Effect of bacterial toxins on the GTPase activity of transducin from bovine rod outer segments

The effects of choleragen- and pertussis toxin (PT)-induced ADP-ribosylation on the GTP-binding protein transducin (TD) from retinal rod outer segments (ROS) have been studied. It has been shown that both toxins cause inhibition of the TD GTPase activity. PT inhibited the GTPase by 30-40% in "native" ROS and by 70-80% in homogeneous TD. Choleragen, in contrast with PT, had no effect on the GTPase activity of homogeneous TD, but was as effective as PT in membrane preparations. The effects of both toxins on the GTPase activity of TD were found to be dependent on the chemical structure of the guanyl nucleotide present in the vehicle. The data obtained suggest that PT and choleragen differ in their specificity for the TD-guanyl nucleotide complex. The former can interact with free TD as well as with the TD-GDP complex, while the latter affects only the TD-GTP complex.     

Purification and characterization of a GTP-binding protein serving as pertussis toxin substrate in starfish oocytes

In response to a meiosis-inducing hormone, 1-methyladenine (1-MA), starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown. The 1-MA-initiated signal is, however, inhibited by prior microinjection of pertussis toxin into the oocytes (Shilling, F., Chiba, K., Hoshi, M., Kishimoto, T., and Jaffe, L.A. (1989) Dev. Biol. 133, 605-608), suggesting that a pertussis-toxin-sensitive guanine-nucleotide-binding protein (G protein) is involved in the 1-MA-induced signal transduction. Based on these findings, we purified a G protein serving as the substrate of pertussis toxin from the plasma membranes of starfish oocytes. The purified G protein had an alpha beta gamma-trimeric structure consisting of 39-kDa alpha, 37-kDa beta, and 8-kDa gamma subunits. The 39-kDa alpha subunit contained a site for ADP-ribosylation catalyzed by pertussis toxin. The alpha subunit was also recognized by antibodies specific for a common GTP-binding site of many mammalian alpha subunits or a carboxy-terminal ADP-ribosylation site of mammalian inhibitory G-alpha. An antibody raised against mammalian 36-/35-kDa beta subunits strongly reacted with the 37-kDa beta subunit of starfish G protein. The purified starfish G protein had a GTP-binding activity with a high affinity and displayed a low GTPase activity. The activity of the G protein serving as the substrate for pertussis-toxin-catalyzed ADP-ribosylation was inhibited by its association with a non-hydrolyzable GTP analogue. Thus, the starfish G protein appeared to be similar to mammalian G proteins at least in terms of its structure and properties of nucleotide binding and the pertussis toxin substrate. A possible role of the starfish G protein is also discussed in the signal transduction between 1-MA receptors and reinitiation of meiosis with germinal vesicle breakdown.     

IL-3 stimulated haemopoietic stem cell proliferation: evidence for G protein independent mitogenic signalling events.

Interleukin-3 stimulates the survival and proliferation of the FDCP-Mix 1 multipotent stem cell line. We have investigated the possible involvement of a guanyl nucleotide regulatory (G) protein(s) in the IL-3 stimulated proliferative response. We report here that pertussis toxin (PT) can partially inhibit IL-3 stimulated DNA synthesis and that this inhibition is bypassed by TPA. The ADP-ribosylation of the PT substrate G protein in vivo is complete in 2 hours without concomitant inhibition of IL-3 stimulated hexose transport or Na+/H+ exchange. When loaded into FDCP-Mix 1 cells fluoroaluminate and GTP-gamma-S, which can directly activate G proteins, are not capable of mimicking the effects of IL-3. Evidence is also presented that IL-3 does not stimulate a membrane-bound high affinity GTPase activity in the FDCP-Mix 1 cell line. These data suggest that a PT substrate G protein(s) can influence the IL-3 signalling cascade in an indirect or permissive manner, but that the IL-3 receptor does not directly couple to a PT substrate G protein.     

Proliferative effect of PGD2 on osteoblast-like cells; independent activation of pertussis toxin-sensitive GTP-binding protein from PGE2 or PGF2 alpha.

