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.     

Cholera toxin induces cAMP-independent degradation of Gs

Cholera toxin stimulates adenylyl cyclase by catalyzing ADP-ribosylation of the alpha chain (alpha s) of Gs, a guanine nucleotide binding regulatory protein. In a rat pituitary cell line, GH3, the toxin-induced increase in GTP-dependent adenylyl cyclase activity is maximal at 1 h; adenylyl cyclase remains elevated for at least 32 h. Surprisingly, cholera toxin also induces a 74-95% decrease in the amount of immunoreactive alpha s in the same cells, as assessed on immunoblots probed with either of two antisera directed against separate alpha s peptide sequences. The decrease in immunoreactive alpha s, which begins after 1 h of toxin treatment and is complete by 8 h, is accompanied by a comparable decrease in the amount of biochemically active alpha s, as assessed by its ability to complement the biochemical defect of alpha s-deficient S49 cyc- membranes. Cholera toxin induces similar decreases in alpha s in wild type S49 lymphoma cells, in S49 kin- mutants, which lack cAMP-dependent protein kinase, and in S49 H21 a mutants, in which alpha s is unable to assume an active conformation upon binding GTP. The toxin-induced decrease in alpha s is somewhat temperature-dependent, but is not blocked by agents that increase lysosomal pH or by colchicine, which promotes breakdown of microtubules. alpha s in detergent-solubilized GH3 membranes is susceptible to proteolysis by an endogenous protease; this susceptibility is markedly increased in membranes from cells previously exposed to cholera toxin for 1 h. Taken together, these results suggest that cholera toxin-induced covalent modification of alpha s marks the protein for accelerated degradation. In addition, the persistence of elevated GTP-dependent adenylyl cyclase activity despite loss of a substantial fraction of alpha s suggests that the amount of alpha s membranes is greater than the amount necessary for maximal activation of cAMP synthesis by cholera toxin.     

Cholera toxin treatment produces down-regulation of the alpha-subunit of the stimulatory guanine-nucleotide-binding protein (Gs).

The effect of activation of the alpha-subunit(s) of the stimulatory guanine-nucleotide-binding protein, Gs, on levels of this polypeptide(s) associated with the plasma membrane of L6 skeletal myoblasts was ascertained. Incubation of these cells with cholera toxin led to a time- and concentration-dependent 'down-regulation' of both 44 and 42 kDa forms of Gs alpha as assessed by immunoblotting with an anti-peptide antiserum (CS1) able to identify the extreme C-terminus of Gs. The effect of cholera toxin was specific for Gs; levels of Gi alpha in membranes of cholera toxin-treated cells were not different from untreated cells. Down-regulation of Gs was absolutely dependent upon prior ADP-ribosylation, and hence activation of Gs and was not mimicked by other agents which elevate intracellular levels of cyclic AMP. Pretreatment with pertussis toxin, which catalyses ADP-ribosylation of Gi but not of Gs, did not down-regulate either Gi or Gs, demonstrating that covalent modification by ADP-ribosylation is alone not a signal for removal of G-proteins from the plasma membrane.     

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.     

Inhibitory regulation of adenylyl cyclases. Evidence inconsistent with beta gamma-complexes of Gi proteins mediating hormonal effects by interfering with activation of Gs

The possible effect of cholera toxin (CTX) on hormonal inhibition of adenylyl cyclase in somatostatin (SST)-sensitive GH3 cells was quantitatively evaluated. The toxin treatment employed led to an essentially complete ADP ribosylation of all alpha s subunits of the stimulatory regulatory component (Gs) of the system and to ca. 5- to 7-fold increases in the activity measured, yet it failed to affect the inhibitory action of SST regardless of whether analyzed in terms of degree of inhibition (ca. 60%) that is attainable or in terms of the apparent Kact with which the inhibitory hormone elicits its action. In absolute terms the activity inhibited after CTX was ca. 6 times larger than that inhibited under control conditions, indicating that SST is equally effective in regulating control and CTX-stimulated adenylyl cyclase system and that interpretations are independent of possible intramembraneous compartmentalizations of adenylyl cyclase and its various regulatory components. Since CTX-mediated ADP ribosylation of the alpha-subunits of Gs has been demonstrated to result in an at least 10-fold decrease in the potency (i.e. EC50) with which the beta gamma-complexes of G proteins act to stabilize preactivated purified alpha-subunits of Gs and in an approximately 300-fold decrease in the potency with which exogenously added beta gamma-complexes act to prevent activation of Gs in intact membranes, the present data indicate that beta gamma-complexes cannot be mediating the inhibitory effects of hormones by interfering with activation of the Gs of adenylyl cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mutation in the putative Mg(2+)-binding site of Gs alpha prevents its activation by receptors.

