The guanine nucleotide-binding regulatory component of adenylate cyclase in human erythrocytes

The guanine nucleotide-binding regulatory component of adenylate cyclase (G/F) has been purified from human erythrocyte membranes. It is composed of two major polypeptides with molecular weights of 35,000 and 45,000. When cyc- S49 lymphoma cell plasma membranes are reconstituted with purified human erythrocyte G/F, stimulation of adenylate cyclase by beta-adrenergic agonists, guanine nucleotides, and fluoride is restored. Binding of GTP gamma S to human erythrocyte G/F and GTP gamma S-mediated activation of the protein are closely correlated. The agreement between the apparent dissociation constants for these two reactions suggests that the measured binding site is identical to the site responsible for activation. A 41,000-dalton protein has been identified as a contaminant of preparations of G/F that have been purified by four successive chromatographic steps. This protein serves as a specific substrate for ADP-ribosylation and labeling by islet activating protein (IAP) and [32P]NAD, and it appears to contribute an additional high-affinity guanine nucleotide binding site to such preparations.     

Release of guanyl nucleotides from the regulatory subunit of adenylate cyclase

Choleragen and beta-adrenergic agonists, both of which activate turkey erythrocyte adenylate cyclase, have been reported to accelerate release of bound [3H]guanyl nucleotides from turkey erythrocyte membranes. We have now obtained evidence that choleragen- or isoproterenol-stimulated release reflects a change in the affinity of the regulatory subunit (G/F) of adenylate cyclase for guanyl nucleotides. Solubilized preparations of turkey erythrocytes that had bound radiolabeled GTP were chromatographed on Ultrogel AcA 34. The protein from which guanyl nucleotide was released upon incubation with choleragen or isoproterenol was co-eluted with G/F activity. Furthermore, this protein appears to be the same size as the complex containing the 42,000-dalton peptide, ADP*-ribosylated by choleragen, which is presumably a subunit of G/F. ADP ribosylation of the 42,000-dalton subunit of G/F by choleragen occurred with a half-time of about 5 min, whereas choleragen-stimulated release of guanyl nucleotides was much slower (t1/2 greater than or equal to 60 min). When membranes were treated with choleragen and NAD, the delay in activation of adenylate cyclase by guanylyl imidodiphosphate was decreased but not abolished, a finding consistent with the idea that release of endogenously bound nucleotide (and subsequent binding of the nonhydrolyzable GTP analog) occurs only slowly following ADP ribosylation. In contrast, activation of the adenylate cyclase of either toxin-treated or untreated membranes in the presence of isoproterenol and guanylyl imidodiphosphate was very rapid. These data support the hypothesis that isoproterenol and choleragen may activate adenylate cyclase, at least in part, by increasing the rate of release of guanyl nucleotides from G/F.     

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

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

The regulatory component of adenylate cyclase. Purification and properties

The regulatory component (G/F) of adenylate cyclase, which has been purified previously, contains three putative subunits with molecular weights of 52,000, 45,000, and 35,000 (Northup, J. K., Sternweis, P. C., Smigel, M. D., Schleifer, L. S., Ross, E. M., and Gilman, A. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6516-6520). The published procedure has been modified to reduce the time required for preparation and to increase the yield. Application of the improved procedure allows purification of .5 to 1.0 mg of purified G/F from 1.5 kg of frozen rabbit liver. Greater than 95% of the protein observed on sodium dodecyl sulfate polyacrylamide gels is found in the three bands mentioned above. Purified G/F has the following properties: 1. Hydrodynamic measurements in cholate indicate that purified hepatic G/F has a molecular weight of about 70,000. If G/F is activated with either fluoride or GTP analogs, its apparent molecular weight is reduced to 50,000. 2. The measurement of G/F by reconstitution with the catalytic moiety of adenylate cyclase is dependent on the concentrations of both G/F and catalytic moiety. This interaction is consistent with a model derived from a simple bimolecular binding equilibrium. 3. Purified G/F can be activated by fluoride and guanine nucleotide analogs in a Mg2+-dependent reaction. The rate of activation by guanine nucleotides is markedly stimulated by high concentrations of Mg2+, indicating a site of action of divalent metallic cations on G/F. 4. The 52,000- and 45,000-dalton polypeptides can be partially resolved by heptylamine-Sepharose chromatography. G/F fractions that are enriched in the 52,000-dalton protein reconstitute hormone-stimulated adenylate cyclase activity more efficiently and are activated by GTP analogs more rapidly than are fractions that are essentially free of this polypeptide. The 35,000-dalton protein is present in all cases.     