PGD2 stimulated DNA synthesis and decreased alkaline phosphatase activity dose-dependently between 10 nM and 10 microM in osteoblast-like MC3T3-E1 cells. PGD2 had little effect on cAMP production, but caused very rapid enhancement of phosphoinositide (PI) hydrolysis dose-dependently between 10 nM and 10 microM. The formation of inositol trisphosphate (IP3) induced by PGD2 reached the peak within 1 min and decreased thereafter, which is more rapid than that induced by PGE2 or PGF2 alpha and both PGE2 and PGF2 alpha affected PGD2-induced IP3 formation additively. Pertussis toxin (PTX) inhibited both PGD2-induced formation of inositol phosphates and DNA synthesis. The degree of these PTX (1 micrograms/ml)-induced inhibitions was similar. In addition, neomycin, a phospholipase C inhibitor, inhibited PGD2-induced DNA synthesis as well as the formation of IP3, and the patterns of both inhibitions were similar. In the cell membranes, PTX-catalyzed ADP-ribosylation of a 40-kDa protein was significantly attenuated by pretreatment of PGD2. Time course of the attenuation of PTX-catalyzed ADP-ribosylation by PGD2 was apparently different from that by PGE2 or PGF2 alpha. These results indicate that PGD2 activates PTX-sensitive GTP-binding protein independently from PGE2 or PGF2 alpha and stimulates PI hydrolysis resulting in proliferation of osteoblast-like cells.     

ADP-ribosylation of 24-26-kDa GTP-binding proteins localized in neuronal and non-neuronal cells by botulinum neurotoxin D

Clostridium botulinum D (strain South Africa) produces ADP-ribosyltransferase which modifies eukaryotic 24-26-kDa proteins. ADP-ribosyltransferase activity was associated with a neurotoxin of 150 kDa (Dsa toxin) as confirmed by the elution profile of Dsa toxin from high performance anion-exchange column. The 24-kDa substrate of Dsa toxin-catalyzed ADP-ribosylation was detected in several tissues examined including rat brain, heart, and liver; bovine adrenal medulla; sea urchin eggs; electric organs of electric fish; and cell lines of neural (N18, N1E115, NS20Y, NG108, PC12, and C6) and non-neural (3T3) origins, suggesting its ubiquitous localization in eukaryotic cells. On the other hand, the 26-kDa substrate was detected only in membrane fractions of neural tissues and neuronal cells, suggesting its specific localization in membrane of nerve terminals. ADP-ribosylation of both the 24-kDa substrate in PC12 membrane and the 24-26-kDa substrates in rat brain membrane was potentiated by either divalent cations or guanine nucleotides, whereas adenine nucleotides did not affect the ADP-ribosylation reaction. Trypsin digestion of the 24-kDa substrate in PC12 membrane and the 24-26-kDa substrates in rat brain membrane extract produced different tryptic fragments indicative of the structural difference between the 24- and 26-kDa substrates. Both the 24- and 26-kDa substrates were less sensitive to trypsin digestion before being ADP-ribosylated by Dsa toxin than after, suggesting the conformational alterations of the 24-26-kDa proteins induced by ADP-ribosylation. These results suggest that Dsa toxin modifies two distinct low molecular mass GTP-binding proteins by ADP-ribosylation to alter their putative function(s).     

Effect of pertussis toxin treatment on the down-regulation of opiate receptors in neuroblastoma X glioma NG108-15 hybrid cells