The properties of a Gs alpha mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn alpha s, were studied after expression in S49 alpha s-deficient (cyc-) cells. Ser-54 in alpha s is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn alpha s did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn alpha s, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP gamma S. Guanine nucleotide regulation of mutant alpha s suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn alpha s for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells.     

Mutations of GS alpha designed to alter the reactivity of the protein with bacterial toxins. Substitutions at ARG187 result in loss of GTPase activity

We have introduced two types of mutations into cDNAs that encode the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase. The arginine residue (Arg187) that is the presumed site of ADP-ribosylation of Gs alpha by cholera toxin has been changed to Ala, Glu, or Lys. The rate constant for hydrolysis of GTP by all of these mutants is reduced approximately 100-fold compared with the wild-type protein. As predicted from this change, these proteins activate adenylyl cyclase constitutively in the presence of GTP. Despite these substitutions, cholera toxin still catalyzes the incorporation of 0.2-0.3 mol of ADP-ribose/mol of mutant alpha subunit. The sequence near the carboxyl terminus of Gs alpha was altered to resemble those in Gi alpha polypeptides, which are substrates for pertussis toxin. Despite this change, the mutant protein is a poor substrate for pertussis toxin. Although this protein has unaltered rates of GDP dissociation and GTP hydrolysis, its ability to activate adenylyl cyclase in the presence of GTP is enhanced by 3-fold when compared with the wild-type protein but only when these assays are performed after reconstitution of Gs alpha into cyc- (Gs alpha-deficient) S49 cell membranes.     

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.     

Coupling of Corticotropin-Releasing Hormone Receptors to Adenylyl Cyclase in Human Y-79 Retinoblastoma Cells

Abstract: In human Y-79 retinoblastoma cells, corticotropin-releasing hormone (CRH) stimulates adenylyl cyclase activity and increases cyclic AMP accumulation. Different CRH analogues mimic the CRH stimulation of adenylyl cyclase and show similar sensitivity to the CRH receptor antagonist α-helical CRH_9–41. Vasoactive intestinal peptide (VIP) also increases the enzyme activity but less potently than CRH, and its effect is counteracted by the VIP receptor antagonist [ d - p -Cl-Phe⁶,Leu¹⁷]VIP. The VIP antagonist does not affect the response to CRH. The CRH-stimulated adenylyl cyclase activity is amplified by Mg²⁺, is inhibited by submicromolar concentrations of Ca²⁺, and requires GTP. Moreover, the CRH stimulation is reduced by pretreatment of cells with cholera toxin and by incubation of membranes with the RM/1 antibody, which recognizes the C-terminus of the α subunit of G_s. In immunoblots, the RM/1 antibody identifies a doublet of 45 and 52 kDa. Two proteins of similar molecular weights are ADP-ribosylated by cholera toxin. These data demonstrate that in human Y-79 retinoblastoma cells, specific CRH receptors stimulate cyclic AMP formation by interacting with G_s and by affecting a Ca²⁺-inhibitable form of adenylyl cyclase.     

Incubation of bovine thyroid slices with thyrotropin is associated with a decrease in the ability of pertussis toxin to adenosine diphosphate-ribosylate guanine nucleotide regulatory component(s)

