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Laboratoire de Biologie et de Pharmacologie de l'Heemostase et de la Thrombose, Institut National de la Santee et de la Recherche Meedicale U.311, Etablissement Franais du Sang-Alsace, Strasbourg Cedex, France (A.B., A.E., B.H., G.K., J.-P.C., C.L., C.G.)
Deepartement Reecepteurs et Proteeines Membranaires, Unitee Propre de Recherche Centre National de la Recherche Scientifique 9050, Institut Feedeeratif Gilbert Laustriat, l'Institut Feedeeratif de Recherches 85, Ecole Supeerieure de Biotechnologie de Strasbourg, Illkirch, France (J.-L.G.)
Abstract
In the present study, we investigated the desensitization and trafficking of the P2Y1 and P2Y12 receptors after agonist-induced stimulation of platelets or astrocytoma cells transfected with the P2Y1 or P2Y12 receptors fused to green fluorescent protein. In platelets and in transfected cells, exposure to 10 e ADP caused desensitization of the P2Y1 receptor-driven calcium signal, whereas the P2Y12 receptor-mediated inhibition of cAMP formation was not affected. Plasma membranes from ADP-stimulated platelets also retained P2Y12 activity. Agonist-induced P2Y1 receptor desensitization was accompanied by its internalization in platelets and transfected cells. In contrast, although a substantial fraction of P2Y12 receptors was rapidly and transiently internalized, most of the P2Y12 receptors remained at the plasma membrane. Activated P2Y1 receptors were internalized through a clathrin-dependent pathway in cells and platelets, whereas the P2Y12 receptors seemed to use a distinct, clathrin-independent pathway. Together, these data indicate that the P2Y1 and P2Y12 receptors are differentially regulated upon activation. The absence of desensitization of the Gi protein-coupled P2Y12 receptor-dependent responses could represent a mechanism to preserve the hemostatic properties of otherwise unresponsive platelets.
ADP plays a central role in hemostasis and in arterial thrombosis. Stored in the platelet dense granules and released upon activation by a number of stimuli, including vessel wall collagen, thrombin, and thromboxane A2, it participates in all stages of platelet activation and enhances thrombus growth (Gachet, 2001). Two G protein-coupled receptors mediate platelet aggregation in response to ADP. The Gq-coupled P2Y1 receptor is responsible for the mobilization of intracellular calcium stores, shape change, and transient aggregation, whereas the Gi2-coupled P2Y12 receptor is responsible for inhibition of cyclic AMP production, amplification of the response and stabilization of the aggregates. Based on pharmacological studies as well as on studies with P2 receptor knockout animals, it is now well established that both receptors are necessary for normal platelet activation by ADP (Hechler et al., 1998b; Fabre et al., 1999; Leon et al., 1999; Foster et al., 2001). The P2Y12 receptor mostly supports the enhancing cofactor role of ADP when platelets are activated by other stimuli through activation of transduction pathways downstream of Gi, including phosphoinositide 3-kinases, rap-1b, and vasodilator-stimulated phosphoprotein dephosphorylation (Hechler et al., 1998a; Schwarz et al., 1999; Trumel et al., 1999; Gachet, 2001; Kauffenstein et al., 2001; Conley and Delaney, 2003; Kim et al., 2004). This receptor is thus an attractive target for potent antiplatelet drugs, which is indeed the case with the thienopyridine compound clopidogrel (Herbert and Savi, 2003) and with several classes of competitive antagonists such as the AR-C compounds (Humphries, 2000; Hollopeter et al., 2001).
To date, little is known concerning the regulation of these receptors after their activation. It has been known for a long time that platelets can become refractory to activation by ADP in vitro (O'Brien, 1965; Spaet and Lejnieks, 1966) or after major surgical operations (O'Brien et al., 1971), which could represent a postsurgical bleeding risk. ADP accumulates extracellularly in suspensions of washed platelets and is sufficient to desensitize the aggregation response to ADP (Ardlie et al., 1971; Holme and Holmsen, 1975; Fijnheer et al., 1992). Addition of apyrase (ATP-diphosphohydrolase; EC 3.6.1.5 [EC] ), an ATP/ADP degrading enzyme extracted from potatoes, protects the cells from desensitization and enhances their aggregability (Ardlie et al., 1971; Holme and Holmsen, 1975; Fijnheer et al., 1992). Thus, it seems that a nucleotide scavenger mechanism is necessary not only to prevent irrelevant cell activation but also to preserve cell responsiveness. When such a system is present, platelets activated by ADP are only transiently refractory to a second challenge with the agonist, whereas in its absence, their refractoriness is stable. This refractoriness could be mimicked in vitro by the use of nonhydrolyzable agonists such as ADPS (Poole et al., 1993; Baurand et al., 2000). In vivo, such a scavenger enzyme system has been discovered to be the CD39/ATP diphosphohydrolase or E-NTDPase-1, which is expressed on endothelial cells and is responsible not only for inhibition of platelet activation at the surface of healthy vessels (Marcus et al., 1997; Robson et al., 1997) but also for maintenance of platelet responsiveness, because the response to ADP is desensitized in CD39-deficient mouse platelets (Enjyoji et al., 1999).
