Phospholipase C-β1 is regulated by a pertussis toxin-insensitive G-protein

Regulation of phospholipase C (PLC) by receptors is mediated either through protein tyrosine phosphorylation or by activation of GTP-binding proteins (Gp). For the latter, pertussis toxin (PT)-sensitive and -insensitive pathways have been described, indicating PLC regulation by at least two types of G-proteins. The identity of PLC isoenzymes which are regulated by either type of Gp remains to be determined. Thyrotropin-releasing hormone stimulates a PLC in GH3 cells via a PT-insensitive Gp. Reconstitution methods for the assay of the GH3-cell Gp were developed. Previously, the membrane PLC was found to be reversibly extracted from membranes by high salt and to be activated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) only when membrane-associated, suggesting that Gp was retained in salt-extracted membranes. In the present work, Gp was cholate-solubilized from PLC-deficient membranes and incorporated into phospholipid vesicles, which were found to confer GTP[S]- and AlF4(-)-stimulated activity on a solubilized membrane PLC. The reconstitution provided a direct assay for the GH3-cell Gp which was shown to be distinct from Gi, Go and Gs proteins by immunodepletion studies. Incorporation of G-protein beta-gamma subunits into phospholipid vesicles with Gp inhibited GTP[S]-stimulated activity in the reconstitution. The results indicated that Gp is a heterotrimeric G-protein with the properties expected for the PT-insensitive GH3-cell Gp protein. PLC-beta 1 was fully purified and shown to be regulated by Gp in the reconstitution. In contrast, PT-sensitive G-proteins failed to affect the activity of PLC-beta 1. The results indicate (1) that a PT-insensitive Gp regulates PLC-beta 1 and (2) that PT-sensitive and -insensitive pathways of PLC regulation employ different PLC isoenzymes as well as different G-proteins.

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Dual Regulation of Phospolipase C Activity by G Proteins

Physiology ◽

10.1152/physiologyonline.1993.8.2.61 ◽

1993 ◽

Vol 8 (2) ◽

pp. 61-63

Author(s):

H Deckmyn ◽

C Van Geet ◽

J Vermylen

Keyword(s):

Adenylate Cyclase ◽

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Pertussis Toxin ◽

Inhibitory Pathway ◽

Close Parallelism

Some subtypes of phosphatidylinositide-specific phospholipase C (PLC) are activated via pertussis toxin-sensitive or -insensitive G proteins. However, a G protein-dependent PLC inhibitory pathway also may exist. The resultant picture is of dual regulation of PLC, showing a close parallelism with the dual regulation of adenylate cyclase.

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G-proteins are not directly involved in the CD3-antigen-mediated production of inositol phosphates in HPB-ALL T-leukaemia cells expressing phospholipase C isoforms γ1 and β3

Biochemical Journal ◽

10.1042/bj2890387 ◽

1993 ◽

Vol 289 (2) ◽

pp. 387-394 ◽

Cited By ~ 2

Author(s):

M Biffen ◽

M Shiroo ◽

D R Alexander

Keyword(s):

Phospholipase C ◽

Tyrosine Phosphorylation ◽

G Protein ◽

G Proteins ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Cross Linking ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine ◽

