v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine.

Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with arachidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG production and enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.     

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.     

Effect of nonhydrolyzable guanosine phosphate on IgE-mediated activation of phospholipase C and histamine release from rodent mast cells

Rat mast cells and bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE mAb, and permeabilized by ATP to introduce guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) and/or guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) into the cells. After ATP-induced lesions were resealed with Mg2+, the cells were challenged by Ag to determine the effect of the nonhydrolyzable guanosine phosphate on Ag-induced hydrolysis of phosphoinositides and histamine release. Introduction of GTP gamma S into permeabilized rat mast cells or BMMC, followed by exposure of the cells to extracellular Ca2+, resulted in histamine release, but failed to induce hydrolysis of phosphoinositides. It was also found that introduction of GTP gamma S into the cells did not synergistically enhance Ag-induced histamine release. Introduction of GDP beta S into sensitized BMMC inhibited the GTP gamma S-dependent, Ca2+-induced histamine release but failed to inhibit Ag-induced histamine release. The results suggest that GTP gamma S-dependent, Ca2+-induced histamine release and Ag-induced histamine release go through independent biochemical pathways. It was also found that introduction of GTP gamma S or GDP beta S into sensitized BMMC neither enhanced nor inhibited Ag-induced formation of inositol phosphates. These results together with previous findings that pretreatment of BMMC with either pertussis toxin or cholera toxin does not affect Ag-induced hydrolysis of phosphoinositides, indicate that a G protein is not involved in the transduction of IgE-mediated triggering signals to phospholipase C in rodent mast cells.     

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: possible involvement of an inhibitory GTP-binding protein

Cultured pituitary cells prelabeled with myo-[2-3H] inositol were permeabilized by ATP4-, exposed to guanine nucleotides and resealed by Mg2+. Addition of guanosine 5'-0-(3-thio triphosphate) (GTP gamma S) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5'-0-(2-thiodiphosphate) (GDP beta S) or by neomycin. Surprisingly, addition of GDP beta S (100-600 microM) on its own resulted in a dose-related increase in [3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP greater than GTP gamma S greater than ATP greater than CTP. The stimulatory effect of UTP, ATP and CTP, but not GTP gamma S or GDP beta S, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTP gamma S decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.     

Guanosine 5'-O-(3-thiotriphosphate) and guanosine 5'-O-(2-thiodiphosphate) activate G proteins and potentiate fibroblast growth factor-induced DNA synthesis in hamster fibroblasts

Addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to intact Chinese hamster lung fibroblasts (CCL39) depolarized by high K+ concentrations results in activation of phosphoinositide-specific phospholipase C (PLC) (at GTP gamma S concentrations greater than 0.1 mM), inhibition of adenylate cyclase (between 10 microM and 0.5 mM), and activation of adenylate cyclase (above 0.5 mM). Since GTP gamma S-induced activation of PLC is dramatically enhanced upon receptor-mediated stimulation of PLC by alpha-thrombin, we conclude that in depolarized CCL39 cells GTP gamma S directly activates various guanine nucleotide-binding regulatory proteins (G proteins) coupled to PLC (Gp(s)) and to adenylate cyclase (Gi and Gs). Pretreatment of cells with pertussis toxin strongly inhibits GTP gamma S-induced activation of PLC and inhibition of adenylate cyclase. GTP gamma S cannot be replaced by other nucleotides, except by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which mimics after a lag period of 15-20 min all the effects of GTP gamma S, with the same concentration dependence and the same sensitivity to pertussis toxin. We suggest that GDP beta S is converted in cells into GTP beta S, which acts as GTP gamma S. Since cell viability is not affected by a transient depolarization, these observations provide a simple method to examine long-term effects of G protein activation on DNA synthesis. We show that a transient exposure of G0-arrested CCL39 cells to GTP gamma S or GDP beta S under depolarizing conditions is not sufficient by itself to induce a significant mitogenic response, but markedly potentiates the mitogenic action of fibroblast growth factor, a mitogen known to activate a receptor-tyrosine kinase. The potentiating effect is maximal after 60 min of pretreatment with 2 mM GTP gamma S. GDP beta S is equally efficient but only after a lag period of 15-20 min. Mitogenic effects of both guanine nucleotide analogs are suppressed by pertussis toxin. Since the activation of G proteins by GTP gamma S under these conditions vanishes after a few hours, we conclude that a transient activation of G proteins facilitates the transition G0----G1 in CCL39 cells, whereas tyrosine kinase-induced signals are sufficient to mediate the progression into S phase.     