In a previous work, we showed that platelet refractoriness to ADP, illustrated by complete absence of shape change and aggregation in response to ADP, was caused entirely by selective desensitization of the P2Y1 receptor, whereas the P2Y12 receptor remained functional (Baurand et al., 2000). Our hypothesis, based on functional and radioligand binding studies, was that the P2Y1 receptor could be desensitized and internalized, whereas the P2Y12 receptor persisted at the plasma membrane. The aim of the present study was to further investigate the desensitization and the trafficking of the P2Y1 and P2Y12 receptors both in platelets and in cells separately transfected with the receptors fused to the enhanced green fluorescent protein (eGFP). We report here that, either in platelets or in the heterologous systems, the P2Y1 receptor-induced intracellular calcium increase could be fully desensitized, whereas the P2Y12 receptor-induced inhibition of cAMP production remains functional after either short- or long-term stimulation. Both receptors were found to be internalized, however, with different kinetics and through distinct pathways, resulting in a permanent presence of the P2Y12 receptors at the plasma membrane, whereas the P2Y1 receptors remained inside the cells as long as the nucleotide was present in the medium.
Materials and Methods
Materials. ADP, ADPS, U46619 , 2MeSADP, adenylyl 5'-imidodiphosphate, GTP, forskolin, prostaglandin E1 (PGE1), adrenalin, isobutylmethyl xanthine, and essentially fatty acid-free human serum albumin were from Sigma (Saint Quentin-Fallavier, France). Thrombin-receptor-activating peptide (TRAP) was purchased from Neosystem (Strasbourg, France). AR-C69931MX was a generous gift from Astra (Charnwood, UK). Human fibrinogen was from Kabi (Stockholm, Sweden), and Fura-2/acetoxymethyl ester (Fura-2/AM) was from Calbiochem (Meudon, France). The cAMP assay kit was from Amersham Biosciences (Les Ulis, France). Apyrase (ATP-diphosphohydrolase; EC 3.6.1.5 [EC] ) was purified from potatoes (Cazenave et al., 1983). Dulbecco's modified Eagle's medium, G418 (Geneticin), and PBS were from Invitrogen (Paris, France), and FuGENE 6 transfection reagent was from Roche Diagnostics (Mannheim, Germany). Texas Red-transferrin conjugates and Lysotracker were from Molecular Probes (Eugene, OR).
Synthesis of eGFP-P2Y Fusion Proteins. eGFP was fused to the C-terminal and N-terminal ends of the human P2Y1 and P2Y12 receptors, respectively, to generate the construct P2Y1-eGFP and P2Y12-eGFP. The chimera N was constructed by polymerase chain reaction amplification of the coding sequence of the P2Y12 and in frame cloning downstream of the eGFP coding sequence into the pCDNA3 vector (Invitrogen, Groningen, The Netherlands), preceded by a signal sequence derived from the 7-5-hydroxytryptamine3. Chimera C construct was obtained by cloning the coding sequence of the P2Y1 receptor into the pEGFP-N3 vector (BD Biosciences Clontech, Palo Alto, CA). Characterization of these cells is shown in supplemental data.
Cell Culture. Human astrocytoma 1321N1 cells (reference no. 80630402, European Collection of Cell Cultures, UK) were cultured as described previously (Kauffenstein et al., 2004). Cells were grown to 50 to 80% confluence overnight in 35-mm culture dishes and transfected with 1 to 2 e/dish plasmid DNA by noneCliposome-mediated transfer using FuGENE 6 transfection reagent at a ratio of 1:3 (micrograms of DNA/microliters of FuGENE6). After 24 to 48 h, the cells were washed and stable transfectants were selected in the presence of G418 (0.8 mg/ml). Twelve hours before the experiment, apyrase (0.5 U/ml) was added to the cell culture to prevent desensitization of the receptors by naturally occurring nucleotide release from cells.
