THROMBOSIS RESEARCH 67; 559-567,1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
PERTUSSIS
TOXIN SENSITIVE G-PROTEIN COUPLING OF HDL RECEPTOR TO PHOSPHOLIPASE C IN HUMAN PLATELETS
H. Nazih*, D. Devred*, F. Martin-Nizard*, V. Clavey*+, J.C. Fruchart*+, C. Delba.rt*+ *Serlia : Institut Pasteur, 1 rue du Pr Calmette, B.P. 245 - 59019 LILLE Cklex, France +UFR de Pharmacie, 3 rue du Professeur Laguesse, 59045 LILLE Cedex, France.
(Received 10.3.1991; accepted in revised form 7.7.1992 by Editor J. Soria) (Received by Executive Editorial Office 11.8.1992)
ABSTRACT Initially we established that, in human platelets, low concentrations of HDL3 stimulate phosphatidylcholine (PC) hydrolysis and a transient increase im 1,2diacylglycerol (DAG). In (3H) PC prelabelled platelets, phosphocholine is released into the medium during HDL3 induced PC turnover with a 1.5 to 2 fold increment, indicating that HDL3 stimulated DAG generation in platelets is likely due to phospholipase C (PLC). GTP or GTP-y-S augments, and pertussis toxin inhibits HDL3 stimulated DAG production. Treatment of p~latelet membranes with HDL3 or with proteoliposome containing apo A-I ot A-II substantially prevents 41 kDa protein ADP-ribosylation that was induced by pertussis toxin, with apo A-II having an inhibitory potency greater than apo AI. These data provide strong evidence that the pertussis sensible G protein (Go or Gi) is directly involved in coupling PLC to HDL3 receptor in platelets.
UCTION
Initially we established that the binding of low concentrations of HDL3 to human blood platelets stimulates both the rapid loss of phosphatidylcholine (PC) and the biphasic generation of 1,2diacylglycerol (1,2-DAG) [ 1,2]. These results indicated that the binding of HDL3 stimulates PC breakdown by activating membrane phospholipase. Agonist induced generation of DAG has been reported to occur via PLC or PLD catalyzed hydrolysis of PC [3]. PC hydrolysis by either pathway was also described in human platelets [4, 51. To clarify the interpretation of our data, it was necessary to define the contribution of PLC or Key words : platelets, high density lipoproteins, phospholipase C, GTP binding protein.
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PLD to PC turnover and DAG formation under our experimental conditions. Moreover, platelet responses to agonist are believed to be mediated by at least two pertussis toxin-sensitive Gproteins : Gi which inhibits adenylyl cyclase and Gp which stimulates phospholipase C [6]. The effect of pertussis toxin on platelets is accompanied by the ADP-ribosylation of a protein with an apparent molecular mass of 40-42 KDa [7]. The present studies examine the coupling of PLC to the HDL3 receptor, and the ability of G-protein to stimulate PC breakdown in HDL3 stimulated platelets.
Materials. Pertussis toxin (islet activating protein) and cholera toxin, Thrombin, Guanosine 5’-kthiol m phosphate (GTP [T-S]), and GTP were from Sigma Chemical Co. [Adenylate 32Pl NAD. L3-phosphatidyt[Nmeu@H] choline-1,2dipalmitoyl(3H Chol-PC) and phosphatidylcholine L-a-dipalmitoyl [2-palmitoyl-9.10-3H (N)] (3H Cl6:0-PC) were from Amersham. Silica gel TLC plates were purchased from Merck. Other biochemical reagents were from Sigma Lipoprotein isolation. HDL3 were isolated from human serum by standard differential ultracentrifugal flotation [8] (HDL3 d = 1.125-1.210 g/cm3). Its protein content was measured by the procedure of Petersen et al. 191.The apolipoprotein E (apo E) constituted less than 0.2 % of the total HDL3 protein. Apo AI and ape AH were isolated [lo] inorder toprepareproteolipos ill]. Human platelet membrane isolation. Platelets were isolated from titrated blood drawn freshly from healthy human donors. Following centrifugation at 15Oxgfor 20 min., the platelet rich plasma (PKP) was centrifuged for 15 min. at 9ooxg and the platelets resuspended in a 0.35 % Bovine Serum Albumin (w/v) buffer (BSA-Buffer) containing 137 mM NaCl, 2.7 mM KCl, 0.32 mM NaH2PO4.5.6 mM glucose, 11.9 mM NaHCO3 and 1.3 mM Hepes pH 7.4. Platelet membranes were prepared according to the procedure of Barber et al. [12] and resuspended in an incubation buffer containing 15 mM Tris-HCl, pH7.5,0.125 M sucrose, 75 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1 mM ATP, 5 mM MgC12 and 0.05 mM G’IP. (3H)-phospbatidylcholine prelabelled platelet membranes. 