Pretreatment of bovine thyroid slices with TSH resulted in desensitization of TSH-sensitive adenylyl cyclase activity but no change in stimulatory nucleotide binding regulatory component of adenylyl cyclase (Gs) activity assessed by reconstitution of the Gs-defective cyc-S49 adenylyl cyclase system. Possible changes in substrates for pertussis toxin (PT)-induced ADP ribosylation due to TSH treatment and/or in endogenous ADP ribosylation of membrane proteins were explored. Using 10 microM [32P]NAD+ as substrate, endogenous ADP ribosylation was not observed in membranes from control or TSH-treated slices. ADP ribosylation of alpha-subunits of Gs by cholera toxin was also unaffected by incubation of thyroid slices with TSH. In contrast, ADP ribosylation of 40 kilodalton (kDa) substrates for PT was decreased between 40% and 60% by TSH treatment. This effect of TSH was dependent on its concentration and the time of incubation of the slices and was specific for labeling of the 40 kDa PT substrate. Prostaglandin E1 treatment of thyroid slices, which results in a much smaller homologous desensitizing effect, did not result in changes in ADP ribosylation by PT. The effect of incubation of slices with TSH was abolished by pretreatment of the membranes with 0.3-1.0% Lubrol PX, which increased the labeling of the 40 kDa polypeptides. The data suggests that TSH induces in thyroid tissue a redistribution of 40 kDa polypeptides changing their availability to PT.     

Activation of the alpha subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity

Binding of GTP induces alpha subunits of heterotrimeric G proteins to take on an active conformation, capable of regulating effector molecules. We expressed epitope-tagged versions of the alpha subunit (alpha s) of Gs in genetically alpha s-deficient S49 cyc- cells. Addition of a hemagglutinin (HA) epitope did not alter the ability of wild type alpha s to mediate hormonal stimulation of adenylyl cyclase or to attach to cell membranes. The HA epitope did, however, allow a mAb to immunoprecipitate the recombinant protein (HA-alpha s) quantitatively from cell extracts. We activated the epitope-tagged alpha s in intact cells by: (a) exposure of cells to cholera toxin, which activates alpha s by covalent modification; (b) mutational replacement of arginine-201 in HA-alpha s by a cysteine residue, to create HA-alpha s-R201C; like the cholera toxin-catalyzed modification, this mutation activates alpha s by slowing its intrinsic GTPase activity; and (c) treatment of cells with the beta-adrenoceptor agonist, isoproterenol, which promotes binding of GTP to alpha s, thereby activating adenylyl cyclase. Both cholera toxin and the R201C mutation accelerated the rate of degradation of alpha s (0.03 h-1) by three- to fourfold and induced a partial shift of the protein from a membrane bound to a soluble compartment. At steady state, 80% of HA-alpha s- R201C was found in the soluble fraction, as compared to 10% of wild type HA-alpha s. Isoproterenol rapidly (in < 2 min) caused 20% of HA-alpha s to shift from the membrane-bound to the soluble compartment. Cholera toxin induced a 3.5-fold increase in the rate of degradation of a second mutant, HA-alpha s-G226A, but did not cause it to move into the soluble fraction; this observation shows that loss of membrane attachment is not responsible for the accelerated degradation of alpha s in response to activation. Taken together, these findings show that activation of alpha s induces a conformational change that loosens its attachment to membranes and increases its degradation rate.     

Deletion within the amino-terminal region of Gs alpha impairs its ability to interact with beta gamma subunits and to activate adenylate cyclase.

Proteolytic experiments performed on transducin and Go alpha subunit strongly suggest that the amino-terminal residues of the alpha chain are involved in the interaction with beta gamma subunits. To test the possibility that the same region in Gs may fulfill a similar function, we introduced a deletion in the amino-terminal domain of Gs alpha. The properties of the wild type and the deleted alpha chains were characterized on in vitro translated proteins or after reconstitution of cyc- membranes by in vitro-translated alpha subunits. The mutant (delta 2-29) Gs alpha could still bind guanosine 5'-3-O-(thio)triphosphate, as revealed by its resistance to trypsin proteolysis and was still able to interact with the membrane. However, (delta 2-29) Gs alpha was not ADP-ribosylated by cholera toxin. In contrast to Gs alpha, addition of beta gamma subunits did not increase the rate of sedimentation of (delta 2-29) Gs alpha in sucrose gradients. Binding experiments on reconstituted membranes showed that the coupling to beta-adrenergic receptors was very low with (delta 2-29) Gs alpha. Finally, the mutant did not restore activation of adenylate cyclase of cyc- membranes. We propose that the primary functional defect is the loss of interaction with beta gamma subunits, which secondarily impairs beta gamma-dependent properties such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation. However, it remains to be established that the lack of adenylate cyclase activation also results from this impaired interaction with beta gamma subunits.     