Site of pertussis toxin-induced ADP-ribosylation on Gi is critical for receptor modulation of GDP interaction with Gi

In this study, the influence of the inhibitory mu-opioid receptor on the potencies of 5'-guanosine alpha-thiotriphosphate (GTP gamma S) and GDP at the inhibitory GTP-binding protein (Gi) were investigated in an adenylyl cyclase system. It was hoped that a receptor-mediated change in the potency of either GTP gamma S or GDP in affecting adenylyl cyclase activity may elucidate how a receptor alters cyclase activity via its G-protein. In an adenylyl cyclase system employing 5'-adenylyl imidodiphosphate as substrate, GTP gamma S, a nonhydrolyzable analog of GTP, inhibited forskolin-stimulated adenylyl cyclase activity in the absence of morphine; morphine failed to significantly affect the apparent potency of GTP gamma S. GDP blocked the GTP gamma S-induced inhibition of adenylyl cyclase; morphine profoundly diminished the ability of GDP to block the inhibitory effect of GTP gamma S. The IC50 values of GTP gamma S were 0.02 +/- 0.01, 0.18 +/- 0.04, and 2.2 +/- 0.5 microM in the absence of other drugs, in the presence of a combination of 100 microM GDP and morphine, and in the presence of 100 microM GDP, respectively. GDP blocked the inhibitory effect of GTP gamma S (0.3 microM) in a concentration-dependent manner; the EC50 for GDP was 16 +/- 2.6 microM in the absence of morphine and 170 +/- 32 microM in the presence of morphine. Exposure of 7315c cells to pertussis toxin for 3 h resulted in a small decrease in the potency of GTP gamma S in inhibiting cyclase. However, the relative potency of GDP in blocking the GTP gamma S-mediated inhibition of cyclase was increased: the EC50 values of GDP were 11 +/- 4 and 0.81 +/- 0.2 microM in untreated and pertussis toxin-treated membranes, respectively. In untreated membranes, there was a brief lag in the GTP gamma S-induced inhibition of adenylyl cyclase; morphine diminished this lag. In membranes treated with pertussis toxin, there was an exaggerated lag in the onset of GTP gamma S inhibition of adenylyl cyclase activity; morphine could no longer affect this lag. Thus, uncoupling the mu-opioid receptor from Gi appeared to increase the affinity of Gi for GDP. These data suggest that the effect of an inhibitory receptor is to decrease the affinity of Gi for GDP by virtue of its interaction with the carboxy-terminal region of Gi alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin-1 signal transduction. Increased GTP binding and hydrolysis in membranes of a murine thymoma line (EL4)

The post-receptor events which follow the binding of interleukin 1 (IL1) to cells are unclear. The present studies provide evidence for the activation of a guanine nucleotide binding protein (G protein) by IL1 in the membranes of an IL1 receptor-rich strain (NOB-1) of the EL4 murine thymoma line. IL1 alpha and beta increased the binding of the GTP analogue [35S]guanosine 5'-[gamma-thiol]trisphosphate (GTP gamma S) to membranes prepared from these cells. By 1 min after addition of IL1 there was a 2-fold enhancement in binding which was dose dependent in the range 0.1-100 ng/ml. A qualitatively similar result was obtained with IL1 beta although it was 10 times less potent. Specific neutralizing antisera to IL1 alpha and IL1 beta abolished the response. Experiments in which the concentration of [35S]GTP gamma S was varied revealed that IL1 increased the affinity of the binding sites for [35S]GTP gamma S and not their number. IL1 alpha was shown to stimulate GTPase activity in the membranes, the time and concentration dependence of this was similar to that observed for increased [35S]GTP gamma S binding. Half-maximal enhancement of [35S]GTP gamma S binding by IL1 alpha, measured after 4 min, occurred at 5% IL1 receptor occupancy. Maximal stimulation was achieved when 30% of receptors were occupied. Experiments with pertussis and cholera toxins revealed that pretreating membranes with pertussis toxin (100 ng/ml) inhibited by 50% the IL1-induced [35S]GTP gamma S binding and [gamma-32P]GTP hydrolysis. Cholera toxin (100 ng/ml) was without effect. However, both pertussis and cholera toxins at concentrations of 100 ng/ml inhibited IL1-induced IL2 secretion in EL4 NOB-1 cells. These results show that the IL1 receptor of a responsive thymoma line activates, and may be coupled to, a G protein(s). This is a possible mechanism of IL1 signal transduction.     

