scholarly journals Norepinephrine exocytosis stimulated by α–latrotoxin requires both external and stored Ca 2+ and is mediated by latrophilin, G proteins and phospholipase C

1999 ◽  
Vol 354 (1381) ◽  
pp. 379-386 ◽  
Author(s):  
M. Atiqur Rahman ◽  
Anthony C. Ashton ◽  
Frédéric A. Meunier ◽  
Bazbek A. Davletov ◽  
J. Oliver Dolly ◽  
...  
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Fluorescent Dyes ◽  
Transmembrane Protein ◽  
Clostridial Neurotoxins ◽  
Dependent Component ◽  
Intracellular Ca ◽  
Botulinum Neurotoxins ◽  
Vesicular Exocytosis

α–latrotoxin (LTX) stimulates massive release of neurotransmitters by binding to a heptahelical transmembrane protein, latrophilin. Our experiments demonstrate that latrophilin is a G–protein–coupled receptor that specifically associates with heterotrimeric G proteins. The latrophilin–G protein complex is very stable in the presence of GDP but dissociates when incubated with GTP, suggesting a functional interaction. As revealed by immunostaining, latrophilin interacts with Gα q/11 and Gα o but not with Gα s , Gα i or Gα z , indicating that this receptor may couple to several G proteins but it is not promiscuous. The mechanisms underlying LTX–evoked norepinephrine secretion from rat brain nerve terminals were also studied. In the presence of extracellular Ca 2+ , LTX triggers vesicular exocytosis because botulinum neurotoxins E, C1 or tetanus toxin inhibit the Ca 2+ –dependent component of the toxin–evoked release. Based on (i) the known involvement of Gα q in the regulation of inositol–1,4,5–triphosphate generation and (ii) the requirement of Ca 2+ in LTX action, we tested the effect of inhibitors of Ca 2+ mobilization on the toxin–evoked norepinephrine release. It was found that aminosteroid U73122, which inhibits the coupling of G proteins to phospholipase C, blocks the Ca 2+ –dependent toxin's action. Thapsigargin, which depletes intracellular Ca 2+ stores, also potently decreases the effect of LTX in the presence of extracellular Ca 2+ . On the other hand, clostridial neurotoxins or drugs interfering with Ca 2+ metabolism do not inhibit the Ca 2+ –independent component of LTX–stimulated release. In the absence of Ca 2+ , the toxin induces in the presynaptic membrane non–selective pores permeable to small fluorescent dyes; these pores may allow efflux of neurotransmitters from the cytoplasm. Our results suggest that LTX stimulates norepinephrine exocytosis only in the presence of external Ca 2+ provided intracellular Ca 2+ stores are unperturbed and that latrophilin, G proteins and phospholipase C may mediate the mobilization of stored Ca 2+ , which then triggers secretion.

Stimulation by G Protein βγ Subunits of Phospholipase C β Isoforms in Human Platelets

1998 ◽  
Vol 79 (05) ◽  
pp. 1008-1013 ◽  
Author(s):  
Yoshiko Banno ◽  
Tomiko Asano ◽  
Yoshinori Nozawa
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Human Platelet ◽  
Minor Component ◽  
Similar Efficacy ◽  
Human Platelets ◽  
Bovine Brain ◽  
Γ Subunit ◽  
A Minor

SummaryDifferent phospholipase C (PLC) isoforms were located in human platelet cytosol and membranes. PLCγ2 and PLCβ3b were mainly located in the cytosol and PLCβ2 and PLCβ3a were in both cytosol and membranes by using specific antibodies against PLC isozymes (Banno Y, Nakashima S, Ohzawa M, Nozawa Y. J Biol Chem 1996; 271: 14989-94). Three PLC fractions activated by G protein βγ subunits were purified from human platelet cytosol and membrane fractions. Two PLC fractions from membranes were identified as PLCβ2 and PLCβ3a, and one from cytosol was PLCβ3b. These PLCβ isoforms were activated by the purified βγ subunits of brain G proteins in the order PLCβ3b > PLCβ3a > PLCβ2. Western blot analysis of γ subunits of the purified platelet G proteins with antibodies against various standard γ subunits revealed that the major component of the γ subunit of Gi2 and Gq was γ5, and that γ7 was a minor component. Studies using various subtypes of βγ subunits, βγ2, βγ3, and βγ7 purified from bovine brain, βγ5 from bovine lung, or βγ12 from bovine spleen, failed to show differences in their ability to stimulate the isolated platelet PLCβ isoforms. These results suggest that the βγ subunits of Gi2 and Gq have similar efficacy in regulation of effectors in human platelets.