Determination of G-protein levels, ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db) mice

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of Gi alpha-2, Gi alpha-3 and G-protein beta-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of Gs alpha-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of Gs alpha-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 microM), GTP (100 microM), p[NH]ppG (100 microM), NaF (10 mM) and glucagon (10 microM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 microM) was lower by about 22%. Lower concentrations (EC50-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC50-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional Gi activity. The lower levels of expression of G-protein beta-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

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.     

Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Putative effect of C kinase on alpha s-GTP-catalytic subunit interaction

Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to S49 lymphoma cells (wild type and a cyclic AMP-dependent protein kinase-lacking clone) has little effect alone but doubles accumulation of cyclic AMP in response to isoproterenol. The effect is immediate and has an apparent affinity and order of potency characteristic of the activation of protein kinase C by phorbol esters. Enhancement does not reflect an altered time course of the beta-adrenergic response, enhanced affinity of the cellular beta-receptor for agonist, or decreased degradation and export of cellular cyclic AMP. Reduction of the beta-adrenergic response by somatostatin does not remove the effect of TPA nor does TPA abolish the effect of somatostatin. Phorbol ester enhances cyclic AMP accumulation in response to cholera toxin in wild type and UNC clones but not in H21a or cyc-. TPA also enhances cAMP accumulation in response to forskolin in wild type cells. The effect of TPA is stable to rapid preparation of membranes. In adenylate cyclase assays on membranes from cells treated with TPA, the activation by guanosine 5'-(beta, gamma-imino)triphosphate is enhanced by 40% with no change in lag time; the effect of beta-agonist plus Gpp(NH)p is similarly enhanced; activation by Mn2+ is unchanged. We conclude that phorbol ester facilitates the productive interaction of the alpha subunit of the transducer protein Gs with the catalytic unit of adenylate cyclase, hypothetically via an action of protein kinase C.     

The effect of iloprost on the ADP-ribosylation of Gs alpha (the alpha-subunit of Gs).

Treatment of platelets with a prostacyclin analogue, iloprost, decreased the cholera-toxin-induced ADP-ribosylation of membrane-bound Gs alpha (alpha-subunit of G-protein that stimulates adenylate cyclase; 42 kDa protein) and a cytosolic substrate (44 kDa protein) [Molina y Vedia, Reep & Lapetina (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5899-5902]. This decrease is apparently not correlated with a significant change in the quantity of membrane Gs alpha, as detected by two Gs alpha-specific antisera. This finding contrasts with the suggestion in a previous report [Edwards, MacDermot & Wilkins (1987) Br. J. Pharmacol. 90, 501-510], indicating that iloprost caused a loss of Gs alpha from the membrane. Our evidence points to a modification in the ability of the 42 kDa protein to be ADP-ribosylated by cholera toxin. This modification of Gs alpha might be related to its ADP-ribosylation by endogenous ADP-ribosyltransferase activity. Here we present evidence showing that Gs alpha was ADP-ribosylated in platelets that had been electropermeabilized and incubated with [alpha-32P]NAD+. This endogenous ADP-ribosylation of Gs alpha is inhibited by nicotinamide and stimulated by iloprost.     

Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs.

Coupling of the three alpha 2-adrenergic receptor (alpha 2AR) subtypes to Gi and Gs was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the alpha 2AR agonist UK-14304, alpha 2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, alpha 2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of alpha 2C10 to both Gi and Gs. To isolate functional Gs and Gi coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only alpha 2C10-mediated inhibition (approximately 50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only alpha 2C10-mediated stimulation (approximately 60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl-terminal portion of Gs alpha blocked this functional alpha 2AR.Gs coupling to the same extent as that found for beta 2AR.Gs coupling. In addition to functional Gs coupling, we also verified direct, agonist-dependent, physical coupling of alpha 2AR to Gs alpha. In agonist-treated membranes, an agonist-receptor-Gs alpha complex was immunoprecipitated with a specific alpha 2C10 antibody, and the Gs component identified by both western blots using Gs alpha antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the alpha 2AR subtypes, we investigated whether G-protein coupling was subtype-selective, using UK-14304 and cells with the same alpha 2AR expression levels (approximately 5 pmol/mg). Coupling to Gi was equivalent for alpha 2C10, alpha 2C4, and alpha 2C2: 53.4 +/- 8.8% versus 54.9 +/- 1.0% versus 47.6 +/- 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to Gs were found between the three alpha 2AR subtypes: stimulation of adenylyl cyclase was 57.9 +/- 6.3% versus 30.7 +/- 1.1% versus 21.8 +/- 1.7% for alpha 2C10, alpha 2C4, and alpha 2C2, respectively. Thus, alpha 2AR have the potential to couple physically and functionally to both Gi and Gs; for Gi coupling we found a rank order of alpha 2C10 = alpha 2C4 = alpha 2C2, while for Gs coupling, alpha 2C10 greater than alpha 2C4 greater than alpha 2C2.     