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with 10 nM [D-Ala2,D-Leu5] enkephalin (DADLE) results in a reduction of cell-surface opiate delta receptors. Whether opiate receptor internalization requires the activation of the guanine nucleotide-binding protein (Ni) is unclear. Hence, activation of Ni was attenuated by treating hybrid cells with 100 ng/ml pertussis toxin (PT) for 3 h, which resulted in a decrease in DADLE's ability to inhibit adenylate cyclase activity. Despite this prior treatment with PT, chronic exposure of these cells to 10 nM DADLE resulted in a time-dependent decrease in both [3H]diprenorphine and [3H]DADLE binding. This reduction in 3H-ligand binding in cells previously treated with PT represented internalization of the receptors because translocation was observed of bound [3H]DADLE from plasma membrane fractions to the lysosomal fractions in the Percoll gradients. Thus, opiate receptors internalize without activation of Ni. The internalization of opiate receptors was not accompanied by Ni. By measuring the amount of the 41-kDa alpha subunit being labeled by PT with [32P]NAD+, it was determined that plasma membrane preparations, of both the control cells and cells treated with 10 nM of DADLE for 4 h, contained equal concentrations of Ni, 2 pmol of Ni/mg of protein. Additionally, there was no measurable alteration in the amount of PT substrate in the lysosomal fractions of the DADLE-treated cells as compared to that of control cells. Chronic DADLE treatment resulted in a decrease in Km value of NAD+ in the ADP-ribosylation of 41-kDa subunit of Ni. In summary, opiate receptors internalize as agonist-receptor complexes without the guanine nucleotide-binding component.     

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)     

Identification of a new GTP-binding protein. A Mr = 43,000 substrate for pertussis toxin

In purified preparations of human erythrocyte GTP-binding proteins, we have identified a new substrate for pertussis toxin, which has an apparent molecular mass of 43 kDa by silver and Coomassie Blue staining. Pertussis toxin-catalyzed ADP-ribosylation of the 43-kDa protein is inhibited by Mg2+ ion and this inhibition is relieved by the co-addition of micromolar amounts of guanine nucleotides. GTP affects the ADP-ribosylation with a K value of 0.8 microM. Addition of a 10-fold molar excess of purified beta gamma subunits (Mr = 35,000 beta; and Mr = 7,000 gamma) of other GTP-binding proteins results in a significant decrease in the pertussis toxin-mediated ADP-ribosylation of the 43-kDa protein. Treatment of the GTP-binding proteins with guanosine 5'-O-(thiotriphosphate) and 50 mM MgCl2 resulted in shifting of the 43-kDa protein from 4 S to 2 S on sucrose density gradients. Immunoblotting analysis of the 43-kDa protein with the antiserum A-569, raised against a peptide whose sequence is found in the alpha subunits of all of the known GTP-binding, signal-transducing proteins (Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 265-259) showed that the 43-kDa protein is specifically recognized by the common peptide antiserum. A pertussis toxin substrate of similar molecular weight was observed in human erythrocyte membranes, bovine brain membranes, membranes made from the pituitary cell line GH4C1, in partially purified GTP-binding protein preparations of rat liver, and in human neutrophil membranes. Treatment of neutrophils with pertussis toxin prior to preparation of the membranes resulted in abolishment of the radiolabeling of this protein. From these data, we conclude that we have found a new pertussis toxin substrate that is a likely GTP-binding protein.     

Activation of Galpha (i) and subsequent uncoupling of receptor-Galpha(i) signaling by Pasteurella multocida toxin

Bacterial protein toxins are powerful tools for elucidating signaling mechanisms in eukaryotic cells. A number of bacterial protein toxins, e.g. cholera toxin, pertussis toxin (PTx), or Pasteurella multocida toxin (PMT), target heterotrimeric G proteins and have been used to stimulate or block specific signaling pathways or to demonstrate the contribution of their target proteins in cellular effects. PMT is a major virulence factor of P. multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. PMT modulates various signaling pathways, including phospholipase Cbeta and RhoA, by acting on the heterotrimeric G proteins Galpha(q) and Galpha(12/13), respectively. Here we report that PMT is a powerful activator of G(i) protein. We show that PMT decreases basal isoproterenol and forskolin-stimulated cAMP accumulation in intact Swiss 3T3 cells, inhibits adenylyl cyclase activity in cell membrane preparations, and enhances the inhibition of cAMP accumulation caused by lysophosphatidic acid via endothelial differentiation gene receptors. PMT-mediated inhibition of cAMP production is independent of toxin activation of Galpha(q) and/or Galpha(12/13). Although the effects of PMT are not inhibited by PTx, PMT blocks PTx-catalyzed ADP-ribosylation of G(i). PMT also inhibits steady-state GTPase activity and GTP binding of G(i) in Swiss 3T3 cell membranes stimulated by lysophosphatidic acid. The data indicate that PMT is a novel activator of G(i), modulating its GTPase activity and converting it into a PTx-insensitive state.     