Platelet Preparation. Washed human platelets were prepared as described previously (Cazenave et al., 1983) and resuspended in Tyrode's buffer containing 2 mM CaCl2, at a density of 3 x 105 platelets/e, in the presence of 0.02 U/ml apyrase, a concentration sufficient to prevent the desensitization of platelet ADP receptors during storage.
Desensitization Procedures. Two different procedures were used to study the desensitization of the P2 receptors. First, the washed platelets or the cells were incubated with ADP (10 e) for 1, 5, or 15 min, followed by apyrase (0.2 U/ml for 30 s) to remove the ADP. Platelets or cells were then restimulated with ADPS (10 e), the nonhydrolyzable analog of ADP. The functionality of the P2Y1 and P2Y12 receptors was determined by intracellular calcium measurements and intracellular cAMP assays, respectively, as described below. The second procedure was already reported (Baurand et al., 2000). In brief, the platelets or the cells were incubated with ADPS (1 mM) or with 2MeSADP (1 mM) for 1 h at 37°C. The agonist was then removed by centrifugation (Baurand et al., 2000). Under these experimental conditions, washed platelet suspensions, with no added fibrinogen and in the absence of stirring, do not aggregate during the ADP-induced desensitization.
Preparation of Human Platelet Membranes. Platelet plasma membranes were prepared essentially as described previously (Barber and Jamieson, 1970; Gachet et al., 1995). Washed platelets were loaded with glycerol by centrifugation through a 0 to 30% glycerol gradient and lysed in a hypotonic 10 mM Tris/HCl buffer, pH 7.5. After lysis, broken platelets were layered onto a 30% sucrose cushion and centrifuged for 4 h at 60,000g. The floating plasma membranes were removed and pelleted by centrifugation at 100,000g and resuspended in 10 mM Tris/HCl buffer, pH 7.5, and stored at eC80°C. For desensitization conditions, washed platelets were first treated with ADP (10 e) for 15 min, and ADP (10 e) was added during subsequent centrifugation through the glycerol gradient.
Adenylyl Cyclase Assay on Human Platelet Plasma Membranes. The reaction mixture for adenylyl cyclase assay contained 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol, 1 mM isobutylmethyl xanthine, 2 mg/ml bovine serum albumin (BSA), 30 e adenylyl 5'-imidodiphosphate (a nonhydrolyzable ATP analog used as adenylyl cyclase substrate), 10 e GTP, and 30 e of membrane protein. The following reagents were added: 1 e PGE1, 10 e AR-C69931MX, 1 e 2MeSADP, or 10 e adrenalin in a total volume of 250 e. Incubation was started by addition of platelet membrane suspension to the reaction mixture and carried out in duplicate for 10 min at 37°C. Reaction was stopped by addition of 25 e of ice-cold 6.6 N perchloric acid, and cAMP was extracted and quantified using the commercial radioimmunoassay kit as described previously (Baurand et al., 2000).
Intracellular Signaling. For intracellular calcium measurements, platelets were loaded with Fura-2/AM as described previously (Hechler et al., 1998b). Suspension of 1321N1 cells (15 x 106 cells/ml) was loaded with 5 e Fura-2/AM for 30 min at 37°C, and cells were resuspended at a density of 2 x 106 cells/ml for intracellular calcium measurements. cAMP measurements of adherent cells or platelets were performed as described previously (Hechler et al., 1998b).
Confocal Microscopy. 1321N1 cells were grown to about 50% confluence on fibronectin-coated coverslips. The medium was replaced with PBS containing calcium and 0.1% fatty acid-free human serum albumin, and the cells were incubated at 37°C in the presence of the agonist or the vehicle. Reactions were stopped by washing the dishes with ice-cold PBS. After fixation with 2% paraformaldehyde in PBS for 15 min at room temperature, the cells were examined by confocal microscopy using an inverted microscope Zeiss LSM510. The amount of internalized eGFP-receptor was determined by quantifying the intracellular fluorescence using an image analysis software (MetaMorph; Universal Imaging Corporation, Downingtown, PA). At each time point, at least 25 cells were analyzed. In double-labeling experiments, the 1321N1 cells were either pretreated with 50 e/ml Texas Red-conjugated transferrin for 45 min before addition of ADPS (1 mM), or they were incubated with 75 nM Lysotracker Red after cell fixation. In some experiments, sucrose (0.45 M) was added during the incubation with the agonist.