A cellular activator of catecholamine-sensitive adenylate cyclase in rat reticulocytes and erythrocytes: changes during reticulocyte development and effects on the beta receptor

Rat reticulocytes contain an isoproterenol-sensitive adenylate cyclase activity which is lost with maturation to erythrocytes despite no change in the density of β-adrenergic receptors. To explore this observation, a cytosol factor, previously shown to be important in the expression of catecholamine-sensitive adenylate cyclase in the reticulocyte, was compared to a cytosol factor obtained in a similar manner from mature erythrocytes. The cytosol factor from reticulocytes augmented isoproterenol-responsive adenylate cyclase activity in reticulocyte and erythrocyte membranes half-maximally at 0.7 ± 0.1 (SEM) and 1.1 ± 0.3 μg/ml, respectively. These concentrations of reticulocyte-derived cytosol factor were significantly lower (P < 0.01) than those concentrations of the factor from erythrocytes necessary to augment isoproterenol-responsive adenylate cyclase activity in reticulocyte (9.7 ± 2.3) and erythrocyte (7.5 ± 1.0) membranes. Cytosol factor from reticulocytes also caused greater total isoproterenol responsiveness than that from erythrocytes both in reticulocyte (784 ± 107 vs 525 ± 65 pmol/mg protein) and in erythrocyte membranes (54 ± 6 vs 36 ± 3); P < 0.05. Neither reticulocyte nor erythrocyte cytosol factor affected the concentration at which isoproterenol half-maximally stimulated adenylate cyclase in either set of membranes. However, the cytosol factor from reticulocytes markedly decreased the binding affinity of isoproterenol for β receptors in reticulocytes from 0.8 ± 0.2 to 6.9 ± 1.4 μm; P < 0.001. This reticulocyte factor had no significant effect on the binding affinity of isoproterenol for erythrocyte membranes. Erythrocyte factor did not change the binding affinity for isoproterenol in either reticulocyte or erythrocyte membranes.     

Evidence that a beta-adrenergic receptor-associated guanine nucleotide regulatory protein conveys guanosine 5'-O-(3-thiotriphosphate)- dependent adenylate cyclase activity

The guanine nucleotide regulatory protein component (N) of the frog erythrocyte membrane adenylate cyclase system appears to form a stable complex with the beta-adrenergic receptor (R) in the presence of agonist (H). This agonist-promoted ternary complex HRN can be solubilized with Lubrol. The guanine nucleotide regulatory protein associated with the solubilized complex can be adsorbed either to GTP-Sepharose directly or to wheat germ lectin-Sepharose via its interaction with the receptor which is a glycoprotein. Guanosine 5'-O-(3-thiotriphosphate)(GTP gamma S) can be used to elute the guanine nucleotide regulatory protein from either Sepharose derivative. The resulting N.GTP gamma S complex conveys nucleotide-dependent adenylate cyclase activity when combined with a Lubrol-solubilized extract of turkey erythrocyte membranes. The ability to observe GTP gamma S-dependent reconstitution of adenylate cyclase activity in the eluate from either resin required the formation of the HRN complex prior to solubilization. The N protein can be identified by its specific [32P]ADP ribosylation catalyzed by cholera toxin in the presence of [32P]NAD+. The existence of a stable HRN intermediate complex is supported by the observation that agonist pretreatment of frog erythrocyte membranes results in a 100% increase in the amount of 32P-labeled N protein eluted from the lectin-Sepharose in the presence of GTP gamma S compared to membranes pretreated with either antagonist or agonist plus GTP. Our results therefore provide evidence that the same guanine nucleotide-binding protein that associates with the beta-adrenergic receptor in the presence of agonist mediates adenylate cyclase activation.     

