Pertussis and cholera toxin ADP-ribosylation in Dictyostelium discoideum membranes

We report a 39 kDa substrate for cholera and pertussis toxins is present in D. discoideum membranes. This protein did not co-migrate with alpha subunits of either Gs (45 kDa and 52 kDa) or Gi (41 kDa) from control mammalian cells. The presence of GTP or its non-hydrolyzable analogs enhanced the ADP-ribosylation in response to cholera toxin, but did not significantly alter ADP-ribosylation by pertussis toxin. Divalent cations inhibited the ADP-ribosylation by both toxins. The possible association of this novel G-protein with D. discoideum adenylate cyclase may underlie some of the unique regulatory features of this enzyme. Alternatively, this G-protein may regulate one of several other cellular responses mediated by the cAMP receptor.     

Two distinct Gi-proteins mediate formyl peptide receptor signal transduction in human leukemia (HL-60) cells

In membranes of myeloid differentiated HL-60 cells, the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine stimulates phospholipase C via a pertussis toxin-sensitive G-protein but does not inhibit adenylyl cyclase. In these membranes, the chemotactic peptide markedly stimulates the cholera toxin-dependent [32P]ADP-ribosylation of two proteins with approximate molecular masses of 40 and 41 kDa, respectively. The radiolabeled proteins comigrate on sodium dodecyl sulfate-polyacrylamide gels with the two pertussis toxin substrates present in HL-60 membranes, alpha i2 and alpha i3. The effect of the chemotactic peptide is blocked by treatment of intact HL-60 cells with pertussis toxin. Peptide mapping studies using Staphylococcus aureus protease V8 reveal that the two radiolabeled proteins are structurally distinct. Thus, the agonist-activated formyl peptide receptor functionally interacts with two distinct pertussis toxin substrates, most likely with Gi2 and Gi3. As the third Gi protein, Gi1, appears to be absent from both HL-60 cells and from systems that clearly reveal hormonal inhibition of adenylyl cyclase, the results strongly suggest that primary structure alone does not suffice to determine which effector mechanism is regulated by a given Gi-protein.     

Guanine nucleotide regulation of the pertussis and cholera toxin substrates of rat glioma C6 BU1 cells

Rat glioma C6 BU1 cells contain a pertussis toxin substrate of 40 kDa which does not appear to be identical with Gi,Go or transducin. The GTP analogue, GTP[gamma S], inhibited the rate of pertussis toxin-catalysed ADPribosylation of this protein, while the GDP analogue GDP[beta S] stimulated this reaction. A protein of the same kDa value was ADPribosylated by cholera toxin in the absence of added guanine nucleotides. It is suggested that this 40 kDa protein can be a substrate for both cholera and pertussis toxins under appropriate conditions.     

The phosphorylation of p68, a calcium-binding protein associated with the human syncytiotrophoblast submembranous cytoskeleton, is modulated by growth factors, activators of protein kinase C and cyclic AMP

The phosphorylation of the lipocortin-related protein, p68, found in Ca2+-dependent association with the submembranous cytoskeleton has been studied using isolated human placental syncytiotrophoblast plasma membrane vesicles. p68 undergoes rapid, cation-independent phosphorylation in unstimulated membrane vesicles which was inhibited, in a dose-dependent manner, by insulin, platelet-derived growth factor, macrophage colony stimulating factor, protein kinase C-activating phorbol esters and phosphatidylinositol-specific phospholipase C. Epidermal growth factor had no effect on overall p68 phosphorylation. Transferrin induced an increase in p68 phosphorylation. However, phosphotyrosine was detected in p68 after treatment with epidermal growth factor, macrophage colony stimulating factor or transferrin, whereas a reduction in p68 phosphorylation appeared to be restricted to serine. cAMP and both cholera and pertussis toxins inhibited p68 phosphorylation. Both toxins were synergistic with the effects of insulin and platelet-derived growth factor whilst being antagonistic to the effect of transferrin. Epidermal growth factor and both human and equine immunoglobulin G, all of which alone did not affect overall p68 phosphorylation, reduced cholera or pertussis toxin-induced inhibition of p68 phosphorylation. Several phosphatase inhibitors failed to prevent macrophage colony stimulating factor-induced reduction of p68 phosphorylation. These results indicate that (i) p68 is a potential substrate of receptor tyrosyl kinases, (ii) p68 is not phosphorylated by protein kinase C or cAMP-dependent kinase and (iii) p68 phosphorylation is inhibited by activation of multiple pathways including those employing diacylglycerol or cAMP as second messengers.     