Dual Regulation of Phospolipase C Activity by G Proteins

Physiology ◽  
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.

scholarly journals The ubiquitin-related protein PLIC-1 regulates heterotrimeric G protein function through association with Gβγ

2003 ◽  
Vol 163 (5) ◽  
pp. 1157-1165 ◽  
Author(s):  
Elsa-Noah N'Diaye ◽  
Eric J. Brown
Keyword(s):  
Wound Healing ◽  
Phospholipase C ◽  
G Proteins ◽  
Protein Function ◽  
Proteasome Inhibition ◽  
Selective Inhibition ◽  
Related Protein ◽  
Dependent Component ◽  
Direct Association ◽  
Signaling Components

PLIC-1, a newly described ubiquitin-related protein, inhibited both Jurkat migration toward SDF-1α and A431 wound healing, but the closely related PLIC-2 did not. PLIC-1 prevented the SDF-1α–induced activation of phospholipase C, decreased ligand-induced internalization of SDF-1α receptor CXCR4 and inhibited chemotaxis signaled by a transfected Gi-coupled receptor. However, PLIC-1 had no effect on Gs-mediated adenylyl cyclase activation, and inhibited only the Gβγ-dependent component of Gq-initiated increase in [Ca2+]i, which is consistent with selective inhibition of Gβγ function. PLIC-1 colocalized with G proteins in lamellae and pseudopods, and precipitated Gβγ in pull downs. Interaction with Gβγ did not require PLIC-1's ubiquitin-like or ubiquitin-associated domains, and proteasome inhibition had no effect on SDF-1α activation of phospholipase C, indicating that PLIC-1's inhibition of Gβγ did not result from effects on proteasome function. Thus, PLIC-1 inhibits Gi signaling by direct association with Gβγ; because it also interacts with CD47, a modulator of integrin function, it likely has a role integrating adhesion and signaling components of cell migration.

Diacylglycerol and ceramide formation induced by dopamine D2S receptors via Gβγ-subunits in Balb/c-3T3 cells

2003 ◽  
Vol 284 (3) ◽  
pp. C640-C648 ◽  
Author(s):  
Gele Liu ◽  
Mohammad H. Ghahremani ◽  
Behzad Banihashemi ◽  
Paul R. Albert
Keyword(s):  
Cell Growth ◽  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Second Messengers ◽  
Mitogen Activated Protein Kinase ◽  
Fibroblast Cells ◽  
Induced Responses ◽  
3T3 Cells ◽  
Mitogen Activated Protein

Diacylglycerol (DAG) and ceramide are important second messengers affecting cell growth, differentiation, and apoptosis. Balb/c-3T3 fibroblast cells expressing dopamine-D2S (short) receptors (Balb-D2S cells) provide a model of G protein-mediated cell growth and transformation. In Balb-D2S cells, apomorphine (EC50= 10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at 1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicating Gi/Goproteins. To address which G proteins are involved, Balb-D2S clones expressing individual PTX-insensitive Gαiproteins were treated with PTX and tested for apomorphine-induced responses. Neither PTX-insensitive Gαi2nor Gαi3rescued D2S-induced DAG or ceramide formation. Both D2S-induced DAG and ceramide signals required Gβγ-subunits and were blocked by inhibitors of phospholipase C [1-(6-[([17β]-3-methoxyestra-1,2,3[10]-trien- 17yl)amino]hexyl)-1H-pyrrole-2,5-dione (U-73122) and partially by D609]. The similar G protein specificity of D2S-induced calcium mobilization, DAG, and ceramide formation indicates a common Gβγ-dependent phospholipase C-mediated pathway. Both D2 agonists and ceramide specifically induced mitogen-activated protein kinase (ERK1/2), suggesting that ceramide mediates a novel pathway of D2S-induced ERK1/2 activation, leading to cell growth.

scholarly journals 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

1993 ◽  
Vol 289 (2) ◽  
pp. 387-394 ◽  
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.

scholarly journals Phospholipase C in Dictyostelium discoideum. Identification of stimulatory and inhibitory surface receptors and G-proteins

1994 ◽  
Vol 297 (1) ◽  
pp. 189-193 ◽  
Author(s):  
A A Bominaar ◽  
P J M Van Haastert
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Alpha Subunit ◽  
Wild Type ◽  
Inhibitory Pathway ◽  
Surface Receptors ◽  
G Protein Alpha Subunit ◽  
Camp Receptor ◽  
Protein Alpha Subunit