Generation of P2Y1 and P2Y12 Polyclonal Antibodies. The antibody against P2Y1 was generated by immunizing rabbits with a peptide of 15 N-terminal amino acids of human P2Y1 (GTDAAFLAG-PGSSWG). The anti-P2Y12 antibody was a rabbit polyclonal IgG raised against the second extracellular domain (TNRQPRGKN-VKKC) and the C-terminal domain of human P2Y12 (CKKEQDGG-DPNEETPM). The characterization of the antibodies was performed by flow cytometry using 1321N1-transfected cells expressing the P2Y1 receptors or HEK-293 cells expressing the P2Y12 receptors (kindly provided by Sylvie Reigner-Meyer, Roche Diagnostics, Basel, Switzerland).
Transmission Electron Microscopy and Immunolabeling. For transmission electron microscopy, two types of approaches were used, a post- and a pre-embedding method. In the postembedding method, platelets were fixed at several time points after activation, and immunogold labeling was performed on sections. This approach allows quantifying the distribution of the receptors at defined time points. In brief, washed platelets (1 ml) were fixed with an equal volume of 2.5% paraformaldehyde and 0.5% glutaraldehyde in 0.2 M sodium cacodylate buffer for 1 h. The fixed platelets were infiltrated with 2.3 M sucrose and frozen in liquid nitrogen. Ultrathin sections (70 nm) were prepared, incubated in PBS containing 1% BSA for 10 min and then in 0.02 M glycine in PBS for 10 min. Samples were incubated at 25°C for 1 h with anti-P2Y1 (10 e/ml), anti-P2Y12 (3 e/ml), or the corresponding nonimmune antibodies and then washed in PBS-BSA. After further incubation with protein A-10-nm gold for 20 min at 25°C, the samples were examined under a Philips CM120 electron microscope at 120 kV (Eindhoven, The Netherlands). Immunogold particles on platelets were counted manually and assigned to various subcellular compartments, including 1) the platelet surface, 2) the inner compartments, and 3) the cytosol. At each time point, assignment of 450 to 800 gold particles on about 30 to 50 platelets enabled calculation of the percentage of labeling of each compartment. The total number of particles per platelet obtained with each antibody was considered to be the total labeling (100%). The number of particles corresponding to each antibody was expressed as the percentage of total labeling.
We also used the pre-embedding approach that allows studying the dynamic of the receptor internalization from the external membranes to cellular compartments. In this method, gold-labeled antibodies were directly coupled to gold particles and were incubated with the platelets during activation, and the samples were fixed at indicated time points. In brief, washed platelets (1 ml) were incubated with a mixture of anti-P2Y1 antibodies labeled with 15-nm gold particles (5 e) and anti-P2Y12 antibodies labeled with 10-nm gold particles (5 e), 1 min before addition of ADP (5 e). The platelets were fixed for 1 h by adding an equal volume of 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. The samples were then incubated at room temperature with 1% tannic acid and processed for transmission electron microscopy as described previously (Eckly et al., 2001).
Results
Desensitization of P2Y1-Dependent ADP-Induced Calcium Increase. Platelets were stimulated with ADP (10 e) for 1, 5, or 15 min, followed by 0.2 U/ml apyrase treatment for 30 s to remove ADP and subsequent restimulation with 10 e ADPS, a nonhydrolyzable ADP analog, under stirring. Under these conditions, the intracellular calcium increase induced by ADPS (Fig. 1A, a) was abolished at all time points tested (Fig. 1A, b). ADP-pretreated platelets were still responsive to 10 e serotonin and 1 U/ml thrombin (data not shown), indicating that a general desensitization/refractoriness of the calcium response is unlikely to occur and is not responsible for the absence of response to ADP. Likewise, in P2Y1-eGFP transfected cells, the calcium signal induced by ADPS (10 e) (Fig. 1B, a) was completely inhibited after a first challenge with ADP for up to 15 min (Fig. 1B, b). However, the desensitization of the P2Y1-driven calcium signal was transient because the calcium response reappeared after 5 min when ADP was removed by three washes (data not shown). Similar experiments were performed in platelets and in transfected cells pretreated with 1 mM ADPS or with 1 mM 2MeSADP for 1 h (second procedure, see Materials and Methods) with identical results (data not shown). Thus, the P2Y1 receptor-mediated increase in intracellular calcium was desensitized both in platelets and in transfected cells.