Inositol Phosphate Production

The possible involvement of G-proteins in T cell antigen-receptor complex (TCR)-mediated inositol phosphate production was investigated in HPB-ALL T-cells, which were found to express the phospholipase C gamma 1 and beta 3 isoforms. Cross-linking the CD3 antigen on streptolysin-O-permeabilized cells stimulated a dose-dependent increase in inositol phosphate production, as did addition of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) or vanadate, a phosphotyrosine phosphatase inhibitor. It was possible, therefore, that the CD3-antigen-mediated production of inositol phosphates was either via a G-protein-dependent mechanism or by stimulation of protein tyrosine phosphorylation. The CD3-induced inositol phosphate production was potentiated by addition of vanadate, but not by addition of GTP[S]. Guanosine 5′-[beta-thio]diphosphate (GDP[S]) inhibited the rise in inositol phosphates induced by GTP[S], vanadate or cross-linking the CD3 antigen. The increase in protein tyrosine phosphorylation stimulated by vanadate or the OKT3 monoclonal antibody was not observed in the presence of GDP[S], showing that in permeabilized HPB-ALL cells, GDP[S] inhibits the actions of tyrosine kinases as well as G-protein function. Addition of either ADP[S] or phenylarsine oxide inhibited CD3- and vanadate-mediated increases in both tyrosine phosphorylation and inositol phosphate production, but did not inhibit GTP[S]-stimulated inositol phosphate production. On the other hand, pretreatment of cells with phorbol 12,13-dibutyrate inhibited subsequent GTP[S]-stimulated inositol phosphate production but did not inhibit significantly inositol phosphate production stimulated by either OKT3 F(ab')2 fragments or vanadate. Our results are consistent with the CD3 antigen stimulating inositol phosphate production by increasing the level of protein tyrosine phosphorylation, but not by activating a G-protein.

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Activation of BK Channels in GH3 Cells by a c-PLA2-Dependent G-Protein Signaling Pathway

Journal of Neurophysiology ◽

10.1152/jn.00865.2004 ◽

2005 ◽

Vol 93 (6) ◽

pp. 3146-3156 ◽

Cited By ~ 7

Author(s):

D. D. Denson ◽

J. Li ◽

X. Wang ◽

D. C. Eaton

Keyword(s):

Arachidonic Acid ◽

G Protein ◽

G Proteins ◽

Pertussis Toxin ◽

Adrenergic Receptor ◽

Bk Channels ◽

Bk Channel ◽

Gh3 Cells ◽

Channel Activity ◽

Excised Patches

BK-channels in GH3 cells are activated by arachidonic acid produced by c-PLA2. β-adrenergic agonists also activate BK channels and were presumed to do so via production of cAMP. We, however, show for the first time in GH3 cells that a β-adrenergic agonist activates a pertussis-toxin-sensitive G protein that activates c-PLA2. The arachidonic acid produced by c-PLA2 then activates BK channels. We examined BK channels in cell-attached patches and in excised patches from untreated GH3 cells and from GH3 cells exposed to c-PLA2 antisense oligonucleotides. For the cell-attached patch experiments, physiologic pipette and bath solutions were used. For the excised patches, 150 mM KCl was used in both the pipette and bath solutions, and the cytosolic surface contained 1 μM free Ca2+ (buffered with 5 mM K2EGTA). Treatment of GH3 cells with the G protein activator, fluoroaluminate, (AlF4−) produced an increase in the Po of BK channels of 177 ± 41% (mean ± SD) in cell-attached patches. Because G proteins are membrane associated, we also added an activator of G proteins, 100 μM GTP-γ-S, to the cytosolic surface of excised patches. This treatment leads to an increase in Po of 50 ± 9%. Similar treatment of excised patches with GDP-β-S had no effect on Po. Isoproterenol (1 μM), an activator of β-adrenergic receptors and, consequently, some G proteins, increased BK channel activity 229 ± 37% in cell-attached patches from cultured GH3 cells. Western blot analysis showed that GH3 cells have β-adrenergic receptor protein and that isoproterenol acts through these receptors because the β-adrenergic receptor antagonist, propanolol, blocks the action of isoproterenol. To test whether G protein activation of BK channels involves c-PLA2, we studied the effects of GTP-γ-S on excised patches and isoproterenol on cell attached patches from GH3 cells previously treated with c-PLA2 antisense oligonucleotides or pharmacological inhibitors of c-PLA2. Neither isoproterenol nor GTP-γ-S had any effect on Po in these patches. Similarly, neither isoproterenol nor GTP-γ-S had any effect on Po in cultured GH3 cells pretreated with pertussis toxin. Isoproterenol also significantly increased the rate of arachidonic production in GH3 cells. These results show that some receptor-linked, pertussis-toxin-sensitive G protein in GH3 cells can activate c-PLA2 to increase the amount of arachidonic acid present and ultimately increase BK-channel activity.