1.25 pCi of (3H C16:O)PC or 2 pCi of (3H Chol)-PC were pipetted into vials and the solvent was evaporated off to leave a thin film. 100 pg of membranes were then added and incubated for 60 min. at room temperature. The specific activity of the preparation was 3.105 cpm/lOU pg membrane proteins. Less than l/200 of the radioactivity was associated with free lipids, and nearly all the radioactivity with the PC fraction. [2.13,14]. Stimulation by HDL3. (3H)-PC prelabelled human platelet membranes (100 l.tg protein) were resuspended in 1 ml of incubation buffer and exposed to HDL3 (50 pg) unless otherwise specified. Studies with pertussis or cholera toxin were performed in incubation buffer containing 20 @I NAD. The incubations were terminated by immediately adding 4 ml of ice cold CHC13/CH30H (2/l v/v). The lipids were extracted as described by Folch et al. [ 151. Lipid analysis. Lipids were spotted on silicagel 60 TLC plates which were developed with petroleum ether/ethylether/acetic acid (90-10-5 v/v/v) to separate DAG. The radioactive spots corresponding to DAG and PC were cut and processed for radioactivity counting. A good correlation for DAG release was found between the (3H) PC method and the DAG Kinase assay 1131.(3H)choline metabolites were determined after lyophilisation of the aqueous phase of a Folch extract. Choline and phosphocholine were separated according to the method outlined by Martin and Michaelis 1161with the following solvant mixture 0.5 46 NaCl/ethanol/CH30H/NH40H (50 - 30 - 20 - 5 v/v/v/v). Areas corn&rating with authentic standards of choline (Bf 0.09) and phosphocholine (Bf 0.39) were scraped and transferred to scintillation vials for radioactivity counting. ADP-ribosylation. Pertussis or cholera toxins were preactivated just before use, by incubation (250 pg/ml) in a freshly prepared solution containing 200 mM sodium phosphate, pH 7.4,0.5 mM dithiothreitol, and 100 pM ATP for 10 min at 37°C 1171.ADP-ribosylation was determined by the incorporation of 32~ from [adenylate - 32P]-NAD into membrane proteins. Membrane suspension (100 ug) was added to 300 pl of freshly prepared incubation mixture
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[17] containing 10 pg pertussis (or 10 pg cholera) toxin, 50 mM Tris, 10 mM MgC12, 100 mM KH2PG4, 1 mM EDTA, 1 mM ATP. 20 mM Arginine, 0.1 mM BSA, 5 mM ADP ribose, 100 mM GTP. Labelling reactions were initiated by adding 5 pM 32P-NAD (specific activity 15-150 pCi/mmol). ADP-ribosylation was also performed on membranes (100 pg) stimulated by HDL3 (50 pg), thrombin (7 units/ml) or proteoliposomes containing ape A-I ( (50 pg) or apo A-II ( (50 pg) added simultaneously with the toxins. After incubating at 37°C for 1 h, reactions were stopped by addition of five volumes of cold acetone and incubation at -20°C for 10 min. Membrane proteius were pelleted by centrifugation(10 000 x g ; 5 mitt). After two mpeated precipitations and dryings, the protein pellet was dissolved in 30 p.l of Laemmli sample buffer [18] at 100°C for 3-5 mm. SDS-PAGE was performed on 5-19 8 gradient gels using the discontinuous system of Laemmli [lg]. Gels were stained with Coomassie Brillant Blue Radiolabelled bands were located by l-2 days exposure with Kodak XOMAT K film at - WC. Data presentation. Data points are the average of at least duplicate determinations which were within 5 % of the mean.
HDL3 induced hydrolysis of PC by PLC. As shown previously [l, 21, incubation of (3H Cl6 : 0)PC - platelets with HDL3 induced a biphasic accumulation of (3H)DAG. The early peak appeared rapidly reaching a maximal 2-3 fold increment at 20-30 seconds. Thereafter a second phase of DAG generation occurred at 60 seconds and returned to baseline level within 2 minutes. The selective incorporation of (3H-Chol)PC into platelet membranes permits a specific evaluation of HDL3 elicited changes in PC metabolism through the release of water soluble choline metabolites. As early as 30 seconds, HDL3 treatment caused an increase in the release of phosphocholine into the medium which could be indicative of PLC involvement in PC hydrolysis (Fig. 1). Changes in choline content were not statistically significant. Production of (3H)-DAG in HDL3 stimulated platelets is likely due to enhanced PLC activity.
0
30
60
Tim.
( au.
)
Fig. 1 : Release of choline metabolites : 100 pg platelet membranes were prelabelled with (3HChol)PC and incubated at 37°C in the presence of 50 pg protein HDL3. Aliquots were taken to determine by TLC the release of water soluble choline metabolite into the medium. Data are the average value of 4 separate preparations in duplicate.
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Effects of GTP and GTP yS on DAG production in HDL3 stimulated platelet membranes. We have examined the effect of guanine nucleotide on PLC activity as measured by (3H)-DAG production in (3H - Cl6 : 0)PC prelabelled platelet membranes when challenged with HDL3. As shown in Fig. 2, either GTP or the GTP analog, GTP-y-S, stimulated a 1.5 fold increase in DAG formation, and displayed a stimulatory potency order similar to that observed for other receptors coupled to G-proteins ie. GTP-y-S > GTP.
100
Fig. 2 : Effects of guanine nucleotides
on DAG production membranes.
300
200
in HDLJ
stimulated
platelet
(3H - Cl6 : 0)pC prelabellcd platelet membranes (100 pg protein) were incubated in the presence of 0.05 mM GTP (x ) or GTP-y-S (0) and stimulated by 50 pg protein HDL3. Aliquots were taken to determine DAG release. Data representative of at least 3 separate preparations in duplicate.