Dependence on pH of substrate binding to a mutant lactose carrier, lacYun, in Escherichia coli. A model for H+/lactose symport.

The potential role of guanine nucleotide regulatory proteins (G-proteins) in acute insulin regulation of glucose transport was investigated by using bacterial toxins which are known to modify these proteins. Cholera-toxin treatment of isolated rat adipocytes had no effect on either 2-deoxyglucose transport or insulin binding. Pertussis-toxin treatment resulted in an inhibition of both insulin binding and glucose transport. Insulin binding was decreased in pertussis-toxin-treated cells by up to 40%, owing to a lowering of the affinity of the receptor for hormone, with no change in hormone internalization. The dose-response curve for insulin stimulation of glucose transport was strongly shifted to the right by pertussis-toxin treatment [EC50 (half-maximally effective insulin concn.) = 0.31 +/- 0.04 ng/ml in control cells; 2.29 +/- 1.0 in treated cells), whereas cholera toxin had only a small effect (EC50 = 0.47 +/- 0.02 ng/ml). Correcting for the change in hormone binding, pertussis toxin was found to decrease the coupling efficiency of occupied receptors (50% of maximal insulin effect with 928 molecules bound/cell in control and 3418 in treated cells). Pertussis-toxin inhibition of insulin sensitivity was slow in onset, requiring 2-3 h for completion. Under conditions where pertussis-toxin inhibition of insulin sensitivity was maximal, a 41,000 Da protein similar to the alpha subunit of Gi (the inhibitory G-protein) was found to be fully ribosylated. These results are consistent with the concept that pertussis-toxin-sensitive G-protein(s) can modify the insulin-receptor/glucose-transport coupling system.     

Biphasic regulation of adenylate cyclase by cholera toxin in neuroblastoma x glioma hybrid cells is due to the activation and subsequent loss of the alpha subunit of the stimulatory GTP binding protein (GS)

Exposure of neuroblastoma x glioma hybrid (NG108-15) cells to low concentrations of cholera toxin produced a stimulation of both basal and forskolin-amplified adenylate cyclase activity in membranes prepared from these cells. Higher concentrations of cholera-toxin reversed this effect. Mn2+ activation of adenylate cyclase indicated that this effect was not due to a modification of the intrinsic activity of this enzyme. Cholera toxin was demonstrated to produce a concentration and time-dependent loss of GS alpha from membranes of these cells. Loss of GS alpha from membranes of these cells was preceded by its ADP-ribosylation. The effects of cholera toxin were specific for GS alpha, as no alterations in levels of the pertussis toxin-sensitive G-proteins Gi2, Gi3 and Go, were noted in parallel. Equally, no alteration in levels of G-protein beta-subunit were produced by the cholera toxin treatment. These experiments demonstrate that cholera toxin-catalysed ADP-ribosylation does not simply maintain an activated population of GS at the plasma membrane and that alterations in levels of GS at the plasma membrane can modify adenylate cyclase activity.     

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.     

Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

Prostaglandin E2 (PGE2) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet (Negishi, M., Ito, S., Tanaka, T., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1987) J. Biol. Chem. 262, 12077-12084). In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, we purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its alpha subunit from two known pertussis toxin substrate G-proteins (Gi and Go) purified from bovine brain. The molecular weight of the alpha subunit was 40,000, which is between those of Gi and Go. The purified protein was also distinguished immunologically from Gi and Go and was referred to as Gam. PGE receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and freed from G-proteins by wheat germ agglutinin column chromatography. Reconstitution of the PGE receptor with pure Gam, Gi, or Go in phospholipid vesicles resulted in a remarkable restoration of [3H]PGE2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. The displacement of [3H]PGE2 binding was specific for PGE1 and PGE2. Furthermore, addition of PGE2 stimulated the GTPase activity of the G-proteins in reconstituted vesicles. These results indicate that the PGE receptor can couple functionally with Gam, Gi, or Go in phospholipid vesicles and suggest that Gam may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.     

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.     

G-protein mRNA levels during adipocyte differentiation

G-protein-mediated transmembrane signaling in 3T3-L1 cells is modulated by differentiation. The regulation of G-protein expression in differentiating 3T3-L1 cells was probed at the level of mRNA by DNA-excess solution hybridization. Pertussis toxin-catalyzed ADP-ribosylation of G-protein alpha-subunits increased as fibroblasts differentiate to adipocytes. Steady-state levels of mRNA for Gi alpha 2 and Go alpha, in contrast, declined sharply. Immunoblotting with antipeptide antibodies specific for Gi alpha 2, too, revealed a decline in the steady-state expression of this pertussis toxin substrate. ADP-ribosylation of Gs alpha by cholera toxin was less in the adipocyte than fibroblast. Analysis by immunoblotting revealed only a modest decline in Gs alpha. Analysis of mRNA levels also demonstrated a decline for Gs alpha. mRNA levels for the G beta-subunits rose initially (25%) on day 1, declined from day 1 to day 3, and remained 25% lower in adipocytes than in fibroblasts. In 3T3-L1 adipocytes the molar amounts of subunit mRNAs were: 60.6 (Gs alpha); 2.1 (Gi alpha 2); and 1.5 (Go alpha) amol/microgram total cellular RNA. In rat fat cells these mRNA levels were 19.4 (Gs alpha); 7.0 (Gi alpha 2); and 2.3 (Go alpha). These data demonstrate that for Gi alpha 2 and Go alpha alike mRNA and protein expression decrease, not increase, in differentiation. A substrate for pertussis toxin other than Gi alpha 2 and Go alpha appears to be responsible for the increase in toxin-catalyzed labeling that accompanies differentiation of 3T3-L1 cells.     

Chemotactic peptide receptor-cytoskeletal interactions and functional correlations in differentiated HL-60 cells and human polymorphonuclear leukocytes

We studied the chemotactic peptide receptor/cytoskeletal interactions in HL-60 cells induced to differentiate with different agents and attempted to correlate these observations with the acquisition of different functional responses. Dibutyryl cyclic AMP-treated cells showed rapid superoxide anion production in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) and slow, sustained response to phorbol myristate acetate (PMA). Retinoic acid-induced cells showed a slow, sustained response to both FMLP and PMA. Interferon-gamma-treated cells produced no superoxide anion on stimulation with FMLP, whereas tumor necrosis factor (TNF)-treated cells showed a slight response. Chemotactic peptide receptor association was the same in the HL-60 cells treated with different agents, despite marked differences in the superoxide anion generation and actin polymerization responses to FMLP and PMA in these cells. In mature neutrophils chemotactic peptide receptor association with the cytoskeleton was not affected by either pertussis or cholera toxin. However, both toxins inhibited FMLP-induced actin polymerization and superoxide anion generation. This suggested involvement of a G-protein similar to Gt, rather than Gi or Gs. Neither toxin had any effect on PMA-induced superoxide anion generation. These observations indicate that receptor association with the cytoskeleton may not have a significant role in affecting signal recognition and response. Among the several possible roles suggested, clearance of the occupied receptors may be the most important role of the cytoskeletal association. HL-60 cells induced to differentiate with different agents (because of their varied functional responses) might prove very useful in dissecting the molecular mechanisms regulating stimulus-induced activation of neutrophils.     