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Membrane-binding properties of phospholipase C-β1 and phospholipaseC-β2: role of the C-terminus and effects of polyphosphoinositides, G-proteins and Ca2+

Biochemical Journal ◽

10.1042/bj3270431 ◽

1997 ◽

Vol 327 (2) ◽

pp. 431-437 ◽

Cited By ~ 22

Author(s):

M. John JENCO ◽

P. Kevin BECKER ◽

J. Andrew MORRIS

Keyword(s):

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Membrane Binding ◽

Molar Ratio ◽

Phospholipid Vesicles ◽

C Terminus ◽

Catalytically Active ◽

Membrane Bound ◽

Active Fragments

We have studied the binding of two G-protein-regulated phospholipase C (PLC) enzymes, PLCs-β1 and -β2, to membrane surfaces using sucrose-loaded bilayer phospholipid vesicles of varying compositions. Neither enzyme binds appreciably to pure phosphatidylcholine vesicles at lipid concentrations up to 10-3 M. PLC-β1 and PLC-β2 bind vesicles composed of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine (molar ratio 1:1:1) with an approximate Kd of 10-5 M. Inclusion of 2% PtdIns(4,5)P2 in these vesicles had no effect on the affinity of this interaction. As reported by others, removal of the C-terminus of PLC-β1 and PLC-β2 produces catalytically active fragments. The affinity of these truncated proteins for phospholipid vesicles is dramatically reduced suggesting that this region of the proteins contains residues important for membrane binding. Inclusion of G-protein α- and βγ-subunit activators in the phospholipid vesicles does not increase the binding of PLC-β1 or PLC-β2, and the magnitude of G-protein-mediated PLC activation observed at low phospholipid concentrations (10-6 M) is comparable to that observed at concentrations at which the enzymes are predominantly membrane-bound (10-3 M). PLC-β1 and -β2 contain C2 domains but Ca2+ does not enhance binding to the vesicles. Our results indicate that binding of these enzymes to membranes involves the C-temini of the proteins and suggest that activation of these enzymes by G-proteins results from a regulated interaction between the membrane-bound proteins rather than G-protein-dependent recruitment of soluble enzymes to a substrate-containing phospholipid surface.

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Interferon-γ-stimulated and GTP-binding-proteins-mediated phospholipase A2 activation in human neuroblasts

Biochemical Journal ◽

10.1042/bj2940893 ◽

1993 ◽

Vol 294 (3) ◽

pp. 893-898 ◽

Cited By ~ 16

Author(s):