Absence of Desensitization of P2Y12-Dependent ADP-Induced Inhibition of cAMP Formation. The P2Y12-mediated inhibition of cAMP production both in platelets and in P2Y12-transfected cells was studied as described above after stimulation with ADP for 1, 5, or 15 min, followed by apyrase treatment (0.2 U/ml, 30 s) and subsequent restimulation with 10 e ADPS. As shown in Fig. 2, ADPS-induced inhibition of cAMP accumulation was not affected by 1, 5, or 15 min ADP pretreatment in either platelets (Fig. 2A) or transfected cells (Fig. 2B). It is noteworthy that forskolin-induced cAMP accumulation in P2Y12-transfected cells decreased with the time of ADP-pretreatment (Fig. 2B), whereas cAMP levels induced by PGE1 stimulation of platelets were not affected by ADP pretreatment (Fig. 2A). This difference between 1 min ADP pretreatment versus 15 min (Fig. 2B, black columns) most probably results from the constitutive release of nucleotides by the cultured cells in the medium. These nucleotides may act on the P2Y12 receptor to inhibit the adenylyl cyclase, thus counteracting the effect of forskolin, because addition of the selective P2Y12 antagonist AR-C69931MX 10 e completely restored the effect of forskolin (Fig. 2B, gray columns). Likewise, AR-C69931MX reversed the inhibitory effect of ADPS on cAMP accumulation in platelets (Fig. 2A). These results indicated that the P2Y12 receptor-dependent inhibition of cAMP production remains functional upon ADP stimulation and is not desensitized. Similar results were obtained with platelets or transfected cells pretreated with ADPS (1 mM) or with 2MeSADP (1 mM) for 1 h (data not shown), a procedure previously shown to induce a stable desensitization state of the P2Y1 receptor for several hours (Baurand et al., 2000).
To avoid the possibility that resensitization of the P2Y12 receptor after ADP pretreatment might be occurring during subsequent adenylyl cyclase assays, plasma membranes from vehicle- or ADP-pretreated platelets (15 min) were prepared (see Materials and Methods), and cAMP levels were measured. As shown in Fig. 2C, 2MeSADP-induced inhibition of cAMP accumulation in platelet membranes was not affected by the ADP pretreatment, whereas the P2Y12 antagonist AR-C69931MX inhibited the effect of ADP. These data add strong evidence that the P2Y12 receptor remains functional upon ADP stimulation and is not desensitized. Together, these results demonstrate both in a native and in a heterologous model of transfected cells that the P2Y1 receptor can be desensitized, whereas the P2Y12 is not.
P2Y12-Dependent Amplification of Gq-Coupled Responses Are Conserved in ADP-Prestimulated Platelets. To further establish functionality of the P2Y12 receptor, we checked for its capacity to potentiate responses to agonists of other Gq-coupled receptors such as the PAR-1 agonist peptide SFFLRN (TRAP) or the TP receptor agonist U46619 . As shown in Fig. 3, AR-C69931MX (10 e) inhibited the PAR-1 and the TP receptor-mediated platelet aggregation to a similar extent in control or ADP-pretreated platelets, demonstrating that the P2Y12 receptor activation pathway remained functional. Again, these results were confirmed after pretreatment with 1 mM ADPS or 1 mM 2MeSADP for 1 h (data not shown).
Internalization of P2Y1- and P2Y12-eGFP Receptors in Transfected Cells. Our previous radioligand binding studies suggested that the P2Y1 receptor could undergo internalization, whereas the P2Y12 receptor was retained at the plasma membrane (Baurand et al., 2000). The intracellular trafficking was investigated in more detail by confocal microscopy on eGFP-P2Y1 and P2Y12 receptors in individually transfected cell lines. The cells have been examined at different time points after agonist incubation. As shown in Fig. 4A, ADPS (1 mM) induced internalization of the P2Y1 receptor within minutes, and which remained intracellularly located for at least 1 h. By contrast, only a small fraction of the P2Y12 receptors was found intracellularly, whereas most of the P2Y12 receptors remained at the plasma membrane (Fig. 4A). The intracellular fluorescence (expressed as the percentage of total fluorescence) was quantified using an image analysis software (MetaMorph) with a minimum of 25 cells examined at each time point (Fig. 4B). After a 15-min stimulation of P2Y1-expressing cells with ADPS, 50% of the total fluorescence was located intracellularly (Fig. 4B) and increased to 75% after 1-h stimulation (Fig. 4B). Concerning the P2Y12 receptor, stimulation with ADPS (1 mM) for 30 s, 60 s, 2 min, or 5 min did not induce intracellular relocation of the P2Y12 receptor (Fig. 4B; data not shown). After 15 min, the P2Y12 receptor was found to enter the cells transiently, because only a small increase in intracellular fluorescence was observed compared with a longer time point (Fig. 4B). Moreover, clear labeling of the plasma membrane was observed at all time points, indicating that either a large proportion of the P2Y12 receptors was not internalized or a very rapid turnover of receptor recycling occurred, allowing the maintenance of the receptors at the cell surface.