Oligomerization of neuropeptide Y (NPY) Y2 receptors in CHO cells depends on functional pertussis toxin-sensitive G-proteins

Human neuropeptide Y Y2 receptors expressed in CHO cells are largely oligomeric, and upon solubilization are recovered by density gradient centrifugation as approximately 180 kDa complexes of receptor dimers and G-protein heterotrimers. A large fraction of the receptors is inactivated in the presence of pertussis toxin, in parallel with inactivation of Gi alpha subunits (with half-periods of about 4 h for both). This is accompanied by a very long-lasting loss of receptor dimers and of masked surface Y2 sites (an apparent receptor reserve pre-coupled mainly to Gi alpha subunit-containing G-proteins). However, surface Y2 receptors accessible to large peptide agonists are much less sensitive to the toxin. All surface Y2 receptors are rapidly blocked by Y2 antagonist BIIE0246, with a significant loss of the dimers, but with little change of basal Gi activity. However, both dimers and Y2 receptor compartmentalization are restored within 24 h after removal of the antagonist. In CHO cells, the maintenance and organization of Y2 receptors appear to critically depend on functional pertussis toxin-sensitive G-proteins.     

Characterization of a G-protein from the mandibular organ of the lobster Homarus americanus (Nephropidae, Decapoda).

1. GTP-binding activity was found in both calf brain and male lobster mandibular organ (MO). There was approximately two to three times as much binding in the calf brain. 2. The GTP-binding activity could be extracted from the calf brain with sodium cholate, but not from the MOs. 3. Using ADP-ribosylation catalyzed by pertussis toxin, GTP-binding was shown to be the result of the presence of G-protein. In the lobster MO the G-protein alpha subunit has a molecular weight of about 42 kDa and may be of the Go or Gi varieties.     

Pertussis toxin-sensitive Gi protein involvement in epidermal growth factor-induced activation of phospholipase C-gamma in rat hepatocytes.

Treatment of rat hepatocytes with epidermal growth factor (EGF) produced an enhanced tyrosine phosphorylation of the EGF receptor and phospholipase C-gamma (PLC-gamma) in conjunction with the mobilization of Ca2+. Approximately 30% of the total PLC-gamma was tyrosine-phosphorylated with a maximum being reached after 30 s of incubation with EGF. Pretreatment of the rats with pertussis toxin prior to isolation of the hepatocytes blocked EGF-induced tyrosine phosphorylation of PLC-gamma and Ca2+ mobilization but had no effect on autophosphorylation of the EGF receptor or Ca2+ responses elicited by angiotensin II or phenylephrine. Under these conditions Gi protein alpha subunits were fully ADP-ribosylated. A 41-kDa Gi protein alpha subunit was found to be present in the anti-PLC-gamma immune complex after EGF stimulation as shown by in vitro ADP-ribosylation using [32P]NAD+ and activated pertussis toxin. The kinetics of association between PLC-gamma with Gi alpha protein reached a maximum after 1 min of incubation with EGF. Antibodies specific for the EGF receptor also coimmunoprecipitated a Gi protein alpha subunit. Treatment of hepatocytes with EGF caused first an increase and then a decrease in the amount of Gi protein alpha subunit associated with the EGF receptor. In contrast, studies with cultured rat liver (WB) cells, a cell line in which EGF stimulation of phosphoinositide hydrolysis is not inhibited by pertussis toxin, showed that a stable complex of Gi alpha was not formed with either PLC-gamma or EGF receptor immunoprecipitates. These results indicate that a pertussis toxin-sensitive Gi protein is uniquely involved in the signal transduction pathway mediating EGF-induced activation of PLC-gamma and Ca2+ mobilization in hepatocytes.     

Regulation of the mannose 6-phosphate/IGF II receptor expression at the cell surface by mannose 6-phosphate, insulin like growth factors and epidermal growth factor.