Guanine nucleotide activation of adenylate cyclase in saponin permeabilized glioma cells

We have compared the regulation of adenylate cyclase activity in membrane fractions from C6 glioma cells and in monolayer cultures of C6 cells that had been permeabilized with saponin. Guanine nucleotides (GTP and GTP gamma S) and isoproterenol increase adenylate cyclase activity in C6 membranes and in permeabilized C6 cells. In C6 membranes, guanine nucleotides activate adenylate cyclase in the presence or absence of isoproterenol; in permeabilized cells, however, guanine nucleotides increase adenylate cyclase activity only in the presence of isoproterenol. We suggest that the properties of the permeabilized cells more closely resemble those of intact cells, and that some component which is present in permeabilized cells but is lost following cell disruption may be important for the normal regulation of adenylate cyclase activity.     

Regulation of catecholamine-responsive adenylate cyclase activity in rat reticulocyte membranes by endogenous factors General characteristics and resolution into protein and nucleotide components

A 100 000 × g soluble, supernatant fraction obtained from the hemolysate of rat reticulocytes was studied for its effect upon catecholamine-sensitive adenylate cyclase activity in reticulocyte membranes. The supernatant material, devoid of adenylate cyclase activity itself, amplified isoproterenol-dependent activity in responsive membranes and was an essential requirement for the expression of hormone sensitivity in membranes rendered unresponsive to isoproterenol alone. The increment in catecholamine-associated activity conferred upon reticulocyte membranes by the supernatant material was β-adrenergic because it did not affect basal or fluoride-related activity and was completely inhibited by propranolol. Guanine nucleotides were present in the supernatant but could account for only a fraction of the total activity because the supernatant was able to cause greater stimulation than maximal concentrations of GTP and when specified concentrations of exogenous GTP were compared with equivalent nucleotide concentrations in the supernatant, the supernatant always led to greater activity. The supernatant was resolved into protein- and nucleotide-containing components by ion-exchange chromatography. Each component was approximately one-half as active in amplifying catecholamine-dependent adenylate cyclase as the unresolved, crude supernatant material. The activity eluted in the first peak of the DEAE chromatogram was resistant to alkaline phosphatase, sensitive to trypsin, not dialyzable and contained no detectable concentrations of GTP or GDP. In contrast, the activity eluted in the second peak of the DEAE chromatogram was sensitive to alkaline phosphatase, resistant to trypsin, completely dialyzable and contained both GTP (30 μM) and GDP (10 μM) in significant concentrations. Neither the crude supernatant not its two active components affected the binding of [¹²⁵I]-iodohydroxybenzylpindolol to reticulocyte membranes. These observations establish in rat reticulocytes the presence of protein and guanine nucleotide constituents which have independent influences upon the catecholamine-responsive adenylate cyclase of reticulocyte membranes.     

Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system

The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O-(thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neither alpha T . GTP gamma S nor beta gamma T having any direct effect on the intrinsic activity of the catalytic moiety. Increasing the MgCl2 concentration in the assay incubations significantly decreases the inhibitions by both alpha T . GTP gamma S and beta gamma T. Similarly, when the pure hamster lung beta-adrenergic receptor is included in the lipid vesicles with Ns and C, the levels of inhibition of the GppNHp-stimulated activity by both alpha T . GTP gamma S and beta gamma T are reduced compared to those obtained in vesicles containing just Ns and C (but not stimulatory receptor). These inhibitions are reduced still further under conditions where the agonist stimulation of adenylate cyclase activity is maximal, i.e. when stimulating with isoproterenol plus GTP. In these cases the alpha T . GTP gamma S inhibitory effects are completely eliminated and the inhibitions observed with holotransducin can be fully accounted for by the beta gamma T complex. The ability of the beta-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to Ns (or alpha s) during the activation of the regulatory protein and the stimulation of adenylate cyclase. These results also suggest that under physiological conditions the beta gamma subunit complex is primarily responsible for mediating the inhibition of adenylate cyclase activity.     

