Guanosine Thiotriphosphate Research Articles

Regulation of phospholipase C-delta by GTP-binding proteins-rhoA as an inhibitory modulator

The regulation of Phospholipase C (PLC) δ activity remains obscure. These studies show that PLC δ 1 activity is significantly enhanced by both guanosine thiotriphosphate (GTP γS) and Clostridium botulinum exoenzyme C3 (C3) but not by aluminium fluoride. C3 ADP ribosylated a 21-kDa protein in the PLC δ 1 preparation and Western blotting identified rhoA in these samples. RhoA acts as an inhibitory modulator of PLC δ activity.

Further characterisation of [ 3H]8-hydroxy-2-(di-N-propylamino)tetralin binding sites in human brain postmortem

The saturation parameters and the pharmacological characteristics of the binding of the serotonin 1A (5-HT 1A) receptor agonist [ 3H]8-hydroxy-2-(di-N-propylamino)tetralin ([ 3H]8-OH-DPAT), as well as the effects of nucleotides and divalent cations (Mg 2+, Mn 2+) on it, were compared in some human postmortem brain regions: the main cortical areas, hippocampus and striatum. [ 3H]8-OH-DPAT labelled a single population of recognition sites with the highest maximal capacity (B max) in the hippocampus and the lowest affinity in the striatum. Among the various cortical areas, the frontal cortex exhibited the highest B max. The pharmacological profile of the [ 3H]8-OH-DPAT binding sites was consistent with the labelling of the 5-HT 1A receptor in the hippocampus and cortex, whereas the striatal site shared strong similarity to the presynaptic serotonin transporter. Modulation of [ 3H]8-OH-DPAT binding by divalent cations and nucleotides was detectable and stable in autopsy brains. In particular, nucleotide effects were area-dependent: guanosine thiotriphosphate (GTPγS) reduced [ 3H]8-OH-DPAT binding to the same extent in the hippocampus and frontal cortex, while having no effect in the striatum. Divalent cation effects depended also upon the brain area: in the striatum, they inhibited [ 3H]8-OH-DPAT binding, while stimulating it in the hippocampus and, with less extent, in the frontal cortex. In summary, these findings suggest that the [ 3H]8-OH-DPAT binding and its modulatory parameters in human brain tissues seem to show similarities but also some differences with respect to those determined in the rat brain. Furthermore, postmortem stability of GTP and divalent cation sensitive 5-HT 1A receptors underlines the need for further studies on the regulatory and functional properties of this receptor in the human brain.

G i protein activation of gonadotropin-releasing hormone–mediated protein dephosphorylation in human endometrial carcinoma

OBJECTIVE: Gonadotropin-releasing hormone receptor is demonstrated in uterine endometrial carcinomas. This study was performed to determine gonadotropin-releasing hormone receptor–mediated membrane events and to identify the guanosine triphosphate binding protein (G protein) subtypes linked to gonadotropin-releasing hormone receptor in the tumors. STUDY DESIGN: Endometrial carcinomas surgically removed had been screened for gonadotropin-releasing hormone receptor expression before plasma membrane isolation. The phosphoprotein level was observed in the phosphorus 32–labeled incorporation from [γ- 32P]adenosine triphosphate into the isolated plasma membranes. The G i (α subunit) protein was detected by immunoblotting and pertussis toxin–catalyzed adenosine diphosphate ribosylation. RESULTS: Incubation of phosphorus 32–labeled membranes with a gonadotropin-releasing hormone analog in the presence of guanosine thiotriphosphate caused a remarkable loss of phosphoprotein from 35 kd protein. This dephosphorylation action was dose dependent of the gonadotropin-releasing hormone analog, and the maximal effect (90% loss) occurred at 100 nmol/L. Pertussis toxin brought about adenosine diphosphate ribosylation of an immunodetected Gα i. Gonadotropin-releasing hormone analog alone or guanosine thiotriphosphate alone had no effect. Pretreatment of the membrane with the pertussis toxin completely inhibited gonadotropin-releasing hormone–mediated dephosphorylation of the 35 kd protein. CONCLUSION: These data demonstrate the coupling of gonadotropin-releasing hormone receptor to protein dephosphorylation through G i, raising the possibility that the antimitogenic action of gonadotropin-releasing hormone may occur by release of the action of protein phosphorylation to promote cell growth.(Am J Obstet Gynecol 1997;176:371-6.)