The epidermal growth factor receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rat hepatocytes.

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.     

Muscarinic-agonist and guanine nucleotide stimulation of myo-inositol trisphosphate formation in membranes isolated from bovine iris sphincter smooth muscle: effects of short-term cholinergic desensitization

The effect of short-term cholinergic desensitization on muscarinic acetylcholine receptor (mAChR)-mediated activation of phospholipase C was investigated in membranes isolated from the bovine iris sphincter smooth muscle. Membranes prepared from normal or desensitized muscles, prelabeled with either [3H]myo-inositol or 32P from [gamma-32P]ATP, were incubated with a hydrolysis-resistant analogue of GTP, GTP gamma S, or GTP gamma S plus carbachol (CCh), and the production of [3H]myo-inositol 1,4,5-trisphosphate (IP3) and the breakdown of polyphosphoinositides were assessed. In normal membranes, GTP (greater than or equal to 1 mM), GTP gamma S (greater than 10 microM) and GTP gamma S (1 microM) plus CCh (10 microM), but not GDP or GDP beta S, increased phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and IP3 production. GTP gamma S increased IP3 accumulation in a time- and dose-dependent manner, and CCh, which had no effect on phospholipase C activity in the absence of GTP gamma S, potentiated the effects of GTP gamma S. The effect of CCh plus GTP gamma S on IP3 production was inhibited by atropine, had an absolute requirement for nM amounts of Ca2+ and was not affected by pertussis toxin. At higher concentrations (greater than 1 microM), Ca2+ alone induced PIP2 hydrolysis. Short-term exposure (less than 60 min) of the muscle to CCh (100 microM) did not affect the total number (Bmax) of mAChRs nor their affinity (KD) for [3H]-N-methylscopolamine. Desensitization did, however, result in: (1) a loss of the CCh-high affinity binding state of the sphincter mAChRs in a manner analogous to that produced by GTP gamma S; (2) a loss of the ability of GTP gamma S to affect CCh binding to the receptors; and (3) an attenuation of the GTP gamma S plus CCh-stimulated PIP2 hydrolysis. In conclusion, the data presented suggest that, in the iris smooth muscle, G-proteins are involved in the coupling of mAChRs to phospholipase C and that short-term cholinergic desensitization results in (1) the uncoupling of the receptor-G-protein complex and (2) the attenuation of mAChR-activation of phospholipase C.     

Functional modifications of transducin induced by cholera or pertussis-toxin-catalyzed ADP-ribosylation.