A combined biochemical and genetic approach was used to show that phospholipase C in the cellular slime mould Dictyostelium is under dual regulation by the chemoattractant cyclic AMP (cAMP). This dual regulation involves stimulatory and inhibitory surface receptors and G-proteins. In wild-type cells both cAMP and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated phospholipase C. In contrast, mutant fgd A, lacking the G-protein alpha-subunit G alpha 2, showed no stimulation by either cAMP or GTP[S], indicating that G alpha 2 is the stimulatory G-protein. In mutant fgd C cAMP did not stimulate phospholipase C, but stimulation by GTP[S] was normal, suggesting that the defect in this mutant is upstream of the stimulatory G alpha 2. Inhibition of phospholipase C was achieved in wild-type cells by the partial antagonist 3′-deoxy-3′-aminoadenosine 3′,5′-phosphate (3′NH-cAMP). This inhibition was no longer observed in transformed cell lines lacking either the surface cAMP receptor cAR1 or the G-protein alpha-subunit G alpha 1; in these cells the agonist cAMP still activated phospholipase C. These results indicate that Dictyostelium phospholipase C is regulated via a stimulatory and an inhibitory pathway. The inhibitory pathway is composed of the surface receptor cAR1 and the G-protein G1. The stimulatory pathway consists of an unknown cAMP receptor (possibly the fgd C gene product) and the G-protein G2.

scholarly journals G-protein inhibition of phospholipase C-β1 in membranes: role of G-protein βγ subunits

1996 ◽  
Vol 319 (1) ◽  
pp. 173-178 ◽  
Author(s):  
Irene LITOSCH
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Plasma Membranes ◽  
Bovine Brain ◽  
Protein Inhibition ◽  
Low Concentrations ◽  
Dependent Inhibition ◽  
Cortical Membranes

Rat liver plasma membranes reconstituted with bovine brain phospholipase C β1 (PLC-β1) exhibit a dual regulation of PLC-β1 activity by G-proteins. Guanosine 5´-[γ-thio]triphosphate (GTP[S]; 0.1 nM) produced a 20-25% inhibition of PLC-β1 activity within 7 min of incubation. The addition of vasopressin resulted in near-basal levels of activity in the presence of 0.1 nM GTP[S]. Clonidine had little effect on the net inhibition due to GTP[S]. A similar antagonism between carbachol and GTP[S] occurred in cerebral cortical membranes containing endogenous PLC-β1 activity. αo/i-GDP (a mixture of GDP-liganded Goα and Giα) attenuated the GTP[S]-dependent inhibition of PLC-β1 whereas αo/i-GTP[S] had no effect, suggesting an involvement of G-protein βγ subunits in the inhibition of PLC-β1. Low concentrations of βγ subunits inhibited PLC-β1 activity. Inhibition was followed by reversal to basal activity and onset of stimulation as the βγ concentration was increased. Inhibition by βγ was dependent on the presence of membranes. These results indicate that G-protein βγ subunits constitute a mechanism by which G-proteins mediate a rapid and transient inhibition of PLC-β1.

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

1991 ◽  
Vol 280 (3) ◽  
pp. 753-760 ◽  
Author(s):  
T F J Martin ◽  
J E Lewis ◽  
J A Kowalchyk
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Gh3 Cells ◽  
Phospholipid Vesicles ◽  
Protein Tyrosine Phosphorylation ◽  
Gtp Binding ◽  
Gamma Subunits ◽  
Gh3 Cell

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.

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

1997 ◽  
Vol 327 (2) ◽  
pp. 431-437 ◽  
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.

Involvement of G proteins in the effect of carbachol and cholecystokinin in rat pancreatic islets

1996 ◽  
Vol 271 (1) ◽  
pp. E65-E72 ◽  
Author(s):  
E. J. Verspohl ◽  
K. Herrmann
Keyword(s):  
Pancreatic Islets ◽  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Insulin Release ◽  
Rat Pancreatic Islets ◽  
Inositol 1,4,5 Trisphosphate ◽  
Insulinotropic Effect ◽  
Gamma S

Phospholipase C is involved in the insulinotropic effect of carbachol (CCh) and cholecystokinin octapeptide (CCK-8). The involvement of the type of G protein was investigated in rat pancreatic islets. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; a nonhydrolyzable GTP analogue) increased insulin release in electrically permeabilized islets. Both CCh and CCK-8 increased the GTP gamma S effect indicative of an involvement of G proteins. Pretreatment of the islets with pertussis toxin (PT) impaired the CCh-induced insulin secretion in the presence of 3.0 mM glucose and inhibited the stimulatory CCh effect on inositol 1,4,5-trisphosphate (IP3) levels at low and high glucose. In contrast to CCh, the CCK-8 effect on both insulin release and IP3 levels of islets was not modified by a PT pretreatment at various glucose concentrations. Two types of experiments indicate the type of G protein involved: first, long-term agonistic stimulation by either CCh or CCK-8 led to a downregulation of alpha o and alpha q/11, respectively; second, introduction of specific anti-alpha o or -alpha q/11 antibodies into electrically permeabilized islets nearly completely abolished the effects of CCh and CCK-8, respectively. The data indicate that both CCh and CCK-8 act as insulinotropic agents via the phospholipase C system; in the effect of CCh the PT-sensitive alpha o and in the effect of CCK-8 the PT-insensitive alpha q/11 is involved.

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