Evidence for coupling of phosphotyrosine phosphatase to gonadotropin-releasing hormone receptor in ovarian carcinoma membrane.

Gonadotropin-releasing hormone (Gn-RH) receptor (Gn-RHR) has been demonstrated in epithelial ovarian carcinoma (Imai et al., Cancer 1994; 74:2555-61). To examine whether Gn-RHR mediates direct antiproliferative effects, we attempted to determine stimulatory regulation by Gn-RH of phosphotyrosine phosphatase (PTP) activity in plasma membranes isolated from ovarian carcinoma samples. Surgically removed ovarian carcinomas were screened for Gn-RHR expression prior to plasma membrane isolation. The phosphotyrosine level was observed by: (1) immunoblotting of membrane extracts with antiphosphotyrosine antibodies, and (2) dephosphorylation from 32P-labeled membrane protein. Membrane PTP activity was determined using the synthetic substrate p-nitrophenyl in a spectrophotometric assay. A Gn-RH analog alone, or guanosine thiotriphosphate (GTP-gamma-S) alone, caused a remarkable loss of phosphotyrosine from a 35-kD protein of the membranes; incubation with a Gn-RH analog and GTP-gamma-S produced a further dephosphorylation of this endogenous protein. The Gn-RH analog buserelin stimulated the PTP activity of the membranes in a dose-dependent manner (P < 0.01). GTP-gamma-S enhanced the stimulatory action of Gn-RH on PTP; GDP-gamma-S reversed the Gn-RH action. A similar stimulation of PTP was observed (P < 0.01) when carcinoma tissue slices were exposed to Gn-RH analog in vivo prior to assay in vitro. Activation of PTP by Gn-RH stimulated the loss of phosphotyrosine from endogenous proteins through GTP-binding protein within plasma membrane isolated from Gn-RHR-expressing ovarian carcinoma. The antimitogenic action of the hormone may occur by counteracting tyrosine phosphorylation to promote cell growth.

G-protein regulation of capacitative calcium entry may be mediated by protein kinases A and C in Xenopus oocytes.

Inositol 2,4,5-trisphosphate irreversibly activated capacitative calcium entry in Xenopus oocytes, whereas guanosine thiotriphosphate (GTP[S]) and AIF4- only activated capacitative calcium entry transiently. Both GTP[S] and AIF4- inhibited capacitative calcium entry activated by thapsigargin pretreatment, but guanosine thiodiphosphate (GDP[S]), inositol 2,4,5-trisphosphate and dibutyryl cyclic GMP did not affect capacitative calcium entry. This suggests the involvement of heterotrimeric GTP-binding proteins in the regulation of capacitative calcium entry. Activation of protein kinase C or cyclic-AMP-dependent protein kinase had profound effects on capacitative calcium entry, which were consistent with the hypothesis that the effects of GTP[S] and AIF4- on capacitative calcium entry may be mediated via heterotrimeric GTP-binding protein stimulation of kinases. Further evidence for this hypothesis was derived from the result that the effects of GTP[S] on calcium entry could be inhibited by the application of the protein kinase inhibitor staurosporine.

Thrombin-mediated phosphoinositide hydrolysis in Chinese hamster ovary cells overexpressing phospholipase C-delta 1.

The regulatory mechanism(s) of a phosphoinositide-specific phospholipase C, PLC-delta 1, was investigated using a clone of stably overexpressed PLC-delta 1 (PLC delta 30 cells) in Chinese hamster ovary cells. Thrombin stimulation of PLC delta 30 cells exhibited 6.5-fold increase in total inositol phosphates (InsP), which was significantly higher than that in the vector-transfected (V1) cells (2.0-fold). AIF-4 increased InsP accumulation in both V1 and PLC delta 30 cells, and pertussis toxin partially blocked InsP accumulation in thrombin-stimulated PLC delta 30 cells. Guanosine thiotriphosphate (GTP gamma S) markedly potentiated thrombin-stimulated InsP generation in permeabilized PLC delta 30 cells compared with V1 cells, suggesting possible involvement of a G-protein (s) in the activation of PLC-delta 1. In PLC delta 30 cells, ionomycin-induced significant InsP generation and thrombin-stimulated InsP generation were completely inhibited by addition of EGTA. Furthermore, the stimulatory effects of thrombin plus GTP gamma S in PLC delta 30 cells were more sensitive to change in free calcium concentration than in V1 cells. Suppression by 12-O-tetradecanoylphorbol 13-acetate of thrombin-stimulated InsP accumulation was not affected by increasing Ca2+ concentration. These results indicate that thrombin-induced PLC-delta 1 activation is regulated via both G-protein(s) and calcium.