Induction of desensitization by phorbol ester to beta-adrenergic agonist stimulation in adenylate cyclase system of rat reticulocytes

Treatment of rat reticulocytes with a phorbol ester, tetradecanoyl phorbol acetate (TPA), resulted in the desensitization of adenylate cyclase to the beta-adrenergic agonist stimulation depending on the dose and period of the TPA treatment. Treatment of the reticulocytes with TPA caused approximately 40% reduction in the stimulation by beta-adrenergic agonists of adenylate cyclase activity, whereas the treatment had little effect on the basal activity and the activation by fluoride and guanine nucleotide of the enzyme system. No change in the number of beta-adrenergic receptors was observed after the TPA treatment. Treatment with 1-oleoyl-2-acetyl-glycerol (OAG), an activator of protein kinase C, also caused the desensitization of reticulocyte adenylate cyclase to isoproterenol. On the other hand, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C, prevented the desensitization induced by TPA. These results suggest the involvement of protein kinase C in a process of desensitization of adenylate cyclase system to beta-adrenergic agonists in rat reticulocytes.     

Modulation of the receptor-coupled adenylate cyclase system in HeLa cells by sodium butyrate

Exposure of HeLa cells to 5 mM sodium butyrate, but not 0.6 mM, resulted in a more efficient coupling between their beta-adrenergic receptors and the guanine nucleotide binding stimulatory (Ns) component of adenylate cyclase. Both concentrations of the fatty acid, however, caused an increase in receptor number. beta receptors from control and butyrate-treated cells had the same affinity for isoproterenol. Modulation of this affinity by GTP was greatly enhanced, however, in cells treated with 5 mM butyrate compared to untreated and 0.6 mM butyrate treated cells. The concentration of isoproterenol required to half-maximally stimulate adenylate cyclase (Kact) was reduced in cells treated with 5 mM butyrate. In addition, the Kact for GTP in the presence, but not the absence, of isoproterenol was reduced. The effect of butyrate on the coupling between beta receptors and Ns was analyzed in detail by monitoring the activation of Ns by guanine 5'-O-(3-thiotriphosphate) (GTP gamma S) in a two-step assay. In the absence of isoproterenol, Ns from control and 5 mM butyrate treated cells was activated to the same extent with the same time course and Kact for GTP gamma S. In the presence of isoproterenol, Ns from 5 mM butyrate treated cells was activated more rapidly and extensively than Ns from control cells. The Kact for both GTP gamma S and isoproterenol also was reduced. The rate of agonist-mediated activation of Ns was strongly dependent on temperature, which accentuated the differences between 5 mM butyrate treated and control cells. At 4 degrees C, the difference in rate was 8.8-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic cholinergic receptor modulation of beta-adrenergic receptor affinity for catecholamines.

The effects of the muscarinic cholinergic agonist methacholine on affinity of beta-adrenergic receptors for isoproterenol and on isoproterenol-induced stimulation of adenylate cyclase activity were assessed in canine myocardium. GTP and guanyl-5'-yl imidoiphosphate both decreased the affinity of beta-adrenergic receptors for isoproterenol without altering the affinity of these receptors for propranolol. Methacholine (10 nM to 10 micronM) antagonized the guanine nucleotide-induced reduction in beta-adrenergic receptor affinity for isoproterenol. This effect of methacholine was reversed by atropine. The choline ester had no effect on the affinity of beta-adrenergic receptors for isoproterenol in the absence of guanine nucleotides. Likewise, methacholine had no effect on the affinity of beta-adrenergic receptors for propranolol, either in the presence or absence of guanine nucleotides. Methacholine also attenuated GTP-induced activation of adenylate cyclase or isoproterenol-induced activation of the enzyme in the presence of GTP. The effects of methacholine on myocardial adenylate cyclase activity were apparent only in the presence of GTP. These effects were also reversed by atropine. The choline ester had no effect on adenylate cyclase activity in the presence of guanyl-5'-yl imidodiphosphate or NaF. The results of the present study suggest that muscarinic cholinergic agonists can regulate both beta-adrenergic receptors and adenylate cyclase by modulating the effects of GTP.     