Transducin (T alpha beta gamma), the heterotrimeric GTP-binding protein that interacts with photoexcited rhodopsin (Rh*) and the cGMP-phosphodiesterase (PDE) in retinal rod cells, is sensitive to cholera (CTx) and pertussis toxins (PTx), which catalyze the binding of an ADP-ribose to the alpha subunit at Arg174 and Cys347, respectively. These two types of ADP-ribosylations are investigated with transducin in vitro or with reconstituted retinal rod outer-segment membranes. Several functional perturbations inflicted on T alpha by the resulting covalent modifications are studied such as: the binding of T alpha to T beta gamma to the membrane and to Rh*; the spontaneous or Rh*-catalysed exchange of GDP for GTP or guanosine 5-[gamma-thio]triphosphate (GTP[gamma S]), the conformational switch and activation undergone by transducin upon this exchange, the activation of T alpha GDP by fluoride complexes and the activation of the PDE by T alpha GTP. ADP-ribosylation of transducin by CTx requires the GTP-dependent activation of ADP-ribosylation factors (ARF), takes place only on the high-affinity, nucleotide-free complex, Rh*-T alpha empty-T beta gamma and does not activate T alpha. Subsequent to CTx-catalyzed ADP-ribosylation the following occurs: (a) addition of GDP induces the release from Rh* of inactive CTxT alpha GDP (CTxT alpha, ADP-ribosylated alpha subunit of transducin) which remains associated to T beta gamma; (b) CTxT alpha GDP-T beta gamma exhibits the usual slow kinetics of spontaneous exchange of GDP for GTP[gamma S] in the absence of Rh*, but the association and dissociation of fluoride complexes, which act as gamma-phosphate analogs, are kinetically modified, suggesting that the ADP-ribose on Arg174 specifically perturbs binding of the gamma-phosphate in the nucleotide site; (c) CTxT alpha GDP-T beta gamma can still couple to Rh* and undergo fast nucleotide exchange; (d) CTxT alpha GTP[gamma S] and CTxT alpha GDP-AlFx (AlFx, Aluminofluoride complex) activate retinal cGMP-phosphodiesterase (PDE) with the same efficiency as their unmodified counterparts, but the kinetics and affinities of fluoride activation are changed; (e) CTxT alpha GTP hydrolyses GTP more slowly than unmodified T alpha GTP, which entirely accounts for the prolonged action of CTxT alpha GTP on the PDE; (f) after GTP hydrolysis, CTxT alpha GDP reassociates to T beta gamma and becomes inactive. Thus, CTx catalyzed ADP-ribosylation only perturbs in T alpha the GTP-binding domain, but not the conformational switch nor the domains of contact with the T beta gamma subunit, with Rh* and with the PDE.(ABSTRACT TRUNCATED AT 400 WORDS)     

G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland

The involvement of G regulatory proteins in muscarinic receptor signal transduction was examined in electrically permeabilized rat submandibular acinar cells. The guanine nucleotide analog, GTP gamma S, caused the dose dependent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to release IP3. This response was insensitive to pertussis toxin treatment and was duplicated by NaF but not by GDP beta S. Enhanced IP3 synthesis was observed with a combination of GTP gamma S and carbachol. Exogenous IP3, as well as carbachol and GTP gamma S, provoked the release of sequestered 45Ca2+ from non-mitochondrial stores. In intact cells, carbachol significantly reduced the level of cyclic AMP induced by the beta-adrenergic agonist, isoproterenol, to 69% of its normal value. Pertussis toxin abolished this inhibitory action of carbachol on cyclic nucleotide levels. These results suggest that muscarinic receptors are coupled to two separate G regulatory proteins in submandibular mucous acini-the pertussis toxin-insensitive Gp of the phosphoinositide transduction pathway associated with elevated cytosolic calcium levels, and the pertussis toxin-sensitive Gi inhibitory protein of the adenylate cyclase complex.     

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

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

Phospholipid base exchange activity in rat liver plasma membranes. Evidence for regulation by G-protein and P2y-purinergic receptor.

Phospholipid base exchange activity using choline as substrate was detected in plasma membranes (PM) and other subcellular fractions of rat liver, with microsomes (MS) showing the highest specific activity. In contrast, phospholipase D activity was only detected in PM. In PM, choline exchanged for phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), whereas ethanolamine exchanged for PE and PS, and serine exchanged for PS. Ca2+ (10 microM or higher) stimulated choline incorporation into PC in MS and PM, whereas Mg2+ (10 microM or higher) stimulated it only in PM. Ethanolamine and serine incorporation into PM phospholipids was also stimulated by Ca2+, and inositol incorporation by Mn2+. Phospholipase D activity was substantial in the presence of EGTA and was slightly stimulated by Ca2+ concentrations less than 500 microM. It was undetectable without Mg2+. Low concentrations of oleate (1 mM or less) stimulated phospholipase D activity. These concentrations inhibited choline base exchange activity, whereas higher concentrations (3-8 mM) were stimulatory. Comparison of the subcellular distribution and Ca2+, Mg2+, and oleate effects on choline base exchange and phospholipase D activities supports the view that they are catalyzed by different enzymes. The incorporation of choline, but not ethanolamine or serine, into the phospholipids of PM, but not MS, was stimulated by micromolar concentrations of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) and other slowly hydrolyzable analogues of GTP. GDP, GMP, and other nucleoside triphosphates and their analogues were ineffective. GTP gamma S stimulation of base exchange activity was dependent upon Mg2+ and was inhibited by high concentrations of guanosine 5'-O-2-(thio)diphosphate. In the presence of low concentrations of GTP gamma S, ATP and its slowly hydrolyzable analogues stimulated base exchange activity. Dose-response curves for these nucleotides revealed a potency order consistent with mediation by purinergic receptors of the P2Y type. Base exchange activity stimulated by ATP plus GTP gamma S or GTP gamma S alone was not altered by treatment with pertussis or cholera toxins. These results suggest that the choline base exchange activity of liver PM is regulated by a pertussis toxin-insensitive G-protein linked to P2Y purinergic receptors.     