Growth factor-induced release of placental alkaline phosphatase from human syncytiotrophoblast membranes

The ability of epidermal growth factor, insulin or guanosine thiotriphosphate to induce the release of two glycosyl-phosphatidylinositol-linked proteins from isolated human placental syncytiotrophoblast plasma membrane vesicles was investigated. Epidermal growth factor induced the ATP-dependent release of a fraction of syncytiotrophoblast plasma membrane placental alkaline phosphatase, whereas no release was detected following insulin treatment. This effect of epidermal growth factor was apparent at 30 min but not at 5 min. Guanosine thiotriphosphate stimulated the release of a small amount of syncytiotrophoblast plasma membrane placental alkaline phosphatase and appeared to have an additive effect when applied together with epidermal growth factor. Guanosine thiodiphosphate did not induce phosphatase release, but partially inhibited the epidermal growth factor response. 28.7% of syncytiotrophoblast plasma membrane 5'-nucleotidase was solubilized using glycosyl-phosphatidylinositol-specific phospholipase C. However, unlike placental alkaline phosphatase, no detectable release of 5'-nucleotidase was observed following treatment of syncytiotrophoblast plasma membrane vesicles with epidermal growth factor or guanosine thiotriphosphate. These results indicate (i) the presence of at least two placental alkaline phosphatase-releasing pathways in syncytiotrophoblast plasma membrane vesicles, and (ii) the presence of subpopulations of glycosyl-phosphatidylinositol-linked proteins sensitive to growth factor-induced release.

Modulation of cardiac L-type Ca2+ channels by GTP gamma S in response to isoprenaline, forskolin and photoreleased nucleotides.

1. Using the patch-clamp recording technique, we have investigated the effects of chronic intracellular application of guanosine thiotriphosphate (GTP gamma S) by cell dialysis, on the potentiation of L-type Ca2+ currents (ICa) by isoprenaline and forskolin and also by GTP gamma S and cyclic AMP released intracellularly by flash-photolysis of their caged derivatives. 2. GTP gamma S prevented enhancement of ICa by isoprenaline with an IC50 of approximately 10 microM and considerably reduced the ability of forskolin to increase ICa. In addition GTP gamma S also reduced the time-to-peak response for potentiation of ICa by forskolin. Responses to forskolin were abolished by co-dialysis of cells with the cyclic AMP antagonist, Rp-adenosine-3'-5'-mono-thionophosphate (Rp-cAMPS). 3. Photoreleased GTP gamma S (PR-GTP gamma S; approximately 23 microM) generally induced a biphasic increase in ICa. This response was also inhibited by chronic intracellular dialysis with GTP gamma S with an IC50 of approximately 1 microM. 4. Pretreatment of cells with pertussis toxin (PTX) reversed the inhibitory effect of 100 microM GTP gamma S on isoprenaline-induced stimulation of ICa. However, PTX pretreatment did not restore the activating action of PR-GTP gamma S inhibited by chronic application of GTP gamma S. 5. Photoreleased cyclic AMP (approximately 5 microM; PR-cyclic AMP) increased peak ICa. This effect was inhibited by dialysis of cells with Rp-cAMPS and by stimulation of ICa by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Co-dialysis of cells with uncaged GTP gamma S reduced the time-to-peak for PR-cyclic AMP mediated activation of ICa but did not affect the magnitude of the response. 6. It is concluded that chronically applied GTP gamma S can (i) inhibit activation of ICa by isoprenaline by interacting with a PTX-sensitive guanosine nucleotide binding (G-) protein located upstream of adenylate cyclase (possibly Gi) and (ii) accelerate the response to cyclic AMP dependent phosphorylation possibly by interacting with a G-protein coupled directly to the channel. 7. In view of this diverse range of effects, care should be taken when using GTP gamma S to characterize G-protein-mediated events, since the resulting physiological response may be due to activation of several G-protein containing pathways.