Effects of toxins on DAG production HDL3 activated platelet membranes. (3H Cl6 : 0)-PC prelabelled platelet membranes were preincubated in the presence of pertussis or cholera toxins and then exposed to HDL3. The initial rise in (3H)-DAG (30 set) produced by HDL3 stimulation in pertussis toxin pretreated membranes is lowered to control values represented by PC labelled membranes alone. Pretreatment of membranes with cholera toxin has no influence on HDL3 induced DAG production (Fig. 3). Pertussis toxin inhibited the production of (3H)-DAG in the presence of HDL3, while cholera toxin had no effect on the signaling pathway. The time course of DAG production is presented in Fig. 4. The presence of pertussis toxin in the medium decreased the formation of HDL3 induced DAG in platelet membranes. These data suggest strongly that a pertussis toxin sensible G-protein (Go or Gi) is involved in the HDL3 receptor PLC system in human platelets. Identification of platelet membrane associated G-proteins. For a primary characterization of the G-protein activated by the HDL3 receptors, we used the pertussis toxin mediated [32P]-ADP-ribosylation labelling technique. Labelled proteins migrated as few bands on 5-19 % gradient SDS-PAGE. A strong incorporation of radioactivity into proteins with an apparent molecular mass of 40-41 kDa and 67 kDa as well was obtained indicating that a 41 kDa platelet membrane protein was selectively ADP-ribosylated by pertussis toxin (Fig. 5 lane 5). An
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accompanying ADP ribosylated protein with an aparent molecular mass ranging around 62 kDa has been described (19), but it displayed no immunological cross reactivity with antibodies against CXsubunits of the GTP binding protein. In addition the 41 kDa proteins is often found to be closely associated with other membrane proteins (20). Including HDL3 in the reaction mixture resulted in decreased ADP-ribosylation of the 41 kDa proteins (Fig. 5 lane 2). Platelet membrane stimulation with pmteoliposomes containing apo A-I or apo A-II also decreased pertussis toxin induced ADPribosylation (Fig. 5 lane 3,4). The highest efficiency in lowering ADP-ribosylation was encountered during platelet stimulation with proteoliposomes containing apo A-II. This result coincided with the previous suggestions that apo A-II was able to generate DAG from PC with the highest apparent efficacy. Similarly, treatment of platelets with thrombin substantially prevented subsequent protein ADP-ribosylation which was induced by pertussis toxin (Fig. 5 lane 1).
Fig. 3 : Effects of toxins on DAG production
in HDW
activated
platelet
membranes.
Platelet membranes prelabelled with (3H - Cl6 : O)-PC were incubated in the presence 6 @ml cholera or pertussis toxin for 20 min at 37°C and exposed to 0 (control) or 50 pg HDL3. Aliquots were taken to determine DAG content. Data representative of 3 preparations in duplicate.
E 8
Time ( sec. ) 20 0
I 40
I 80
I 120
I
I
1
I
160
200
240
280
Fig. 4 : Effects of pertussis toxin on time course of DAG production. Platelet membranes prelabelled with (3H - Cl6 : O)-PC were preincubated in the absence (D), or in the presence (0) of 6 pg/ml pertussis toxin for 20 min at 37°C and exposed to 50 pg HDL3. Aliquots were taken to determine DAG content. Values represent the mean ?rSD of three experiments in duplicate.
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--,
67 -+ 41+
Fig. 5 : Pertussis toxin induced ADP-ribosylation of 41 kDa protein in human platelets. ADP ribosylation was performed as described in methods in the presence of pertussis toxin alone or coupled to HDL3 (50 pg). thrombin (7 units/ml) and proteoliposomes containing apo A-I (50 pg) or apo A-II (50 pg). The results are expressed in cpm and represent the mean + SD of 3 separate experiments.
F
I? P
OH
DAG kitmu
Fig. 6 : Metabolic
id WM : DAG fOmatiOn
pathway
catalyzedby
for the formation
of PC-derived
DAG.
PLC, and subsequent phosphorylation into phosphatidic acid (PA) by D:G kinase. m arrows : Pathway of DAG formation via the initial action of PLD and simultaneous hydrolysis of PA by PA phosphohydrolase. so
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QISCussroN
Stimulation of platelets by HDL3 leads to the activation of the phospholipase cascade and subsequently to a biphasic increase in DAG mass [ 11. The first phase of DAG release was shown to result from a direct activation of membrane phospholipase by receptor occupancy [2]. In platelets agonist-induced generation of DAG has been reported to occur via PLC or PLD catalyzed hydrolysis of PC [5]. Moreover both PLC or PLD are stimulated by PKC or bylphorbol esters [3, 21, 221. So differential sensitivity of lipase is much less helpful, than previously imagined, to identify individual phospholipases interacting with and being activated by a;specific receptor. The activation of either PLC or PLD by HDL3 receptor has not yet been directly established. To determine the relative contribution of PLC or PLD to HDL3 induced DAG formation (Pig. 6) platelets were ptelabelled with (3H-Chol)-PC before HDL3 stimulation. Phosphocholine was the main water soluble choline metabolite to be released into the medium. This result indicates that DAG release is catalyzed directly by PLC during the early phase, as the later secondary rise in DAG mass cannot be observed in isolated platelet membranes. These data alone cannot establish the enzymatic pathway of PC degradation. Although formation of phosphocholine and 3H-DAG supports a PLC mechanism Elucidation of a precise reaction pathway is further complicated by the presence within the cells of specific phosphatases and kinases which rapidly interconvert choline/phosphocholine and DAG/phosphatidic acid [16]. However this result is in good agreement with our previous data [2], indicating that platelet pretreatment with 100 uM sphingosine failed to modify (3H)-PA formation and sustained HDL3-stimulated (3H)-DAG mass formation. Sphingosine, in its role of potent inhibitor of phosphatidic acid phosphohydrolase [23], should have increased PA content if a PLD was involved in both the primary and the secondary phase of PC turnover. Moreover platelet pretreatment with R 59022 (a known DAG-Kinase inhibitor [24]) which decreased the rate of DAG phosphorylation by approx 50 % argues [2] also against HDL3 activation of PLD. Our results are also in good agreement with those of Huang et al. [5] providing strong evidence that in human platelets 87 % of PC is hydrolyzed via the initial action of PLC, and the remaining 13 % by PLD. Our initial interest centred around the possible involvement of a GTP-binding protein in the ability of a HDL3 receptor to activate phospholipase C. Much accumulating evidence suggests that one or more guanine nucleotide binding proteins, commonly referred to as Gp, mediate the interaction between platelet receptors for HDL3 and PLC. The addition of GTP-y-S increased PLC activity to a large extent (1.5 fold over basal level). On the contrary, pretreatment of platelet with pertussis toxin leads to inhibition of HDL3 induced responses ie : PLC activation. Pertussis inhibition of agonist induced responses has been largely described [25. 261. In all cases, an association of the Gi-protein with those responses has been indicated. To determine whether any of the known pertussis toxin sensitive a subunits evoke the HDL3 induced PC breakdown, ADPribosylation in non and agonist stimulated platelet membranes were analysed. Usually pertussis toxin catalyses the ADP-ribosylation of the al subunit of Gi [7]. This ADP-ribosylation is inhibited when al is dissociated, as encountered when Gi molecules are coupled to a specific receptor. We demonstrated that ADP-ribosylation labelling techniques identify a single band at 41 kDa as commonly found [7, 261. The partial inhibition of pertussis toxin catalyzed ADPribosylation by HDL may reflect the apo A-Vapo A-II content of the lipoproteins. The highest efficiency to counteract pertussis catalyzed ADP-ribosylation of 41 kDa protein was encountered with proteoliposomes containing apo A-II. This result is in good agreement with previous’findings suggesting that apo A-II was also a potent switch in signal transduction of platelet membranes. Our finding regarding the possible functions of the platelet G-proteins clearly indicates that these
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proteins mediate the activation of HDL induced PC breakdown through a pertussis toxin sensitive pathway.
This work was supported by grants from INSERM U 325. The authors are very grateful to Paul Kelly for his helpful editorial comments and to J. Fremaux for his technical assistance.
l-
DELBART C, THERET N., AILHAUD G., FRUCHART J.C. Phosphatidylcholine breakdown during receptor binding of HDLS. Afherosclerosis, 2, 719a,1989.
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NAZIH H.. DEVRED D., MARTIN-NIZARD F., FRUCHART J.C., DELBART C. Phosphatidylcholine breakdown in HDLS stimulated platelets. Thrombosis Research, s, 913-920,199O.
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VAN DER MEULEN J., HASLAM R.J. Phorbol ester treatment of intact rabbit platelets greatly enhances both the basaland guanosine 5’-[y-thioltriphosphate-stimulated phospholipase D activities of isolated platelet membranes. Biochem. J., 21l, 693-700.1990.
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REMMAL A., KOUTOUZOV S., MARCHE P. Enhanced turnover of phosphatidylcholine in platelets of hypertensive rats. Possible involvement of a phosphatidylcholine-specific phospholipase C. Biochim.
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HUANG R., KUCERA G.L., RITTENHOUSE SE. Elevated cytosolic Ca2+ activates phospholipase D in human platelets. J. Biol. Chem., 266 1652-1655, 1991.
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BRASS L.F., WOOLKALIS M.J., MANNING D.R. Interactions in platelets between G-proteins and the agonists that stimulate phospholipase C and inhibit adenylyl cyclase. J. Biol. Chem.. 262,5348-5355,1988.
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LAPETINA E.G.. REEP B., CHANG K.J. Treatment of human platelets with trypsin, thrombin, or collagen inhibits the pertussis toxin-induced ADP-ribosylation of a 41 lcDa protein. Proc. Natl. Acad. Sci. USA. 82, 5880-5883.1986.
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HAVEL R.J., EDER H., BRADGON J.H. The distribution and chemical composition of uluacentrifugally separated lipoproteins in human serum. J. Clin. Invest., 1,1345-1353,19X5.
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PETERSEN G.L. A simplification of the protein assay of Lowry et al. Ann. Biochem., &$, 346-356,1977.
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MEZDOUR H., CLAVEY V., KORA I., KOFFIGAN M., BARKIA A., FRUCHART J.C. Anion exchange
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CHEN C.H. and ALBERS J.J. Characterization of proteoliposomes containing apoprotein AI : a new substrate for the measurement of lecithin cholesterol acyltransferase activity. J. Lipid Res., 2& 680-691, 1982.
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BARBER A.J., JAMIESON G.A. Isolation and characterization of plasma membranes from human blood platelets. J. Biol. Chem., &z& 6357-6365, 1970.
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THERET N., DELBART C., AGUIE Cl., FRUCHART J.C., VASSAUX G., AILHAUD G. Cholesterol efllux from adipose cells is coupled to diacylglycerol production and protein kinase C activation. Biochem. Biophys. Res. Cornman.. m, 1361-1368, 1990.
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PRICE B.D., MORRIS J.D.H., MARSHALL C.J., HALL A. Stimulation of phosphatidylcholine hydrolysis, diacylglycerol release, and arachidonic acid production by oncogenic Ras is a consequence of protein kinase C activation. J. Biol. Chem., &f, 16638-16643, 1989.