The epidermal growth factor receptor is coupled to a phospholipase A2-specific pertussis toxin-inhibitable guanine nucleotide-binding regulatory protein in cultured rat inner medullary collecting tubule cells

Studies were performed to examine a potential role for a guanine nucleotide-binding protein in epidermal growth factor (EGF)-stimulated phospholipase A2 (PLA2) activity. EGF increased prostaglandin E2 (PGE2) production in intact or saponin-permeabilized rat inner medullary collecting tubule (RIMCT) cells. Incubation of permeabilized cells with guanosine 5'-O-(thiotriphosphate) (GTP gamma S) enhanced and with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) inhibited the response to EGF. GDP beta S had no effect on ionomycin-stimulated PGE2 production. Exposure of intact cells to 25 mM NaF + 10 microM AlCl3 enhanced both basal and EGF-stimulated PGE2 production. Pertussis toxin ADP-ribosylated a 41-kDa protein in RIMCT cell membranes. Pretreatment of cells with pertussis toxin (100 ng/ml for 16 h) eliminated the response to EGF in intact cells and the response to EGF + GTP gamma S in permeabilized cells. Pertussis toxin had no effect on the response to ionomycin. The effect of pertussis toxin was not due to alterations in cAMP as cellular cAMP levels were unaffected by pertussis toxin both in the basal state and in the presence of EGF. PGE2 production in response to EGF was not transduced by a G protein coupled to phospholipase C (PLC) as neomycin, which inhibited PLC, did not decrease EGF-stimulated PGE2 production. Also, PGE2 production was not increased by inositol trisphosphate and did not require the presence of extracellular Ca2+. In contrast to EGF-stimulated PLC activity, stimulation of PLA2 by EGF was not susceptible to inhibition by phorbol 12-myristate 13-acetate. These results clearly demonstrate the existence of a PLA2-specific pertussis toxin-inhibitable guanine nucleotide-binding protein coupled to the EGF receptor in RIMCT cells.     

Phospholipase D prevents apoptosis in v-Src-transformed rat fibroblasts and MDA-MB-231 breast cancer cells

Phospholipase D (PLD) activity is elevated in response to mitogenic and oncogenic signals. PLD also cooperates with overexpressed tyrosine kinases to transform rat fibroblasts. 3Y1 rat fibroblasts overexpressing the tyrosine kinase c-Src undergo apoptosis in response to serum withdrawal. We report here that elevated expression of either PLD1 or PLD2 in these cells prevents apoptosis induced by serum withdrawal. 3Y1 cells transformed by the activated tyrosine kinase v-Src have elevated PLD activity and are resistant to apoptosis induced by serum withdrawal. However, if PLD activity is blocked, the v-Src-transformed cells underwent apoptosis. MDA-MB-231 cells are a human breast cancer cell line with substantially elevated levels of PLD activity. Inhibiting PLD activity in these cells similarly rendered them sensitive to the apoptotic insult of serum withdrawal. These data indicate that elevated PLD activity generates a survival signal(s) allowing cells to overcome default apoptosis programs.     