Depressing Effects of Protein Kinases a &amp; c on the Receptor-Induced K+-Current Responses in the Ganglion Cells of Aplysia

Application of either dopamine (DA), acetylcholine (ACh), histamine (HA), or Phe-Met-Arg-Phe-NH2 (FMRFamide) induces a K+-current response in the ganglion cells of Aplysia under voltage clamp. We have previously reported that these responses are all mediated by a pertussis toxin (PTX)-sensitive G-protcin Gi or Go. Intracellular application of cAMP, an activator of protein kinase A (PKA), or extracellular application of 30 μM phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), markedly suppressed these transmitter-induced K+-current responses. The depressing effect of cAMP was reversible while that of PDBu was irreversible as observed for 1 hour. Intracellular injection of the catalytic subunit of either PKA or PKC mimicked the effects of cAMP or PDBu. Pretreatment of the ganglion cells with 100 μM H-7, an inhibitor of PKC, significantly suppressed the effect of PDBu. Furthermore, an intraccllular injection of okadaic acid (OA), an inhibitor of phosphatases, facilitated the blocking effects of both cAMP and PDBu. The dose-response curve obtained by each transmitter-receptor system shifted downward by application of either cAMP or PDBu without affecting the affinity of the agonist to each receptor. K+-channel opening directly induced by guanosine thiotriphosphate (GTPγS) or by raising the temperature was not depressed by cither cAMP or 100 μM PDBu. From these results, we postulated that the acting sites of both PKA and PKC might be somewhere between the receptors and G-protein, and that the phosphorylation of these sites would impair the functional coupling efficiency between the receptors and G-protein.

Gonadotropin-releasing hormone stimulates phospholipase C but not protein phosphorylation/dephosphorylation in plasma membrane from human epithelial ovarian cancer.

In view of advances in treatment of certain hormone-dependent cancers with analogues of gonadotropin-releasing hormone (Gn-RH), this study was undertaken to establish the signal transduction events interacting with Gn-RH receptor in a cell-free system prepared from human ovarian mucinous cystadenocarcinoma samples. A high affinity specific binding (Kd=8 x 10-9 M) of [3H] Gn-RH was demonstrated in two from two plasma membrane preparations. Gn-RH showed no effects on the rate of protein phosphorylation from [gamma-32P] adenosine triphosphate in the plasma membrane preparations. On the other hand, incubation of plasma membrane isolated form [3H]inositol-labeled specimens with Gn-RH in the presence of guanosine thiotriphosphate resulted in the rapid production of inositol phosphates. The Gn-RH effects was concentration-dependent, and half-maximal activation occurred with 1-3 nm Gn-RH. The Gn-RH-stimulated membrane event was observed in all plasma membrane isolations tested, but not in those from uterine endometrial carcinoma of a given case. These results provide the first direct evidence that Gn-RH receptor is coupled to phosphoinositide hydrolysis but not to certain membrane protein phosphorylation/dephosphorylation in ovarian carcinoma plasma membrane. Though the functional role of this event in human ovarian cancer is still obscure, it might be part of a possible point of attack for therapeutic approaches using Gn-RH analogues in this malignancy.

Differential regulation of phosphoinositide and phosphatidylcholine hydrolysis by protein kinase C-beta 1 overexpression. Effects on stimulation by alpha-thrombin, guanosine 5'-O-(thiotriphosphate), and calcium.