M Ponzoni ◽

P Cornaglia-Ferraris

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase A2 ◽

Phospholipase C ◽

G Proteins ◽

Pertussis Toxin ◽

Ifn Gamma ◽

Permeabilized Cells ◽

Kinase C ◽

Gtp Binding

Interferon-gamma (IFN-gamma) is a potent growth-inhibitory cytokine also endowed with differentiating activity on neural cells. Binding of IFN-gamma to its high-affinity receptor induces a rapid and transient activation of phospholipase A2 (PLA2). The mechanism coupling the IFN-gamma receptor (IFN-gamma-R) to PLA2 activation is not clearly defined, and no information is available on this mechanism in neuroblast cells. We have tested the hypothesis that GTP-binding proteins (G-proteins) may couple the IFN-gamma-R to PLA2 in the human neuroblastoma (NB) cell line LAN-5. Incubation of NB cells with IFN-gamma resulted in a rapid increase in [3H]arachidonic acid (AA) release, and this effect was blocked by pretreatment with anti-IFN-gamma antibodies. IFN-gamma-stimulated AA release was still observed in permeabilized cells that were blocked by pretreatment with anti-IFN-gamma-R antibodies. Exposure of permeabilized LAN-5 cells to guanosine 5′-[gamma-thio]triphosphate (GTP[S]), a non-hydrolysable GTP analogue, induced a dose-dependent release of [3H]AA. A non-specific nucleotide effect was excluded, since similar stimulatory effects on AA mobilization were not observed by GTP, ATP, CTP, ADP and GDP. IFN-gamma-stimulated AA release was completely blocked by the guanine nucleotide analogue that inhibits G-protein function, guanosine 5′-[beta-thio]diphosphate (GDP[S]). A role for G-proteins in IFN-gamma-R coupling to PLA2 was further supported by the inhibition of IFN-gamma-induced [3H]AA release by treatment of permeabilized cells with pertussis toxin and with the antiserum against the common alpha-subunits of G-proteins. To determine a possible contribution to AA mobilization by the phospholipase C and diacyglycerol lipase pathway or by protein kinase C activation, the effects of neomycin, a phospholipase C inhibitor, and PMA (phorbol 12-myristate 13-acetate), a direct activator of protein kinase C, were investigated. Neither neomycin nor PMA affected either basal or IFN-gamma-stimulated AA release. Ca2+ concentration, which has been shown to regulate the activity of some PLA2s, does not appear to play an important role in the regulation of the IFN-gamma-stimulated PLA2 activity, since incubating permeabilized cells in different concentrations of Ca2+ induced AA release without affecting the IFN-gamma response. Altogether, these findings suggest the existence of IFN-gamma-R, which couples a Ca(2+)-independent PLA2 activation via pertussis-toxin-sensitive G-proteins.

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Regulation of glucose carrier activity by AlCl3 and phospholipase C in fat-cells

Biochemical Journal ◽

10.1042/bj2560515 ◽

1988 ◽

Vol 256 (2) ◽

pp. 515-520 ◽

Cited By ~ 23

Author(s):

B Obermaier-Kusser ◽

C Mühlbacher ◽

J Mushack ◽

E Rattenhuber ◽

M Fehlmann ◽

...

Keyword(s):

Plasma Membrane ◽

Phospholipase C ◽

G Protein ◽

Glucose Transport ◽

Pertussis Toxin ◽

Binding Proteins ◽

Fat Cells ◽

Gtp Binding Proteins ◽

Gtp Binding ◽

Glucose Carrier

Recently it was speculated that activation of GTP-binding proteins and of phospholipase is involved in the transmission of a signal from the insulin-receptor kinase to effector systems in the cell. To confirm this hypothesis, we have tested the effect of AlCl3, which has been recently used as an experimental tool to activate GTP-binding proteins, on glucose transport in fat-cells. We found that AlCl3 has a partial insulin-like effect on glucose transport activity (3-O-methylglucose uptake, expressed as % of equilibrium value per 4 s: basal 9.6 +/- 2, AlCl3 29.6 +/- 4, insulin 74.0 +/- 3). The AlCl3 effect is totally blocked by pertussis toxin, whereas the insulin effect was not altered. The effect starts at [AlCl3] greater than 1 fM and reaches its maximum at 0.1 nM. Addition of phospholipase C (PLC; 50 munits/ml) also stimulated glucose transport (maximal 53.0 +/- 5%). Both substances acted faster than insulin itself (maximal values within 1 min for PLC, 2 min for AlCl3 and 5-10 min for insulin). Using the cytochalasin-B-binding assay to determine the effects of AlCl3 and PLC on the distribution of glucose carrier sites in subcellular fractions, we found that their glucose-transport-stimulating effect does not occur through an increase in glucose carrier sites in the plasma-membrane fraction. When PLC was combined with the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate), which increases glucose carrier sites in the plasma membrane, an additive effect on glucose transport was found [PLC (50 munits/ml), 53.0 +/- 5%, TPA (1 nM), 17.3 +/- 2%; PLC + TPA, 68.0 +/- 3%]. In conclusion: (1) the data show that AlCl3, probably through activation of a pertussis-toxin-inhibitable G protein, and PLC are able to modulate the intrinsic glucose carrier activity; (2) as pertussis toxin did not modify the effect of insulin, it seems unlikely that the insulin signal on glucose transport involves activation of this specific G protein.