Evidence that forskolin activates turkey erythrocyte adenylate cyclase through a noncatalytic site

The diterpene forskolin has been reported to activate adenylate cyclase in a manner consistent with an interaction at the catalytic unit. However, some of its actions are more consistent with an interaction at the coupling unit that links the hormone receptor to the adenylate cyclase activity. This report adds support to the latter possibility. Under conditions that lead to stimulation of adenylate cyclase in turkey erythrocyte membranes by GTP, forskolin also becomes more active. Additional evidence to support an influence of forskolin upon adenylate cyclase via the GTP-coupling protein N includes the following: (i) forskolin, at submaximal concentrations, leads to enhanced sensitivity and responsiveness of isoproterenol-dependent adenylate cyclase activity in turkey erythrocyte membranes; (ii) under specified conditions, the nucleotide GDP, an inhibitor of the stimulating nucleotide GTP and its analog, guanyl imidodiphosphate (Gpp(NH)p), also markedly inhibits the action of forskolin; (iii) both Gpp(NH)p and forskolin are associated with a decrease in agonist affinity for the beta-adrenergic receptor. However, actions of forskolin in the turkey erythrocyte are not identical to those of GTP: (i) forskolin is never as potent as Gpp(NH)p in activating adenylate cyclase; (ii) the magnitude of synergism between isoproterenol and forskolin is not equal to that observed with isoproterenol and Gpp(NH)p; (iii) at high concentrations, forskolin inhibits antagonist binding to the beta-receptor. Forskolin appears to have several sites of action in the turkey erythrocyte membrane, including an influence upon the adenylate cyclase regulatory protein N.     

Reconstitution of beta 1-adrenoceptor-dependent adenylate cyclase from purified components.

In continuation of our efforts to reconstitute from purified components into lipid vesicles the signal transmission chain from beta 1-adrenoceptors to adenylate cyclase, we now report on the total reconstitution of the hormone-dependent adenylate cyclase. In these reconstitution experiments we have employed the purified adenylate cyclase (C) from bovine brain and rabbit heart, the stimulatory GTP-binding protein (GS) purified from turkey erythrocytes and rabbit liver and the beta 1-adrenoceptor (R) from turkey erythrocytes. Several detergents were compared with respect to their suitability to allow reconstitution of subunits into phospholipid vesicles. While octyl-polyoxyethylene (octyl-POE) was almost as potent as lauroyl-sucrose for preparation of vesicles containing GS.C, the latter detergent was clearly superior for vesicles enabling productive R.GS and R.GS.C coupling. The catalytic subunit from either bovine brain or rabbit heart was equally efficient in reconstitution. However, GS from turkey erythrocytes and rabbit liver revealed significant differences in RGS and RGS.C containing vesicles. While isoproterenol-induced activation of GS by GTP gamma S was first order in both instances, kon with turkey GS was 0.12 min-1, whereas kon with rabbit liver GS was 0.6 min-1. Moreover, GTP gamma S activation of erythrocyte GS was significantly more dependent on the presence of hormone than that of liver GS, confirming observations made on the native membrane-bound system. Compared with stimulation by isoproterenol (GTP gamma S) (4-fold), stimulation by isoproterenol/GTP was modest (1.3- to 1.6-fold).(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of adenylate cyclase in human keratinocytes by protein kinase C