ADPS-induced internalization of the P2Y1 receptor was significantly inhibited by treatment with sucrose (0.45 M), an inhibitor of the clathrin-coated endocytic pathway (Fig. 4C). In contrast, sucrose had no effect on the early relocalization of the P2Y12 receptors. Double-labeling experiments indicated that the P2Y1-eGFP receptor relocalized into vesicles that overlapped with those stained by Texas Red-transferrin, a marker of early endosomes, contrary to P2Y12-eGFP receptors. These results suggested that P2Y1 receptor sequestration, unlike the P2Y12 receptor, occurred through a clathrin-endocytotic pathway. Finally, the pattern of P2Y1 and P2Y12 fluorescence did not coincide with that of LysoTracker Red, which labels lysosomes, suggesting that these receptors do not enter the lysosomal pathway (Fig. 4C).
Characterization of Polyclonal Antibodies against the P2Y1 and P2Y12 Receptors. To study the cellular distribution of P2Y1 and P2Y12 upon ADP stimulation, polyclonal antibodies were raised as described in Materials and Methods, and characterized by flow cytometry analyses. As shown in Fig. 5A, 1321N1-P2Y1 cells incubated with the P2Y1 antibody exhibited a 10-fold rightward shift of the mean fluorescence intensity compared with cells incubated with nonimmune serum (Fig. 5A, left), whereas HEK-P2Y12 cells incubated with the P2Y12 antibody exhibited a 9-fold rightward shift of the fluorescence intensity (Fig. 5B, left). No fluorescence shift was detected with nontransfected cells (Fig. 5, A and B, right). In addition, no labeling of anti-P2Y12 antibody on 1321N1-P2Y1 cells (Fig. 5A, dotted lines) or of anti-P2Y1 antibody on HEK-P2Y12 cells was detected (Fig. 5B, dotted lines), indicating absence of cross-reactivity between the two antibodies.
Localization of the P2Y1 and P2Y12 Receptors in Platelets by Electron Microscopy. A first approach was to examine the distribution of both receptors by performing postembedding immunostaining of ADP-stimulated platelets. This method allows the quantification of the receptor distribution at defined time points and was performed on cryosections, a procedure preserving the antigenicity of the proteins and the subcellular structures. In resting platelets and 7 s after ADP stimulation, P2Y1 receptors were detected on the surface of the plasma membrane and in the open canalicular system (OCS) (Fig. 6A). After 45 s, at maximum of aggregation, the P2Y1 receptors occurred in inner compartments where they were located in -granules (Fig. 6A). After 4 min, the P2Y1 receptors were detected both on the extracellular membranes and in inner compartments, whereas after 15 min, the majority of the P2Y1 receptors were again located at the surface of the platelets. For the P2Y12 receptors (Fig. 6B), the receptors were located on the external membranes in resting platelets (Fig. 6B, R). Some of the gold particles that seem apparently intracellular are in fact located on the OCS, which is an involution of, and continuous with, the outer platelet plasma membrane and not an inner compartment (Fig. 6B, arrowheads). After 7 s, a small fraction of the P2Y12 receptors was observed in inner compartments (Fig. 6B, arrows). After 45 s, 4 min, or 15 min, most of the P2Y12 receptors were located in external membranes (Fig. 6B). The quantification of the immunostainings corresponding to P2Y1 and P2Y12 receptors is shown in Fig. 6C (average of 40 platelets for each time point). In resting platelets, 50 ± 2% of P2Y1 and 50 ± 2% of P2Y12 receptors were located at the external membranes and weakly in inner compartments (10 ± 2 and 13 ± 1%, for P2Y1 and P2Y12 receptors, respectively). At 45 s and 4 min, the P2Y1 receptors have mostly disappeared from the plasma membrane (Fig. 6C, left, ) and were found in inner compartments (32 ± 3% at 45 s, 26 ± 2% at 4 min) (Fig. 6C, left, ), whereas at 15 min, they were observed on the external membranes (40 ± 2%; Fig. 6C, left, ). On the other hand, the P2Y12 receptors, although transiently internalized at 7 s (25 ± 2%), rapidly returned to the external membrane (42 ± 2% at 45 s; Fig. 6C, right, ). These results indicated, similarly to transfected cells, differential relocation of the P2Y1 and P2Y12 receptors upon platelet activation with ADP.