Reconstitution of cyc- S49 membranes by in vitro translated Gs alpha. Membrane anchorage and functional implications

After ADP-ribosylation by cholera toxin which promotes dissociation of the subunits, the alpha-subunit of Gs (Gs alpha) remained strongly associated with plasma membranes of wild-type S49 cells, since its interaction with the membrane was insensitive to 1 M KCl. Its association with the membrane was partially disrupted by 6 M urea and totally abolished by treatment with alkali at pH greater than or equal to 11.5. In vitro translated Gs alpha could interact with plasma membranes from the cyc- mutant of S49 cells as revealed by its cosedimentation with the membrane fraction and incubation of reconstituted membranes with GTP gamma S did not alter anchorage of Gs alpha. The characteristics of the association of in vitro translated Gs alpha with cyc- membranes after GTP gamma S treatment, i.e. sensitivity to 1 M KCl, 6 M urea and alkali treatment, were very similar to those described for the ADP-ribosylated form in wild-type membranes. Restoration of the coupling between the adrenergic receptor and adenylate cyclase further confirmed the vectorial reconstitution of cyc- membranes by in vitro translated alpha-subunit of Gs.     

Translocation of alpha subunits of stimulatory guanine nucleotide-binding proteins through stimulation of the prostacyclin receptor in mouse mastocytoma cells.

The translocation of the alpha subunits of Gs from the membrane to the cytosol by iloprost, a stable prostacyclin analogue, was studied in mouse mastocytoma P-815 cells. In the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S), iloprost stimulated the adenylate cyclase activity, caused the release of both 42- and 45-kDa proteins reactive with the anti Gs alpha carboxyl-terminal antibody, RM/1, from the membrane and attenuated cholera toxin-catalyzed ADP-ribosylation of the 42- and 45-kDa proteins in the membrane. The iloprost-stimulated adenylate cyclase activity and release of Gs alpha from the membrane were markedly suppressed by RM/1. Cholera toxin treatment also stimulated the adenylate cyclase activity and release of Gs alpha from the membrane, and iloprost synergistically potentiated these actions of cholera toxin. In mastocytoma cells, iloprost induced the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol, 45-kDa Gs alpha remaining in the cytosol for a longer time than 42- kDa Gs alpha. Whereas 42-kDa Gs alpha in the cytosol was eluted at the position of Mr = approximately 40,000 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 from a Superose 12 gel filtration column. In contrast, both 42- and 45-kDa Gs alpha released in vitro from the membrane by iloprost plus GTP gamma S were eluted at the position of Mr = approximately 40,000, but only 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 when it was incubated with cytosol. These results taken together demonstrate that iloprost induces the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol and that only the 45-kDa Gs alpha released exists in the cytosol as a soluble complex with unidentified component(s) in mastocytoma cells.     

Identification of proteins resembling G-protein alpha subunits in locust muscle

In locust skeletal muscle, FMRFamide-like peptides decrease a K+ conductance. Functional data suggest the involvement of G-proteins. For identification of G-protein alpha-subunits, membranes of locust skeletal muscle were probed with ADP-ribosylating bacterial toxins, the photoreactive GTP analog, [alpha-32P]GTP azidoanilide, and with antibodies against mammalian alpha-subunits. Multiple guanine nucleotide-binding proteins of approximately 24-95 kDa were detected. Pertussis toxin catalyzed the ADP-ribosylation of two proteins comigrating with the ADP-ribosylated alpha-subunits of the mammalian G-proteins Go and Gi. Cholera toxin promoted ADP-ribosylation of a protein comigrating with mammalian cholera toxin substrates (i.e., Gs alpha-subunits). An antibody against mammalian Go alpha-subunits detected a 54-kDa protein. Thus proteins with properties of mammalian G-protein subunits are present in insect muscle.