Rat 6 fibroblasts that stably overexpress cDNA for the beta 1 isozyme of protein kinase C (PKC3 cells) were used to determine the effect of protein kinase C (PKC) overexpression on hormonal stimulation of phospholipid hydrolysis. In control Rat 6 cells, inositol trisphosphate levels (InsP3) were increased 9-fold in 15 s in response to 10 nM alpha-thrombin, compared with only a 2-fold increase in PKC3 cells. PKC overexpression also inhibited thrombin-stimulated production of 1,2-diacylglycerol, the other product of phosphatidylinositol 4,5-bisphosphate hydrolysis, by 73% at 15 s. In permeabilized cells, PKC overexpression greatly reduced guanosine thiotriphosphate-stimulated InsP3 accumulation, but did not affect InsP3 stimulation by increased free calcium concentration. These data suggest that desensitization of thrombin-stimulated phosphoinositide-phospholipase C is enhanced by PKC-beta 1 overexpression and may involve modulation of G-protein/phospholipase C coupling. In contrast, thrombin was 4.5-fold more effective in stimulation of phosphatidylcholine-phospholipase D activity in PKC3 cells than in control cells, as determined by phosphatidylethanol formation. In permeabilized cells, guanosine thiotriphosphate also stimulated phospholipase D activity more effectively in PKC3 cells than in control cells, suggesting that upregulation of phospholipase D activity by PKC overexpression occurs distal to the thrombin receptor. These results suggest that PKC may act as a switch to up-regulate phosphatidylcholine-phospholipase D and down-regulate phosphoinositide-phospholipase C stimulations.

A phosphorothioate at the 3' splice-site inhibits the second splicing step in a group I intron.

RNA polymerases can synthesize RNA containing phosphorothioate linkages in which a sulfur replaces one of the nonbridging oxygens. Only the Rp isomer is generated during transcription. A Rp phosphorothioate at the 5' splice-site of the Tetrahymena group I intron does not inhibit splicing (McSwiggen, J.A. and Cech, T.R. (1989) Science 244, 679). Transcription of mutants in which the first base of the 3' exon, U+1, was mutated to C or G, in the presence, respectively, of either cytosine or guanosine thiotriphosphate, introduced a phosphorothioate at the 3' splice-site. In both cases exon ligation was blocked. In the phosphorothioate substituted U+1G mutant, a new 3' splice-site was selected one base downstream of the correct site; despite the fact that the correct site was selected with very high fidelity in unsubstituted RNA. In contrast, the exon ligation reaction was successfully performed in reverse using unsubstituted intron RNA and ligated exons containing an Rp phosphorothioate at the exon junction site. Chirality was reversed during transesterification as in 5' splice-site cleavage (vide supra). This suggests that one non-bridging oxygen is particularly crucial for both splicing reactions.

Effect of glucocorticoids on α1-adrenergic receptor binding in rat vascular smooth muscle

Glucocorticoids are known to have marked effects on blood pressure regulation, predominantly through altering cardiovascular sensitivity to noradrenaline. However, the molecular mechanisms underlying this action remain unclear. As part of our studies into these we have measured alpha 1-adrenergic receptor binding using the ligand [3H]prazosin in plasma membrane fractions of aortas prepared from control, adrenalectomized and dexamethasone-treated adrenalectomized rats. In controls there were 50 +/- 8 (S.E.M.; n = 6) fmol alpha 1-adrenergic receptors/mg membrane protein (Bmax) with a dissociation constant (Kd) of 0.52 +/- 0.10 nM (n = 6). Adrenalectomy 8 days before tissue preparation caused a 40% decrease in Bmax and a 60% decrease in Kd. Dexamethasone replacement after adrenalectomy returned these values close to those of controls. Noradrenaline competed for the [3H]prazosin-binding sites. Computer analysis by a non-linear curve-fitting program (LIGAND) showed that noradrenaline binding was to a heterogeneous population of high- and low-affinity receptors with Kd values of 1.87 +/- 0.73 microM and 0.48 +/- 0.12 mM (n = 5) respectively. Guanosine thiotriphosphate (GTP[S]) caused the conversion of high-affinity to low-affinity binding, consistent with the model of the high-affinity sites being coupled to a G protein. After adrenalectomy, noradrenaline binding was to a homogeneous population of low-affinity receptors; hence, the effect of GTP[S] was no longer apparent, suggesting that under these conditions the alpha 1-adrenergic receptors were unable to couple to a G protein. The two-site model of binding and GTP[S] effect was returned by dexamethasone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)