Guanine nucleotides induce Ca2+-independent insulin secretion from permeabilized RINm5F cells

The role of guanine nucleotides in insulin secretion was investigated in electrically permeabilized RINm5F cells. Ca2+ stimulated insulin release (EC50 approximately 2 microM Ca2+). The GTP stable analog, GTP gamma S, elicited insulin secretion at vanishingly low Ca2+ concentrations (less than 10(-11) M), slightly potentiated the response to intermediate Ca2+ levels, but exerted less than additive effects at maximal Ca2+ concentrations. The GDP analog, GDP beta S, inhibited both GTP gamma S- and Ca2+-stimulated secretion. The action of GTP gamma S was not mediated by cAMP, as the latter only enhanced Ca2+-induced secretion. In contrast, 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, promoted insulin release at nonstimulatory Ca2+ levels as well as potentiating the Ca2+ response. GTP analogs stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2), as assessed by inositol phosphate generation. However, this could not fully explain guanine nucleotide-induced secretion because: GTP gamma S-stimulated PtdInsP2 breakdown was totally dependent on Ca2+ and abolished at Ca2+ below 10(-11) M; at these Ca2+ levels, activators of protein kinase C were weak or ineffective secretagogues; the GTP analog Gpp(NH)p was much less effective than GTP gamma S in activating PtdInsP2 hydrolysis, while fully mimicking the effect on Ca2+-independent secretion. Both GTP gamma S-induced PtdInsP2 hydrolysis and insulin release were insensitive to pertussis toxin and cholera toxin. The findings point to a guanine nucleotide-regulated site in the activation of insulin secretion different from the known transmembrane signalling systems.     

Ca2+ inhibits guanine nucleotide-activated phospholipase D in neural-derived NG108-15 cells.

We have investigated the regulation of phospholipase D (PLD) activity by guanine nucleotides and Ca2+ in cells of the NG108-15 neuroblastoma X glioma line that were permeabilized with digitonin. The nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) caused a nearly sixfold increase (EC50 = 3 microM) in production of [3H]phosphatidylethanol (specific product of the PLD transphosphatidylation reaction). Other GTP analogues were less effective than GTP gamma S, and guanosine-5'-O-(2-thiodiphosphate) inhibited PLD activation by GTP gamma S. Both basal and GTP gamma S-stimulated PLD activities were potentiated by MgATP and Mg2+. Adenosine-5'-O-(3-thiotriphosphate) and ADP also potentiated the effect of GTP gamma S, but non-phosphorylating analogues of ATP had no such effect. The activation of PLD by GTP gamma S did not require Ca2+ and was independent of free Ca2+ ions up to a concentration of 100 nM (resting intracellular concentration). Higher Ca2+ concentrations (greater than or equal to 1 microM) completely inhibited PLD activation by GTP gamma S. It is concluded that elevated intracellular Ca2+ concentrations may negatively modulate PLD activation by a guanine nucleotide-binding protein, thus affecting receptor-PLD coupling in neural-derived cells.     

Guanine nucleotides regulate beta-adrenergic activation of Na-H exchange independently of receptor coupling to Gs.

We have previously shown that the beta-adrenergic receptor (beta-AR) stimulates activity of the ubiquitous Na-H exchanger (NHE-1) independently of changes in cAMP accumulation and independently of a cholera toxin-sensitive stimulatory GTP-binding protein (Gs). To further investigate the potential role of a GTP-binding protein in coupling the beta-AR to NHE-1, we have used a recently available nonhydrolyzable GTP analog, "caged" guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), to study time-dependent effects of GTP on NHE-1 in intact cells. By monitoring intracellular pH (pHi) in cells loaded with the fluorescent pH-sensitive dye, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein, we determined NHE-1 activity in primary cultures of canine enteric endocrine cells, which express an endogenous beta-AR, and in mouse L cells stably transfected with either the wild type hamster beta 2-AR or a mutant construct of the hamster beta 2-AR containing a deletion in amino acid residues 222-229. This D(222-229)beta 2-AR is functionally uncoupled from Gs and adenylylcyclase. In all three cell types, NaF and GTP gamma S induced an increase in activity of the exchanger, determined by assessing the rate of pHi recovery from an acute intracellular acid load (dpHi/dt). This increase in pHi recovery was dependent on extracellular Na+ and sensitive to the amiloride analog ethylisopropylamiloride. GTP gamma S, but not NaF, also increased beta-adrenergic stimulation of resting NHE-1 activity. The alkalinization in response to isoproterenol was reversed by propranolol in the absence, but not the presence, of GTP gamma S and was completely blocked by GDP beta S. The ability of guanine nucleotides to regulate beta-adrenergic activation of NHE-1 in cells expressing the mutant D(222-229)beta 2-AR suggests that functional coupling of the beta-AR to NHE-1 may be mediated by a GTP-binding protein other than Gs.