Pertussis toxin treatment attenuates some effects of insulin in BC3H-1 murine myocytes

The effects of pertussis toxin (PT) treatment on insulin-stimulated myristoyl-diacylglycerol (DAG) generation, hexose transport, and thymidine incorporation were studied in differentiated BC3H-1 myocytes. Insulin treatment caused a biphasic increase in myristoyl-DAG production which was abolished in myocytes treated with PT. There was no effect of PT treatment on basal (nonstimulated) myristoyl-DAG production. Insulin-stimulated hydrolysis of a membrane phosphatidylinositol glycan was blocked by PT treatment. ADP-ribosylation of BC3H-1 plasma membranes with [32P]NAD revealed a 40-kDa protein as the major PT substrate in vivo and in vitro. The time course and dose dependence of the effects of PT on diacylglycerol generation correlated with the in vivo ADP-ribosylation of the 40-kDa substrate. Pertussis toxin treatment resulted in a 71% attenuation of insulin-stimulated hexose uptake without effect on either basal or phorbol ester-stimulated uptake. The stimulatory effects of insulin and fetal calf serum on [3H]thymidine incorporation into quiescent myocytes were attenuated by 61 and 59%, respectively, when PT was added coincidently with the growth factors. Nonstimulated and EGF-stimulated [3H]thymidine incorporation was unaffected by PT treatment. These data suggest that a PT-sensitive G protein is involved in the cellular signaling mechanisms of insulin.     

Stimulation of macrophage colony-stimulating factor synthesis by interleukin-1.

Interleukin-1 (IL-1), which plays an important role in the inflammatory response, was found to induce colony-stimulating factor-1 (CSF-1) expression in the MIA PaCa-2 cells. IL-1-induced CSF-1 production was markedly suppressed (70%) by pertussis toxin. This inhibition by pertussis toxin was reversed by benzamide, an inhibitor of ADP-ribosylation reactions. Similarly, IL-1-induced CSF-1 production was inhibited by cholera toxin and this inhibition was reversed by an arginine analog, p-methoxy-benzylaminodecamethylene guanidine sulfate. Dibutyryl-cAMP as well as other cAMP elevating agents such as theophylline and forskolin also suppressed IL-1-induced CSF-1 production, suggesting that cAMP concentrations inversely regulate the biosynthesis of CSF-1. Measurement of cAMP concentration indicated that IL-1 treatment of MIA PaCa-2 cells did not change the cAMP level. IL-1-induced CSF-1 production was not suppressed by the protein kinase C (PKC) inhibitor, H7, under conditions in which 12-O-tetradecanoylphorbol-13-acetate-induced CSF-1 production was completely abolished. These data suggest that IL-1-induced CSF-1 production is not mediated via the activation of PKC. Analysis of oncogene c-fos and c-jun expression has shown the enhancement of expression of both protooncogenes prior to CSF-1, suggesting that the expression of these two oncogenes may be the mechanism which triggers CSF-1 gene expression.     

