleotide exchange. Inhibition of oocyte adenylate cyclase by 2',5'-dideoxyadenosine was potentiated by millimolar concentrations of Mn2+, but inhibition by progesterone was abolished. The results indicate that inhibition of Xenopus oocyte adenylate cyclase by progesterone has features in common with both P site and receptor-mediated inhibitory mechanisms.     

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.     

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.