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TSH activates macrophage inflammation by G13 and G15-dependent pathways

Endocrinology ◽

10.1210/endocr/bqab077 ◽

2021 ◽

Author(s):

Chongbo Yang ◽

Zhao He ◽

Qunye Zhang ◽

Ming Lu ◽

Jiajun Zhao ◽

...

Keyword(s):

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Pertussis Toxin ◽

Rho Gtpases ◽

Thyroid Stimulating Hormone ◽

Tsh Receptor ◽

Sirna Interference ◽

Inflammatory Effect ◽

Stimulating Hormone

Abstract Thyroid-stimulating hormone (TSH) treatment activates IκB/NFκB (p65) and ERK, P38 in macrophages, but how these pathways are activated, and how they contribute to the pro-inflammatory effect of TSH on macrophages remain unknown. TSH receptor (TSHR) is coupled to four subfamilies of G proteins (Gs, Gi/o, Gq/11 and G12/13) for its downstream signalling. This study investigated the G protein subtypes responsible for the pro-inflammatory effect of TSH on macrophages. qPCR showed that Gi2, Gi3, Gas, Gq, G11, G12, G13, G15 are abundantly expressed by macrophages. The contribution of different G-protein pathways to the pro-inflammatory effect was studied by the corresponding inhibitors or siRNA interference. While TSH-induced IκB phosphorylation was not inhibited by Gs inhibitor NF449, Gi inhibitor pertussis toxin, Gq or G11 siRNA, it was blocked by phospholipase C inhibitor U73122 or G15 siRNA interference. TSH-induced ERK and P38 phosphorylation was blocked by G13 but not G12 siRNA interference. Interfering either G13 or G15 was able to block the pro-inflammatory effect of TSH on macrophages. The present study demonstrate that TSH activates macrophage inflammation by G13/ERK-P38/Rho GTPases and G15/PLC/PKCs/IκB pathways.

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Platelet adenylate cyclase and phospholipase C are affected differentially by ADP-ribosylation. Effects on thrombin-mediated responses

Biochemical Journal ◽

10.1042/bj2520297 ◽

1988 ◽

Vol 252 (1) ◽

pp. 297-300 ◽

Cited By ~ 17

Author(s):

H S Banga ◽

R K Walker ◽

L K Winberry ◽

S E Rittenhouse

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Pertussis Toxin ◽

Marked Decrease ◽

Human Platelets ◽

Adp Ribosylation

Thrombin stimulates phospholipase C and inhibits adenylate cyclase in human platelets. We have studied the effect of purified S1 monomer, the ADP-ribosylating subunit of pertussis toxin, on these receptor-coupled G-protein-dependent activities. ADP-ribosylation of a 41 kDa protein is associated with a marked decrease in the ability of thrombin to inhibit cyclic AMP formation, but has little effect on phospholipase C. Therefore adenylate cyclase and phospholipase C appear to be modulated by different G-proteins.

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Regulation of Na+-K+-ATPase activity in kidney proximal tubules: involvement of GTP binding proteins

AJP Renal Physiology ◽

10.1152/ajprenal.1989.256.1.f57 ◽

1989 ◽

Vol 256 (1) ◽

pp. F57-F62 ◽

Cited By ~ 3

Author(s):

A. Bertorello ◽

A. Aperia

Keyword(s):