A second approach using a pre-embedding procedure was used to study the dynamic of internalization of the receptors from the external membranes to cellular compartments, with fine identification of the organelles. Gold-labeled anti-P2Y1 and anti-P2Y12 antibodies were coincubated with the platelets before (T0) or during activation with 5 e ADP. At the resting state, 15-nm-labeled P2Y1 and 10-nm-labeled P2Y12 were localized at the cell wall and the OCS (Fig. 7A). At 45 s, the P2Y1 and P2Y12 receptors were still found at the external membranes (Fig. 7B), despite a few gold-labeled P2Y12 in inner compartments (Fig. 7C). These observations, which contrast to the internalization kinetic determined with the postembedding method (Fig. 6C), are most probably caused by the difference of technical approaches. Indeed, the conjugation of the antibodies with the gold particles is known to affect their ability to penetrate into the cells (Robinson et al., 2000). The intracellular P2Y12 receptors were observed in compartments devoid of clathrin coat, even at 19.5°C, temperature at which the internalization is slowed down (Fig. 7C, inset). Four minutes after stimulation, the P2Y12 receptors have returned to the external membranes, whereas almost all the P2Y1 receptors were in inner vesicles (Fig. 7D). These vesicles displayed the classic feature of clathrin-coated vesicles (Fig. 7E, arrowheads), suggesting that the P2Y1 receptors were internalized through a clathrin-endocytic pathway. Ten minutes after stimulation, the P2Y1 receptors were back to the external membrane, with only minor inner labeling (about 14%) (Fig. 7F). Thus, using two different approaches, we found that platelet ADP stimulation induces internalization of the P2Y1 receptors, whereas only one-third of the P2Y12 receptors was rapidly and transiently internalized.
Discussion
The aims of the present study were to assess possible differential desensitization and internalization of the P2Y1 and P2Y12 receptors in platelets and in heterologously transfected cells. The present results unambiguously confirm that the so-called "refractory state" of platelets to ADP is entirely caused by selective desensitization of the P2Y1 receptor, whereas the P2Y12 receptor remains fully functional after either short-(1eC15 min) or long-term (1 h) stimulation (data not shown; Baurand et al., 2000). This was the case not only in intact cells but also on membranes prepared from ADP-treated platelets. The functional and physiological consequences are that under conditions where platelets are rendered refractory to ADP, the P2Y12 receptor is still able to potentiate aggregation induced by other agonists (Fig. 3). Previous radioligand binding studies suggested that desensitization of the P2Y1 receptor was accompanied by internalization of these receptors but not of the P2Y12 receptors (Baurand et al., 2000). In fact, the present study established that both receptors are internalized in platelets or in heterologously transfected cells, but in a different manner. Kinetic analysis using immunoelectron microscopy showed that the platelet P2Y1 receptors were internalized within 1 min and reappeared on the external membranes by 15 min, obviously through recycling, because platelets do not synthesize proteins. This is in line with the fact that desensitization is only transient provided that a scavenger system removes the nucleotide from the medium (Ardlie et al., 1971). In transfected cells, the eGFP-P2Y1 receptor also underwent almost complete internalization. This P2Y1 receptor internalization was transient because 30 min after removal of the agonist the receptors were recycled back to the plasma membrane (data not shown).
On the other hand, the P2Y12 receptor was internalized more rapidly, within seconds in platelets, and seemed to recycle rapidly to the platelet membrane. This may explain why the number of P2Y12 binding sites was not decreased after agonist pretreatment in our previous study (Baurand et al., 2000). In transfected cells, weak internalization of the fluorescent P2Y12 receptor was detected 15 min after agonist stimulation, but the plasma membrane always retained the majority of the fluorescence, suggesting either that only a fraction of the P2Y12 receptors was internalized or that the receptor was rapidly recycled to the plasma membrane. Thus, in platelets and in transfected cells, the fraction of the P2Y12 receptors present at the plasma membrane did not desensitize, or the rapid turnover of the internalized P2Y12 receptors returned to the cell surface in an active form, immediately available for reactivation by agonist, either of which explains the absence of functional P2Y12 desensitization. Our results using plasma membranes of ADP-pretreated platelets favor the first hypothesis.