Mastoparan interacts with the carboxyl terminus of the alpha subunit of Gi

Mastoparan, a peptide toxin from wasp venom, stimulates guanine nucleotide binding and hydrolysis by G proteins. To elucidate the site of mastoparan-G protein interaction, we utilized a polyclonal antibody (R16,17) directed against the carboxyl terminus of the Gi alpha subunit to develop a competitive enzyme-linked immunosorbent assay. We investigated the ability of mastoparan to influence R16,17 antibody binding to G protein alpha subunits in a purified preparation of brain Gi and in neutrophil membrane extracts. Mastoparan antagonized the ability of R16,17 to detect G protein alpha subunits with an IC50 of 15 microM in the purified preparation and with an IC50 of 1 microM for the predominant G protein population in membrane extracts. This reduction was not seen when an unrelated peptide or a peptide of similar charge composition to mastoparan was used in place of mastoparan in the assay. Additionally, antibody R16,17 blocked up to 85% of mastoparan-stimulated GTPase activity. Taken together, these data indicate that the interaction of mastoparan with G protein depends in part on the carboxyl terminus of Gi alpha. Pertussis toxin-catalyzed ADP-ribosylation of Gi alpha markedly inhibited mastoparan-stimulated GTPase activity but only slightly attenuated the ability of mastoparan to recognize G protein. These data suggest that ribosylation inhibits mastoparan-induced G protein activation by a mechanism distinct from the ability of mastoparan to physically interact with G protein. Since mastoparan is thought to mimic hormone-liganded receptors, these findings may be applicable to the mechanism of receptor-Gi protein uncoupling that results from ADP-ribosylation of the G protein.     

Coupling of the guanine nucleotide regulatory protein to chemotactic peptide receptors in neutrophil membranes and its uncoupling by islet-activating protein, pertussis toxin. A possible role of the toxin substrate in Ca2+-mobilizing receptor-mediated signal transduction

A chemotactic peptide stimulated the high-affinity GTPase activity in membrane preparations from guinea pig neutrophils. The enzyme stimulation was inhibited by prior exposure of the membrane-donor cells to islet-activating protein (IAP), pertussis toxin, or by direct incubation of the membrane preparations with its A-protomer (the active peptide) in the presence of NAD. The affinity for the chemotactic peptide binding to its receptors was lowered by guanyl-5'-yl beta, gamma-imidodiphosphate (Gpp(NH)p) reflecting its coupling to the guanine nucleotide regulatory protein in neutrophils. The affinity in the absence of Gpp(NH)p was lower, but the affinity in its presence was not, in the A-protomer-treated membranes than in nontreated membranes. The inhibitory guanine nucleotide regulatory protein of adenylate cyclase (Ni) was purified from rat brain, and reconstituted into the membranes from IAP-treated cells. The reconstitution was very effective in increasing formyl-Met-Leu-Phe-dependent GTPase activity and increasing the chemotactic peptide binding to membranes due to affinity increase. The half-maximal concentration of IAP to inhibit GTPase activity was comparable to that of the toxin to inhibit the cellular arachidonate-releasing response which was well correlated with ADP-ribosylation of a membrane Mr = 41,000 protein (Okajima, F., and Ui, M. (1984) J. Biol. Chem. 259, 13863-13871). It is proposed that the IAP substrate, Ni, couples to the chemotactic peptide receptor and mediates arachidonate-releasing responses in neutrophils, as it mediates adenylate cyclase inhibition in many other cell types.     

Pertussis toxin catalyzed ADP-ribosylation of a 41 kDa G-protein impairs insulin-stimulated glucose metabolism in BC3H-1 myocytes

In this study, we examined the effects of pertussis toxin (PT) on the ADP-ribosylation of guanine nucleotide binding proteins (G-proteins) and various insulin-stimulated processes in cultured BC3H-1 myocytes. Treatment of intact myocytes with 0.1 microgram/ml PT for 24 hours resulted in the complete ribosylation of a 41 kDa protein. The 41 kDa PT substrate was immunoprecipitated with antibodies directed against a synthetic peptide corresponding to a unique sequence in the alpha subunit of Gi-proteins. PT treatment of intact cells had no effect on insulin receptor binding or internalization. However, PT inhibited insulin-stimulated glucose transport at all insulin-concentrations tested (1-100 ng/ml). Maximally stimulated glucose transport was reduced by 50% +/- 15%. Insulin-stimulated glucose oxidation was also decreased by 31% +/- 8%. The toxin had no significant effect on the basal rates of glucose transport and glucose oxidation. The time course of PT-induced inhibition on glucose transport correlated with the time course of the "in vivo" ADP-ribosylation of the 41 kDa protein. The results suggest that a 41 kDa PT-sensitive G-protein, identical or very similar to Gi, is involved in the regulation of glucose metabolism by insulin in BC3H-1 cells.     