Atpase Activity ◽

G Protein ◽

G Proteins ◽

Pertussis Toxin ◽

Rat Kidney ◽

Inhibitory Effect ◽

Proximal Tubules ◽

Regulatory Proteins ◽

Free Access ◽

Gtp Binding

This study evaluates the involvement of GTP-dependent regulatory proteins (G-proteins) in the regulation of Na+-K+-ATPase activity in proximal convoluted tubule (PCT) segments. Single PCT segments were dissected from rat kidney and permeabilized to allow nucleotides and medium free access to the interior of the cell. A GDP analogue that blocks GTP-dependent activation of the G-protein, GDP beta S (400 microM) significantly inhibited PCT Na+-K+-ATPase activity when Na in the medium (Nam) was greater than or equal to 70 mM. The inhibition was attenuated when Nam was 55 and 35 mM and was no longer significant when Nam was 25 mM. GDP beta S had no inhibitory effect on the activity of purified Na+-K+-ATPase. A nonhydrolyzable GTP analogue, GppNHp (50 microM) significantly increased Na+-K+-ATPase activity when Nam was 25 and 35 mM, but not when Nam was 55-140 mM. Dopamine (DA) and DA1 plus DA2 agonists significantly inhibit Na+-K+-ATPase activity. DA inhibition was competitively abolished by GppNHp. In PCT segments from rats pretreated with pertussis toxin, DA and DA1 plus DA2 agonist inhibition of Na+-K+-ATPase activity was abolished. In PCT segments from rats pretreated with cholera toxin, basal Na+-K+-ATPase activity was increased, but DA significantly inhibited Na+-K+-ATPase activity. Na+-K+-ATPase activity in PCT segments is regulated via a G-protein that stimulates Na+-K+-ATPase activity and a DA-activated pertussis toxin-sensitive G-protein that inhibits Na+-K+-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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A slowly ADP-ribosylated pertussis-toxin-sensitive GTP-binding regulatory protein is required for vasopressin-stimulated Ca2+ inflow in hepatocytes

Biochemical Journal ◽

10.1042/bj2990399 ◽

1994 ◽

Vol 299 (2) ◽

pp. 399-407 ◽

Cited By ~ 17

Author(s):

L A Berven ◽

B P Hughes ◽

G J Barritt

Keyword(s):

G Protein ◽

G Proteins ◽

Pertussis Toxin ◽

Single Cells ◽

Regulatory Protein ◽

Regulatory Proteins ◽

Inositol Polyphosphates ◽

Gtp Binding ◽

Inositol 1,4,5 Trisphosphate ◽

Epidermal Growth

The roles of heterotrimeric GTP-binding regulatory proteins (G-proteins) and inositol polyphosphates in the mechanism by which vasopressin stimulates Ca2+ inflow in hepatocytes were investigated by using single cells loaded with fura2 by microinjection. Vasopressin-stimulated Ca2+ inflow was mimicked by microinjection of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) or guanosine 5′-[beta gamma-imido]triphosphate to the cells, but not adenosine 5′-[gamma-thio]triphosphate (ATP[S]) or guanosine 5′-[beta-thio]diphosphate (GDP[S]). Extracellular Gd3+ (5 microM) inhibited both vasopressin- and GTP[S]-stimulated Ca2+ inflow. GDP[S], but not GMP, administered to hepatocytes by microinjection, completely inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. The microinjection of pertussis toxin had no effect either on the release of Ca2+ from intracellular stores or on Ca2+ inflow induced by vasopressin, but completely inhibited changes in these processes induced by epidermal growth factor (EGF). Hepatocytes isolated from rats treated with pertussis toxin for 24 h exhibited no vasopressin- or GTP[S]-stimulated Ca2+ inflow, whereas the vasopressin-stimulated release of Ca2+ from intracellular stores was similar to that observed for control cells. Heparin or ATP[S] inhibited, or delayed the onset of, both vasopressin-induced release of Ca2+ from intracellular stores and vasopressin-stimulated Ca2+ inflow. Vasopressin-induced oscillations in intracellular [Ca2+] were observed in some heparin-treated cells. It is concluded that the stimulation by vasopressin of Ca2+ inflow to hepatocytes requires inositol 1,4,5-trisphosphate (InsP3) and, by implication, the pertussis-toxin-insensitive G-protein required for the activation of phospholipase C beta [Taylor, Chae, Rhee and Exton (1991) Nature (London) 350, 516-518], and another G-protein which is slowly ADP-ribosylated by pertussis toxin and acts between InsP3 and the putative plasma-membrane Ca2+ channel. EGF-stimulated Ca2+ inflow involves at least one G-protein which is rapidly ADP-ribosylated and is most likely required for InsP3 formation.

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