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FOLCH J.. LEES M., SLOANE-STANLEY G.H. A simple method for the isolation and purification total lipids from animal tissues. J. Biol. Chem., 228 497-509. 1957.
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MARTIN T.W.. phosphatidylcholine
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ROTHENBERG P.L., KAHN C.R. Insulin inhibits pertussis proteins. J. Biol. Chem., 261,15546-15552,1988.
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LAEMMLI U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage Nature, mn 680-685,197O.
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MOCHLY-ROSEN D., GORDON A.S. GTP-binding proteins are restricted Biochem. Bioiphys. Res. Comm., m, 388-395, 1990.
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WALKER G., BOURGUIGNON L.Y.W. Membrane-associated induced platelet activation. Faseb J., 4.2924-2933.1990.
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HUANG C.. CABOT M.C. Phorbol diesters stimulate the accumulation of phosphatidate, phosphatidylethanol, and diacylglycerol in three cell types. J. Biol. Chem., m, 14858-14863,199O.
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BILLAH M.M., ANTHES J.C. The regulation and cellular functions of phosphatidylcholine Biochem. J., m, 281-291,199O.
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LAVIE Y., PITERMAN 0.. LISCOVITCH M. Inhibition of phosphatidic by sphingosine. FEBS, Z. 7-10, 1990.
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DE CHAFFOY DE COURCELLES D., ROEVENS P., VAN BELLE H. R 59022, a diacylglycerol inhibitor. J. Biol. Gem., XQ, 15762-15770, 1985.
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WILLIAMS A.G., WOOLKALIS M.J.. PONCZ M., MANNING D.R., GEWIRTZ A.M., BRASS L.F. Identification of pertussis toxin-sensitive G-proteins in platelets megakaryocytes, and human erythioleukemia cells. Blood, 26, 721-730, 1990.
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TAYLOR C.W. The role of G-proteins in transmembrane
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MICHAELIS K.C. Bradykinin stimulates phsophodiesteratic cleavage of in cultured endothelial cells. Biochem. Biophys. Res. Cornman., m, 1271-1~79.1988. toxin-catalyzed
ADP-ribosylation
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T4.
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41 kDa GTP-binding protein in collagen-
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activity
kinase
PERTUSSIS
TOXIN SENSITIVE G-PROTEIN COUPLING OF HDL RECEPTOR TO PHOSPHOLIPASE C IN HUMAN PLATELETS
H. Nazih*, D. Devred*, F. Martin-Nizard*, V. Clavey*+, J.C. Fruchart*+, C. Delba.rt*+ *Serlia : Institut Pasteur, 1 rue du Pr Calmette, B.P. 245 - 59019 LILLE Cklex, France +UFR de Pharmacie, 3 rue du Professeur Laguesse, 59045 LILLE Cedex, France.
(Received 10.3.1991; accepted in revised form 7.7.1992 by Editor J. Soria) (Received by Executive Editorial Office 11.8.1992)
ABSTRACT Initially we established that, in human platelets, low concentrations of HDL3 stimulate phosphatidylcholine (PC) hydrolysis and a transient increase im 1,2diacylglycerol (DAG). In (3H) PC prelabelled platelets, phosphocholine is released into the medium during HDL3 induced PC turnover with a 1.5 to 2 fold increment, indicating that HDL3 stimulated DAG generation in platelets is likely due to phospholipase C (PLC). GTP or GTP-y-S augments, and pertussis toxin inhibits HDL3 stimulated DAG production. Treatment of p~latelet membranes with HDL3 or with proteoliposome containing apo A-I ot A-II substantially prevents 41 kDa protein ADP-ribosylation that was induced by pertussis toxin, with apo A-II having an inhibitory potency greater than apo AI. These data provide strong evidence that the pertussis sensible G protein (Go or Gi) is directly involved in coupling PLC to HDL3 receptor in platelets.
UCTION
Initially we established that the binding of low concentrations of HDL3 to human blood platelets stimulates both the rapid loss of phosphatidylcholine (PC) and the biphasic generation of 1,2diacylglycerol (1,2-DAG) [ 1,2]. These results indicated that the binding of HDL3 stimulates PC breakdown by activating membrane phospholipase. Agonist induced generation of DAG has been reported to occur via PLC or PLD catalyzed hydrolysis of PC [3]. PC hydrolysis by either pathway was also described in human platelets [4, 51. To clarify the interpretation of our data, it was necessary to define the contribution of PLC or Key words : platelets, high density lipoproteins, phospholipase C, GTP binding protein.
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PLD to PC turnover and DAG formation under our experimental conditions. Moreover, platelet responses to agonist are believed to be mediated by at least two pertussis toxin-sensitive Gproteins : Gi which inhibits adenylyl cyclase and Gp which stimulates phospholipase C [6]. The effect of pertussis toxin on platelets is accompanied by the ADP-ribosylation of a protein with an apparent molecular mass of 40-42 KDa [7]. The present studies examine the coupling of PLC to the HDL3 receptor, and the ability of G-protein to stimulate PC breakdown in HDL3 stimulated platelets.