Characterization of cytosolic pertussis toxin-sensitive GTP-binding protein in mastocytoma P-815 cells

We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.     

Gz, a guanine nucleotide-binding protein with unique biochemical properties

Cloning of a complementary DNA (cDNA) for Gz alpha, a newly appreciated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the beta gamma subunit complex common to other G proteins; another 26-kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant Gz alpha (rGz alpha) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rGz alpha has several properties that distinguish it from other G protein alpha subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min-1), which is reduced greater than 20-fold in the presence of mM concentrations of Mg2+. In addition, the rate of the intrinsic GTPase activity of Gz alpha is extremely slow. The hydrolysis rate (kcat) for rGz alpha at 30 degrees C is 0.05 min-1, or 200-fold slower than that determined for other G protein alpha subunits. rGz alpha can interact with bovine brain beta gamma but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family.     

Effect of tumor necrosis factor-alpha on the stimulus-coupled responses of neutrophils and their modulation by various inhibitors.

Preincubation of human peripheral blood polymorphonuclear leukocytes (HPPMN) with recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) enhanced the formylmethionyl-leucylphenylalanine (FMLP)-induced superoxide (O2-.) generation in a concentration- and preincubation time-dependent manner. The enhancement was very high for the FMLP- or opsonized zymosan (OZ)-induced O2-. generation, but was low for arachidonic acid (AA)- and phorbol myristate acetate (PMA)-induced O.2- generation. The rHuTNF-alpha has no effect on the steady state of intracellular calcium ion concentration ([Ca2+]i) nor on the membrane potential of neutrophils. The rHuTNF-alpha-primed FMLP-induced O2-. generation was inhibited by nicotineamide (NA), pertussis toxin (PT), and by the tyrosine kinase (TK) inhibitor, genistein, but was enhanced by the protein kinase C (PKC) inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-3-methyl-piperazine). The inhibitory actions of NA and PT were also observed in in vivo primed guinea pig peritoneal neutrophils (GPtPMN). However, FMLP-induced O2-. generation of GPtPMN was enhanced by genistein, but was inhibited by H-7. These data indicate that TNF-alpha does not induce changes in [Ca2+]i nor in membrane potential of HPPMN, and that TNF-alpha-primed FMLP-induced O.2- generation of HPPMN is coupled with ADP-ribosylation and activation of G-proteins, and that protein kinases, especially TK, seem to exert an important role in the priming action of TNF.     

PTH-dependent adenylyl cyclase activation in SaOS-2 cells: passage dependent effects on G protein interactions

Parathyroid hormone (PTH) sensitive adenylyl cyclase activity (ACA) in SaOS-2 cells varies as a function of cell passage. In early passage (EP) cells (< 6), ACA in response to PTH and forskolin (FOR) was relatively low and equivalent, whereas in late passage (LP) cells (> 22), PTH exceeded FOR dependent ACA. Potential biochemical mechanisms for this passage dependent change in ACA were considered. In EP, prolonged exposure to pertussis toxin (PT) markedly enhanced ACA activity in response to PTH, Isoproterenol and Gpp(NH)p, whereas ACA in response to FOR was decreased. In contrast, the identical treatment of LP with PT diminished all ACA in response to PTH, Gpp(NH)p, and FOR. The dose dependent effects of PT on subsequent [(32)P]ADP-ribosylation of its substrates, GTPase activity, as well as FOR-dependent ACA, were equivalent in EP and LP. The relative amounts of G(alpha)i and G(alpha)s proteins, as determined both by Western blot, PT and cholera toxin (CT) dependent [(32)P]ADP-ribosylation, were quantitatively similar in EP and LP. Western blot levels of G(alpha)s and G(alpha)i proteins were not influenced by prior exposure to PT. Both PT and CT dependent [(32)P]ADP-ribosylation were dose-dependently decreased following exposure to PT. However, the PT-dependent decline in CT-dependent [(32)P]ADP-ribosylation occurred with enhanced sensitivity in LP. The protein synthesis inhibitor cycloheximide partially reversed the PT associated decrease in FOR dependent ACA in EP. In contrast, cycloheximide completely reversed the PT associated decrease in FOR and as well as PTH dependent ACA in LP. G(alpha)s activity, revealed by cyc(-) reconstitution, was not altered either by cell passage or exposure to PT. The results suggest that the coupling between the components of the complex may be pivotally important in the differential responsiveness of early and late passage SaOS-2 cells to PTH.     