The molecular mechanisms of desensitization and internalization have been extensively studied in several G protein-coupled receptors (Hall and Lefkowitz, 2002). Receptors are initially desensitized by rapid phosphorylation allowing the binding of -arrestin followed by internalization via clathrin-coated pits. Once internalized into endosomes, the receptors become dephosphorylated and are recycled back to the plasma membrane ready for reactivation. Electron microscopy clearly showed that the P2Y1 receptor was internalized in vesicles coated with clathrin (Fig. 7E), whereas in eGFP-P2Y1 cells internalization was inhibited in the presence of hypertonic sucrose, which leads to abnormal clathrin polymerization. In addition, sucrose prevented internalization of the P2Y1 receptor in intact platelets, as measured by radioligand binding experiments (A. Baurand, unpublished data), adding evidence that the P2Y1 receptor is internalized through a clathrin-dependent pathway toward early endosomes. The internalization mechanisms are very different for the P2Y12 receptor because the intracellular compartments were devoid of coat (Fig. 7C), and the endocytic process was unaffected by sucrose. In addition, the internalized P2Y12 receptors did not colocalize with transferrin, a marker of early endosomes, suggesting a different mechanism for internalization. Less is known about the mechanisms that link membrane proteins to noneCclathrin-coated vesicles. It will be important to determine whether an adaptor protein for the P2Y12 receptor directs the receptors to caveolae or other endocytic machinery.
Agonist-induced phosphorylation on putative serine, threonine, or tyrosine residues is a common feature of desensitization and internalization. Both the P2Y1 and P2Y12 receptors contain the DRYXXI/VXXP motif at the end of the third transmembrane domain that has been proposed as important for receptor internalization (Moro et al., 1993). In addition, P2Y1 and P2Y12 receptors also possess putative phosphorylated residues in the third intracellular loop and in the C-terminal part. The number of such residues is however higher in the C-terminal part of the P2Y1 than the P2Y12 receptor, and it has been suggested that the phosphorylation density could be more important for functional desensitization than for the internalization process (Hammes et al., 1999). The P2Y1 receptor also possesses three putative protein kinase C phosphorylation sites, of which threonine 339 has been implicated in a protein kinase C-dependent negative feedback mechanism (Fam et al., 2003). Whether the different behavior of the P2Y12 receptor is caused by structural motifs deserves further investigation. Like the P2Y1 receptor, other platelet Gq-coupled receptors such as 5-hydroxytryptamine2A, protease-activated receptor 1, or thromboxane A2 receptors are rapidly desensitized and internalized (Okwu et al., 1992; Roevens and de Chaffoy de Courcelles, 1995; Kawabata et al., 1999). Thus, in view of the central role of the P2Y12 receptor in platelet activation and hemostasis, one may speculate that in vivo, in case of desensitization of Gq-coupled receptors, the Gi-coupled ADP pathway should still be able to potentiate the signals triggered by adhesion receptors such as GPIb, GPVI, or integrin 21, which would represent a way for the cells to maintain their hemostatic properties (Gachet, 2001; Conley and Delaney, 2003; Hardy et al., 2004).
In summary, we have reported that the P2Y1 and P2Y12 receptors are differentially regulated after platelet activation. The P2Y1 receptor is rapidly desensitized and internalized, whereas the P2Y12 receptor remains functional and mainly persists at the plasma membrane. As a consequence, even in platelets refractory to stimulation by ADP, the P2Y12 receptor is able to ensure platelet reactivity at sites of vessel injury, thus preventing loss of the hemostatic function.
Acknowledgements
We thank Dominique Cassel, Meelanie Tunis, Steephanie Magnenat, and Fabienne Proamer for expert technical assistance and Sylvie Grosch for help with confocal microscopy (Plateforme Imagerie in vitro, IFR037, Strasbourg). We thank Astra Charnwood for providing AR-C69931MX. This work was supported by Association de Recherche et Deeveloppement en Medecine et Santee publique (ARMESA).
doi:10.1124/mol.104.004846.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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