Materials. Pertussis toxin (islet activating protein) and cholera toxin, Thrombin, Guanosine 5’-kthiol m phosphate (GTP [T-S]), and GTP were from Sigma Chemical Co. [Adenylate 32Pl NAD. L3-phosphatidyt[Nmeu@H] choline-1,2dipalmitoyl(3H Chol-PC) and phosphatidylcholine L-a-dipalmitoyl [2-palmitoyl-9.10-3H (N)] (3H Cl6:0-PC) were from Amersham. Silica gel TLC plates were purchased from Merck. Other biochemical reagents were from Sigma Lipoprotein isolation. HDL3 were isolated from human serum by standard differential ultracentrifugal flotation [8] (HDL3 d = 1.125-1.210 g/cm3). Its protein content was measured by the procedure of Petersen et al. 191.The apolipoprotein E (apo E) constituted less than 0.2 % of the total HDL3 protein. Apo AI and ape AH were isolated [lo] inorder toprepareproteolipos ill]. Human platelet membrane isolation. Platelets were isolated from titrated blood drawn freshly from healthy human donors. Following centrifugation at 15Oxgfor 20 min., the platelet rich plasma (PKP) was centrifuged for 15 min. at 9ooxg and the platelets resuspended in a 0.35 % Bovine Serum Albumin (w/v) buffer (BSA-Buffer) containing 137 mM NaCl, 2.7 mM KCl, 0.32 mM NaH2PO4.5.6 mM glucose, 11.9 mM NaHCO3 and 1.3 mM Hepes pH 7.4. Platelet membranes were prepared according to the procedure of Barber et al. [12] and resuspended in an incubation buffer containing 15 mM Tris-HCl, pH7.5,0.125 M sucrose, 75 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1 mM ATP, 5 mM MgC12 and 0.05 mM G’IP. (3H)-phospbatidylcholine prelabelled platelet membranes. 1.25 pCi of (3H C16:O)PC or 2 pCi of (3H Chol)-PC were pipetted into vials and the solvent was evaporated off to leave a thin film. 100 pg of membranes were then added and incubated for 60 min. at room temperature. The specific activity of the preparation was 3.105 cpm/lOU pg membrane proteins. Less than l/200 of the radioactivity was associated with free lipids, and nearly all the radioactivity with the PC fraction. [2.13,14]. Stimulation by HDL3. (3H)-PC prelabelled human platelet membranes (100 l.tg protein) were resuspended in 1 ml of incubation buffer and exposed to HDL3 (50 pg) unless otherwise specified. Studies with pertussis or cholera toxin were performed in incubation buffer containing 20 @I NAD. The incubations were terminated by immediately adding 4 ml of ice cold CHC13/CH30H (2/l v/v). The lipids were extracted as described by Folch et al. [ 151. Lipid analysis. Lipids were spotted on silicagel 60 TLC plates which were developed with petroleum ether/ethylether/acetic acid (90-10-5 v/v/v) to separate DAG. The radioactive spots corresponding to DAG and PC were cut and processed for radioactivity counting. A good correlation for DAG release was found between the (3H) PC method and the DAG Kinase assay 1131.(3H)choline metabolites were determined after lyophilisation of the aqueous phase of a Folch extract. Choline and phosphocholine were separated according to the method outlined by Martin and Michaelis 1161with the following solvant mixture 0.5 46 NaCl/ethanol/CH30H/NH40H (50 - 30 - 20 - 5 v/v/v/v). Areas corn&rating with authentic standards of choline (Bf 0.09) and phosphocholine (Bf 0.39) were scraped and transferred to scintillation vials for radioactivity counting. ADP-ribosylation. Pertussis or cholera toxins were preactivated just before use, by incubation (250 pg/ml) in a freshly prepared solution containing 200 mM sodium phosphate, pH 7.4,0.5 mM dithiothreitol, and 100 pM ATP for 10 min at 37°C 1171.ADP-ribosylation was determined by the incorporation of 32~ from [adenylate - 32P]-NAD into membrane proteins. Membrane suspension (100 ug) was added to 300 pl of freshly prepared incubation mixture
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[17] containing 10 pg pertussis (or 10 pg cholera) toxin, 50 mM Tris, 10 mM MgC12, 100 mM KH2PG4, 1 mM EDTA, 1 mM ATP. 20 mM Arginine, 0.1 mM BSA, 5 mM ADP ribose, 100 mM GTP. Labelling reactions were initiated by adding 5 pM 32P-NAD (specific activity 15-150 pCi/mmol). ADP-ribosylation was also performed on membranes (100 pg) stimulated by HDL3 (50 pg), thrombin (7 units/ml) or proteoliposomes containing ape A-I ( (50 pg) or apo A-II ( (50 pg) added simultaneously with the toxins. After incubating at 37°C for 1 h, reactions were stopped by addition of five volumes of cold acetone and incubation at -20°C for 10 min. Membrane proteius were pelleted by centrifugation(10 000 x g ; 5 mitt). After two mpeated precipitations and dryings, the protein pellet was dissolved in 30 p.l of Laemmli sample buffer [18] at 100°C for 3-5 mm. SDS-PAGE was performed on 5-19 8 gradient gels using the discontinuous system of Laemmli [lg]. Gels were stained with Coomassie Brillant Blue Radiolabelled bands were located by l-2 days exposure with Kodak XOMAT K film at - WC. Data presentation. Data points are the average of at least duplicate determinations which were within 5 % of the mean.