Mannose 6-phosphate, insulin like growth factors I and II (IGF I, IGF II), insulin and epidermal growth factor (EGF) induce a 1.5- to 2-fold increase of mannose 6-phosphate binding sites at the cell surface of human skin fibroblasts. The increase is completed within 10-15 min, is dose and temperature dependent, reversible and transient even in the presence of the effectors. It is due to a redistribution of mannose 6-phosphate/IGF II receptors from internal membranes to the cell surface, while the affinity of the receptors is not affected. Combinations of mannose 6-phosphate with IGF I, IGF II or EGF stimulate the redistribution of the receptor to the cell surface in an additive manner, while combinations of the growth factors result in a non-additive stimulation of redistribution. The redistribution is not dependent on extracellular calcium and appears also to be independent of changes of free intracellular calcium. Pre-treatment of fibroblasts with cholera toxin or pertussis toxin increases the number of cell surface receptors 2- and 1.5-fold, respectively. Neither of the toxins affects the redistribution of mannose 6-phosphate/IGF II receptors induced by the growth factors, while both toxins abolish the receptor redistribution induced by mannose 6-phosphate. These results suggest a multiple regulation of the cell surface expression of mannose 6-phosphate/IGF II receptors by Gs- and Gi-like proteins sensitive to cholera toxin and pertussis toxin and by stimulation of mannose 6-phosphate/IGF II, IGF I and EGF receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adipocyte plasma membranes contain two Gi subtypes but are devoid of Go

Antisera generated against synthetic peptides were used to identify G-protein alpha-subunits in plasma membranes from rat adipocytes. Applying the immunoblot technique, we detected two Gs alpha-subunits of 42 and 43 kDa, corresponding to the two cholera toxin substrates, and two Gi alpha-subunits of 40 and 41 kDa, corresponding to the two pertussis toxin substrates present in these membranes. The 40 kDa protein was tentatively identified as the Gi2 alpha-subunit. A serum specific for the Go alpha-subunit failed to detect any immunoreactive protein. Thus plasma membranes of adipocytes possess two forms of Gi but not Go.     

Effect of bacterial toxins on the mitogen-induced increase of the Ca2+ concentration in the cytoplasm of rat thymocytes. The role of N proteins

The effect of bacterial toxins, modifying the activity of regulatory N proteins of adenylate cyclase and probably other systems, on the mitogen-induced changes of cytosolic free Ca2+ concentration ([Ca2+]i) has been studied using Ca2+ fluorescent probe quin-2. It is shown that treatment of thymocytes with cholera toxin, E. coli heat-labile (HL) toxin or pertussis toxin abolishes the concanavalin A (con A)-induced rise of [Ca2+]i. The inhibitory effect of cholera and HL toxins can be explained by the toxin-induced rise of intracellular cAMP. The effect of pertussis toxin indicates the involvement of N proteins in the action of con A receptor and in generation of Ca2+-signal during the mitogenic activation of thymocytes.     

Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human alpha 2-C10 adrenergic receptor into rat 1 fibroblasts. Evidence for the direct interaction of a single receptor with two pertussis toxin-sensitive G-proteins, Gi2 and Gi3.

A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of [3H]yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study. When cholera toxin-catalyzed ADP-ribosylation was performed with [32P]NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha. Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells. Dose response curves for U.K.14304 enhancement of cholera toxin-labeling of the 40-kDa polypeptide(s) and stimulation of high affinity GTPase activity were identical. By contrast, U.K.14304 was ineffective in either assay in membranes from the 4D cells, demonstrating this effect to be dependent upon receptor activation. Furthermore, the alpha 2 receptor antagonist yohimbine blocked all effects of U.K.14304. The agonist promotion of cholera toxin-catalyzed ADP-ribosylation of Gi was completely blocked by guanine nucleotides. Whether GDP or GDP + fluoroaluminate (as a mimic of GTP) was used, blockade of the agonist effect was complete and indeed both conditions prevented agonist-independent labeling by cholera toxin of the 40-kDa polypeptide(s). Mg2+ produced an agonist-independent cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s) but even in the presence of [Mg2+], agonist-stimulation of cholera toxin-labeling of the 40-kDa polypeptide(s) was observed and was additive with the effect of [Mg2+]. Agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi was completely attenuated by pretreatment of the cells with pertussis toxin, which prevents contact between receptors and G-proteins which are substrates for this toxin. By contrast, pretreatment of the cells with concentrations of cholera toxin able to "down-regulate" essentially all of the membrane-associated Gs alpha did not prevent agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prostaglandin E receptors in bovine adrenal medulla are coupled to adenylate cyclase via Gi and to phosphoinositide metabolism in a pertussis toxin-insensitive manner