The amino terminus of G protein alpha subunits is required for interaction with beta gamma

The guanine nucleotide-binding proteins (G proteins), which transduce hormonal and light signals across the plasma membrane, are heterotrimers composed of alpha, beta, and gamma subunits. Activation of G proteins by guanine nucleotides is accompanied by dissociation of the heterotrimer: G + alpha.beta.gamma in equilibrium alpha G + beta.gamma. Brain contains several G proteins of which the most abundant are alpha 39.beta.gamma and alpha 41.beta.gamma. We have used proteolysis by trypsin to study the functional domains of the alpha subunits. In the presence of guanosine 5'-(3-O-thio)triphosphate, trypsin removes a 2-kDa peptide from the amino terminus of these proteins (Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D., and Gilman, A. G. (1984) Science 226, 860-862; Winslow, J. W., Van Amsterdam, J. R., and Neer, E. J. (1986) J. Biol. Chem. 261, 7571-7579). Tryptic cleavage does not affect the GTPase activity of the truncated molecule nor the apparent Km for GTP. However, removal of the 2-kDa amino-terminal peptide prevents association of the alpha subunits with beta.gamma. Since the apparent substrate for pertussis toxin-catalyzed ADP-ribosylation is the alpha.beta.gamma heterotrimer, the trypsin-cleaved alpha subunit is not a substrate for the toxin. Digestion of the carboxyl terminus of alpha 39 with carboxypeptidase A prevents ADP-ribosylation by pertussis toxin but does not interfere with the formation of alpha 39.beta.gamma heterotrimers. We do not yet know whether the amino-terminal region of alpha 39 interacts with beta gamma directly or whether it is necessary to maintain a conformation of alpha 39 which is required for heterotrimer formation. Further studies are needed to define the nature of the contracts between alpha and beta gamma subunits since understanding the structural basis for their reversible interaction is fundamental to understanding their function.     

Pertussis toxin effects on T lymphocytes are mediated through CD3 and not by pertussis toxin catalyzed modification of a G protein

Pertussis toxin (PT) has been shown to have a variety of effects on T lymphocyte function, and its activity has been used to suggest the involvement of a G protein in the early events of T lymphocyte activation. In this report, the effects of PT on T lymphocytes have been investigated in detail. PT at a concentration of 10 micrograms/ml rapidly stimulated early events that are normally induced by occupancy of the TCR complex in Jurkat cells and cloned, murine CTL including increased intracellular Ca2+ concentration, serine esterase release, and induction of Ag non-specific target cell lysis. However, 1-h treatment with this concentration of PT induced a state that was refractory to further receptor stimulation in Jurkat cells but not cloned CTL although substrate membrane proteins were modified to a similar extent in both cell lines. The functional effects of PT were mimicked by the B oligomer of PT which did not, however, catalyze ADP-ribosylation of membrane proteins. In addition, overnight exposure of Jurkat cells to a lower concentration of PT also modified substrate membrane proteins but did not inhibit receptor stimulation. These findings indicate that PT catalyzed ADP-ribosylation of a G protein does not account for the actions of the toxin on T lymphocytes. Finally, direct stimulation of increased intracellular Ca2+ concentration by PT and the B oligomer only occurred in T lymphocytes expressing CD3. This suggests that the mitogenic effect of PT holotoxin is mediated by the interaction of the B oligomer with CD3 and that this may account for many of the effects of PT holotoxin both in vivo and in vitro.