Adenylate cyclase (ATP-pyrophosphate lyase (cyclizing); EC 4.6.1.1) in the human keratinocyte cell line SCC 12F was potentiated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), phorbol-12,13-diacetate, and 1,2-dioctanoylglycerol. Keratinocytes exposed to TPA showed a 2-fold enhancement of adenylate cyclase activity when assayed in the presence of isoproterenol or GTP. The half-maximal effective concentration (EC50) for both isoproterenol and GTP were unaltered by TPA treatment of the cells. Basal adenylate cyclase activity in membranes from TPA-treated cultures was also increased 2-fold relative to activity in control membranes. Potentiation of adenylate cyclase activity was dependent on the concentration of TPA to which the keratinocytes were exposed (EC50 for TPA = 3 nM). TPA actions on adenylate cyclase were maximal after 15 min of incubation of the cells with the compound, correlating well with the time course of translocation of protein kinase C (Ca2+/phospholipid-dependent enzyme) from cytosol to membrane. The action of cholera toxin on adenylate cyclase was additive with TPA. In contrast, pertussis toxin actions on adenylate cyclase were not additive with TPA. Treatment of control cells with pertussis toxin activated adenylate cyclase 1.5-fold, whereas cells exposed to pertussis toxin for 6 h followed by TPA for 15 min showed the same 2-fold increase in adenylate cyclase activity as observed in membranes from cells exposed to TPA without prior exposure to pertussis toxin. Pertussis toxin catalyzed ADP-ribosylation was increased 2-fold in membranes from SCC 12F cells exposed to TPA, indicating an increase in the alpha beta gamma form of Gi. These data suggest that exposure of human keratinocytes to phorbol esters increases adenylate cyclase activity by a protein kinase C-mediated increase in the heterotrimeric alpha beta gamma form of Gi resulting in decreased inhibition of basal adenylate cyclase activity.     

Reticulocyte Cytosol Activator Protein: Its Actions Upon the Beta Adrenergic Receptor and the N-Proteins of Adenylate Cyclase

Abstract

Rat reticulocytes contain a cytosol activator protein (RCAP) that augments catecholamine-sensitive adenylate cyclase activity in reticulocyte membranes. A partially purified preparation of RCAP was obtained by Sephacryl S-200 chromatography and used to elucidate further its mechanism of action. The specific activity of the S-200 fraction to augment isoproterenol responsiveness is increased approximately 1100-fold over the starting material from 1.2 nmoles to 1300 nmoles cyclic AMP formed per milligram of RCAP. The molecular weight is approximately 20,000. In addition to its effects on catecholamine-responsive adenylate cyclase, RCAP is associated with significant increases in basal (0.9 ± 0.2 to 1.5 ± 0.4 nmol/mg; p < 0.02), guanyl-5′-yl imidodiphosphate [Gpp(NH)p]; (3.9 ± 0.9 to 4.4. ± 1.1 nmol/mg; p < 0.005) and fluoride (4.1 ± 0.6 to 4.8 ± 0.6 nmol/mg; p < 0.005) associated activities. RCAP stimulates isoproterenol responsiveness in wild type S49 cell membranes but is inactive in the mutant line, cyc. RCAP alters the characteristics of agonist binding to the beta-adrenergic receptor of reticulocyte and wild S49 cell membranes, causing a significant increase in the IC₅₀ for isoproterenol. Direct assessment of N_s and N_i components of the adenylate cyclase complex demonstrates that RCAP inhibits cholera toxin-specific ADP-ribosylation of the 42K subunit of N_s and stimulates pertussis toxin-specific ADP ribosylation of the 39K subunit of N_i.     