Prostaglandin E (PGE) receptor is coupled to a pertussis toxin-insensitive GTP-binding protein in bovine adrenal medulla, but PGE receptor partially purified from bovine adrenal medulla was functionally reconstituted with Gi into phospholipid vesicles (Negishi, M., Ito, S., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1988) J. Biol. Chem. 263, 6893-6900). We demonstrate here that PGE2 inhibited forskolin-induced accumulation of cAMP in cultured bovine chromaffin cells. In plasma membranes prepared from bovine adrenal medulla, PGE2 inhibited forskolin-stimulated adenylate cyclase activity in a GTP-dependent manner. This inhibitory action of PGE2 was abolished by treatment of the membrane with pertussis toxin. Reconstitution of the membranes ADP-ribosylated by pertussis toxin with Gi purified from bovine brain restored the potency of PGE2 to inhibit the adenylate cyclase activity. Inhibition of forskolin-induced cAMP accumulation by PGE2 was also abolished by exposure to the toxin in the cells, indicating that PGE receptors are coupled to Gi. In contrast, PGE2 stimulated the formation of inositol phosphates in chromaffin cells, but this effect was not affected by treatment of the cells with pertussis toxin, suggesting that the PGE receptors are coupled to phosphoinositide metabolism via a pertussis toxin-insensitive G-protein. Both the inhibitory action of cAMP accumulation and stimulation of phosphoinositide metabolism were specific for PGE1 and PGE2, and the Scatchard plot analysis of PGE2 binding to the membrane showed a single high-affinity binding site (Kd = 2 nM). In bovine adrenal chromaffin cells PGE2 enhanced catecholamine release in the presence of ouabain by stimulation of phosphoinositide metabolism (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). We further examined the modulation of catecholamine release by PGE2 through its inhibitory coupling to the adenylate cyclase system. Prior exposure of chromaffin cells to forskolin or dibutyryl-cAMP reduced nicotine-stimulated catecholamine release, and PGE2 attenuated forskolin-induced inhibition of catecholamine release stimulated by nicotine, but not dibutyryl-cAMP-induced inhibition. In the absence of evidence that PGE receptor subtypes exist, these results suggest that the PGE receptor is coupled to two signal transduction systems leading to inhibition of cAMP accumulation via Gi and to production of inositol phosphates via a pertussis toxin-insensitive G-protein, both of which may modulate catecholamine release from bovine chromaffin cells.     

Adenylate cyclase activity of v-ras-k transformed rat epithelial thyroid cells

The regulation of adenylate cyclase has been analyzed in normal rat thyroid cells as well as in the same cells transformed by the v-ras-k oncogene. In both cell types the adenylate cyclase complex consists of the two GTP-binding proteins, Gi and Gs, as demonstrated by the specific ADP-ribosylation induced by pertussis and cholera toxin, respectively. The response of adenylate cyclase of the transformed cells to forskolin, pertussis toxin and cholera toxin is attenuated with respect to the control cell line. The thyrotropic hormone (TSH), that acts on normal thyroid cells in culture as a growth factor by stimulating the adenylate cyclase activity, is not able to induce DNA synthesis nor does it stimulate adenylate cyclase in v-ras-k transformed cells.