HDL3 induced hydrolysis of PC by PLC. As shown previously [l, 21, incubation of (3H Cl6 : 0)PC - platelets with HDL3 induced a biphasic accumulation of (3H)DAG. The early peak appeared rapidly reaching a maximal 2-3 fold increment at 20-30 seconds. Thereafter a second phase of DAG generation occurred at 60 seconds and returned to baseline level within 2 minutes. The selective incorporation of (3H-Chol)PC into platelet membranes permits a specific evaluation of HDL3 elicited changes in PC metabolism through the release of water soluble choline metabolites. As early as 30 seconds, HDL3 treatment caused an increase in the release of phosphocholine into the medium which could be indicative of PLC involvement in PC hydrolysis (Fig. 1). Changes in choline content were not statistically significant. Production of (3H)-DAG in HDL3 stimulated platelets is likely due to enhanced PLC activity.
0
30
60
Tim.
( au.
)
Fig. 1 : Release of choline metabolites : 100 pg platelet membranes were prelabelled with (3HChol)PC and incubated at 37°C in the presence of 50 pg protein HDL3. Aliquots were taken to determine by TLC the release of water soluble choline metabolite into the medium. Data are the average value of 4 separate preparations in duplicate.
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Effects of GTP and GTP yS on DAG production in HDL3 stimulated platelet membranes. We have examined the effect of guanine nucleotide on PLC activity as measured by (3H)-DAG production in (3H - Cl6 : 0)PC prelabelled platelet membranes when challenged with HDL3. As shown in Fig. 2, either GTP or the GTP analog, GTP-y-S, stimulated a 1.5 fold increase in DAG formation, and displayed a stimulatory potency order similar to that observed for other receptors coupled to G-proteins ie. GTP-y-S > GTP.
100
Fig. 2 : Effects of guanine nucleotides
on DAG production membranes.
300
200
in HDLJ
stimulated
platelet
(3H - Cl6 : 0)pC prelabellcd platelet membranes (100 pg protein) were incubated in the presence of 0.05 mM GTP (x ) or GTP-y-S (0) and stimulated by 50 pg protein HDL3. Aliquots were taken to determine DAG release. Data representative of at least 3 separate preparations in duplicate.
Effects of toxins on DAG production HDL3 activated platelet membranes. (3H Cl6 : 0)-PC prelabelled platelet membranes were preincubated in the presence of pertussis or cholera toxins and then exposed to HDL3. The initial rise in (3H)-DAG (30 set) produced by HDL3 stimulation in pertussis toxin pretreated membranes is lowered to control values represented by PC labelled membranes alone. Pretreatment of membranes with cholera toxin has no influence on HDL3 induced DAG production (Fig. 3). Pertussis toxin inhibited the production of (3H)-DAG in the presence of HDL3, while cholera toxin had no effect on the signaling pathway. The time course of DAG production is presented in Fig. 4. The presence of pertussis toxin in the medium decreased the formation of HDL3 induced DAG in platelet membranes. These data suggest strongly that a pertussis toxin sensible G-protein (Go or Gi) is involved in the HDL3 receptor PLC system in human platelets. Identification of platelet membrane associated G-proteins. For a primary characterization of the G-protein activated by the HDL3 receptors, we used the pertussis toxin mediated [32P]-ADP-ribosylation labelling technique. Labelled proteins migrated as few bands on 5-19 % gradient SDS-PAGE. A strong incorporation of radioactivity into proteins with an apparent molecular mass of 40-41 kDa and 67 kDa as well was obtained indicating that a 41 kDa platelet membrane protein was selectively ADP-ribosylated by pertussis toxin (Fig. 5 lane 5). An
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accompanying ADP ribosylated protein with an aparent molecular mass ranging around 62 kDa has been described (19), but it displayed no immunological cross reactivity with antibodies against CXsubunits of the GTP binding protein. In addition the 41 kDa proteins is often found to be closely associated with other membrane proteins (20). Including HDL3 in the reaction mixture resulted in decreased ADP-ribosylation of the 41 kDa proteins (Fig. 5 lane 2). Platelet membrane stimulation with pmteoliposomes containing apo A-I or apo A-II also decreased pertussis toxin induced ADPribosylation (Fig. 5 lane 3,4). The highest efficiency in lowering ADP-ribosylation was encountered during platelet stimulation with proteoliposomes containing apo A-II. This result coincided with the previous suggestions that apo A-II was able to generate DAG from PC with the highest apparent efficacy. Similarly, treatment of platelets with thrombin substantially prevented subsequent protein ADP-ribosylation which was induced by pertussis toxin (Fig. 5 lane 1).
Fig. 3 : Effects of toxins on DAG production
in HDW
activated
platelet
membranes.
Platelet membranes prelabelled with (3H - Cl6 : O)-PC were incubated in the presence 6 @ml cholera or pertussis toxin for 20 min at 37°C and exposed to 0 (control) or 50 pg HDL3. Aliquots were taken to determine DAG content. Data representative of 3 preparations in duplicate.
E 8
Time ( sec. ) 20 0
I 40
I 80
I 120
I
I
1
I
160
200
240
280
Fig. 4 : Effects of pertussis toxin on time course of DAG production. Platelet membranes prelabelled with (3H - Cl6 : O)-PC were preincubated in the absence (D), or in the presence (0) of 6 pg/ml pertussis toxin for 20 min at 37°C and exposed to 50 pg HDL3. Aliquots were taken to determine DAG content. Values represent the mean ?rSD of three experiments in duplicate.