A reconstitution assay for the guanine nucleotide regulatory protein of the adenylate cyclase system using turkey erythrocyte membranes as the acceptor preparation

The turkey erythrocyte beta-adrenergic receptor-adenylate cyclase system has the unusual property that neither GTP nor Gpp(NH)p are effective in activating adenylate cyclase unless a beta-agonist is present simultaneously. This property results in essentially no basal activity and the inability of GTP or Gpp(NH)p alone to activate the catalytic moiety. In this study, we have exploited these characteristics to utilize turkey erythrocyte membranes as the acceptor preparation in a reconstitution assay. Rat reticulocyte or turkey erythrocyte membranes that have been activated with isoproterenol and Gpp(NH)p followed by solubilization with sodium cholate serve as the donor source of the guanine nucleotide regulatory protein (N). By reconstituting this Gpp(NH)p-activated N protein, it has been found that: (1) exogenous Gpp(NH)p-associated N could activate the catalytic unit of adenylate cyclase in turkey erythrocyte membranes; (2) this system can be used to assay N protein activity; (3) the endogenous pathway for activation of turkey erythrocyte membrane adenylate cyclase by hormones and fluoride remains qualitatively functional; and (4) the effects of combined activation via the endogenous and exogenous pathways are additive and saturable.     

Reduction of GTP activation of adenylate cyclase system by its coupling to hormone receptor.

We have examined the characteristics of the adenylate cyclase system from control and butyrate-treated cells. Butyrate treatment results in both an increased number of catecholamine receptors and an induction of a response to the hormone, as reported previously (Tallman, J.F., Smith, C.C., and Henneberry, R.C. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 873-877); in addition, we found that the same treatment reduces the degree of activation of adenylate cyclase by GTP. We have demonstrated in two cell types that this decrease in GTP activation is inversely related to the degree of induction of the hormone response. Furthermore, in plasma membranes isolated from butyrate-treated cells, the hormone receptor is sensitive to GTP; i.e. GTP reduces the affinity of isoproterenol for the receptor. We propose that these changes reflect an interaction between the beta-adrenergic receptor and the nucleotide regulatory component and that this interaction represents, at least in part, the process of coupling. Several possible mechanisms which can account for the change in GTP activation are discussed in terms of our current understanding of the regulation of the adenylate cyclase system.     

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)     

Expression of Gs alpha in Escherichia coli. Purification and properties of two forms of the protein

Cloning of complementary DNAs that encode either of two forms of the alpha subunit of the guanine nucleotide-binding regulatory protein (Gs) that stimulates adenylyl cyclase into appropriate plasmid vectors has allowed these proteins to be synthesized in Escherichia coli (Graziano, M.P., Casey, P.J., and Gilman, A.G. (1987) J. Biol. Chem. 262, 11375-11381). A rapid procedure for purification of milligram quantities of these proteins is described. As expressed in E. coli, both forms of Gs alpha (apparent molecular weights of 45,000 and 52,000) bind guanosine 5'-(3-O-thio)triphosphate stoichiometrically. The proteins also hydrolyze GTP, although at different rates (i.e. 0.13.min-1 and 0.34.min-1 at 20 degrees C for the 45- and the 52-kDa forms, respectively). These rates reflect differences in the rate of dissociation of GDP from the two proteins. Both forms of recombinant Gs alpha have essentially the same kcat for GTP hydrolysis, approximately 4.min-1. Recombinant Gs alpha interacts functionally with G protein beta gamma subunits and with beta-adrenergic receptors. The proteins can also be ADP-ribosylated stoichiometrically by cholera toxin. This reaction requires the addition of beta gamma subunits. Both forms of recombinant Gs alpha can reconstitute GTP-, isoproterenol + GTP-, guanosine 5'-(3-O-thio)triphosphate-, and fluoride-stimulated adenylyl cyclase activity in S49 cyc- membranes to maximal levels, although their specific activities for this reaction are lower than that observed for Gs purified from rabbit liver. Experiments with purified bovine brain adenylyl cyclase indicate that the affinity of recombinant Gs alpha for adenylyl cyclase is 5-10 times lower than that of liver Gs under these assay conditions; however, the intrinsic capacity of the recombinant protein to activate adenylyl cyclase is normal. These findings suggest that Gs alpha, when synthesized in E. coli, may fail to undergo a posttranslational modification that is crucial for high affinity interaction of the G protein with adenylyl cyclase.