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--,
67 -+ 41+
Fig. 5 : Pertussis toxin induced ADP-ribosylation of 41 kDa protein in human platelets. ADP ribosylation was performed as described in methods in the presence of pertussis toxin alone or coupled to HDL3 (50 pg). thrombin (7 units/ml) and proteoliposomes containing apo A-I (50 pg) or apo A-II (50 pg). The results are expressed in cpm and represent the mean + SD of 3 separate experiments.
F
I? P
OH
DAG kitmu
Fig. 6 : Metabolic
id WM : DAG fOmatiOn
pathway
catalyzedby
for the formation
of PC-derived
DAG.
PLC, and subsequent phosphorylation into phosphatidic acid (PA) by D:G kinase. m arrows : Pathway of DAG formation via the initial action of PLD and simultaneous hydrolysis of PA by PA phosphohydrolase. so
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QISCussroN
Stimulation of platelets by HDL3 leads to the activation of the phospholipase cascade and subsequently to a biphasic increase in DAG mass [ 11. The first phase of DAG release was shown to result from a direct activation of membrane phospholipase by receptor occupancy [2]. In platelets agonist-induced generation of DAG has been reported to occur via PLC or PLD catalyzed hydrolysis of PC [5]. Moreover both PLC or PLD are stimulated by PKC or bylphorbol esters [3, 21, 221. So differential sensitivity of lipase is much less helpful, than previously imagined, to identify individual phospholipases interacting with and being activated by a;specific receptor. The activation of either PLC or PLD by HDL3 receptor has not yet been directly established. To determine the relative contribution of PLC or PLD to HDL3 induced DAG formation (Pig. 6) platelets were ptelabelled with (3H-Chol)-PC before HDL3 stimulation. Phosphocholine was the main water soluble choline metabolite to be released into the medium. This result indicates that DAG release is catalyzed directly by PLC during the early phase, as the later secondary rise in DAG mass cannot be observed in isolated platelet membranes. These data alone cannot establish the enzymatic pathway of PC degradation. Although formation of phosphocholine and 3H-DAG supports a PLC mechanism Elucidation of a precise reaction pathway is further complicated by the presence within the cells of specific phosphatases and kinases which rapidly interconvert choline/phosphocholine and DAG/phosphatidic acid [16]. However this result is in good agreement with our previous data [2], indicating that platelet pretreatment with 100 uM sphingosine failed to modify (3H)-PA formation and sustained HDL3-stimulated (3H)-DAG mass formation. Sphingosine, in its role of potent inhibitor of phosphatidic acid phosphohydrolase [23], should have increased PA content if a PLD was involved in both the primary and the secondary phase of PC turnover. Moreover platelet pretreatment with R 59022 (a known DAG-Kinase inhibitor [24]) which decreased the rate of DAG phosphorylation by approx 50 % argues [2] also against HDL3 activation of PLD. Our results are also in good agreement with those of Huang et al. [5] providing strong evidence that in human platelets 87 % of PC is hydrolyzed via the initial action of PLC, and the remaining 13 % by PLD. Our initial interest centred around the possible involvement of a GTP-binding protein in the ability of a HDL3 receptor to activate phospholipase C. Much accumulating evidence suggests that one or more guanine nucleotide binding proteins, commonly referred to as Gp, mediate the interaction between platelet receptors for HDL3 and PLC. The addition of GTP-y-S increased PLC activity to a large extent (1.5 fold over basal level). On the contrary, pretreatment of platelet with pertussis toxin leads to inhibition of HDL3 induced responses ie : PLC activation. Pertussis inhibition of agonist induced responses has been largely described [25. 261. In all cases, an association of the Gi-protein with those responses has been indicated. To determine whether any of the known pertussis toxin sensitive a subunits evoke the HDL3 induced PC breakdown, ADPribosylation in non and agonist stimulated platelet membranes were analysed. Usually pertussis toxin catalyses the ADP-ribosylation of the al subunit of Gi [7]. This ADP-ribosylation is inhibited when al is dissociated, as encountered when Gi molecules are coupled to a specific receptor. We demonstrated that ADP-ribosylation labelling techniques identify a single band at 41 kDa as commonly found [7, 261. The partial inhibition of pertussis toxin catalyzed ADPribosylation by HDL may reflect the apo A-Vapo A-II content of the lipoproteins. The highest efficiency to counteract pertussis catalyzed ADP-ribosylation of 41 kDa protein was encountered with proteoliposomes containing apo A-II. This result is in good agreement with previous’findings suggesting that apo A-II was also a potent switch in signal transduction of platelet membranes. Our finding regarding the possible functions of the platelet G-proteins clearly indicates that these
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proteins mediate the activation of HDL induced PC breakdown through a pertussis toxin sensitive pathway.
This work was supported by grants from INSERM U 325. The authors are very grateful to Paul Kelly for his helpful editorial comments and to J. Fremaux for his technical assistance.
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DELBART C, THERET N., AILHAUD G., FRUCHART J.C. Phosphatidylcholine breakdown during receptor binding of HDLS. Afherosclerosis, 2, 719a,1989.
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MARTIN T.W.. phosphatidylcholine
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MOCHLY-ROSEN D., GORDON A.S. GTP-binding proteins are restricted Biochem. Bioiphys. Res. Comm., m, 388-395, 1990.
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MICHAELIS K.C. Bradykinin stimulates phsophodiesteratic cleavage of in cultured endothelial cells. Biochem. Biophys. Res. Cornman., m, 1271-1~79.1988. toxin-catalyzed
ADP-ribosylation
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T4.
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41 kDa GTP-binding protein in collagen-
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activity
kinase
