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Pharmacological Characterization of the Human P2Y₁₃ Receptor

Institute for Interdisciplinary Research, School of Medicine (F.M., D.C., D.C., J.-M.B., N.S.G) and Department of Medical Chemistry, Erasme Hospital (J.-M.B.), Université Libre de Bruxelles, Brussels, Belgium; Euroscreen S.A., Gosselies, Belgium (E.L.P.); and Cardiovascular/Thrombosis Research Department, Sanofi-Synthelabo, Toulouse, France (C.L., P.S.)

Received July 12, 2002; accepted March 20, 2003

	Abstract

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The P2Y₁₃ receptor has recently been identified as a new P2Y receptor sharing a high sequence homology with the P2Y₁₂ receptor as well as similar functional properties: coupling to G_i and responsiveness to ADP (Communi et al., 2001). In the present study, the pharmacology of the P2Y₁₃ receptor and its differences with that of the P2Y₁₂ receptor have been further characterized in 1321N1 cells (binding of [³³P]2-methylthio-ADP (2MeSADP) and of GTP[³⁵S]), 1321N1 cells coexpressing G₁₆ [AG32 cells: inositol trisphosphate (IP₃) measurement, binding of GTP[³⁵S]) and Chinese hamster ovary (CHO)-K1 cells (cAMP assay)]. 2MeSADP was more potent than ADP in displacing [³³P]2MeSADP bound to 1321N1 cells and increasing GTP[³⁵S] binding to membranes prepared from the same cells. Similarly, 2MeSADP was more potent than ADP in stimulating IP₃ accumulation after 10 min in AG32 cells and increasing cAMP in pertussis toxin-treated CHO-K1 cells stimulated by forskolin. On the other hand, ADP and 2MeSADP were equipotent at stimulating IP₃ formation in AG32 cells after 30 s and inhibiting forskolininduced cAMP accumulation in CHO-K1 cells. These differences in potency cannot be explained by differences in degradation rate, which in AG32 cells was similar for the two nucleotides. When contaminating diphosphates were enzymatically removed and assay of IP₃ was performed after 30 s, ATP and 2MeSATP seemed to be weak partial agonists of the P2Y₁₃ receptor expressed in AG32 cells. The stimulatory effect of ADP on the P2Y₁₃ receptor in AG32 cells was antagonized by reactive blue 2, suramin, pyridoxal-phosphate-6-azophenyl-2',4'disulfonic acid, diadenosine tetraphosphate, and 2-(propylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (AR-C67085MX), but not by N⁶-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS-2179) (up to 100 µM). The most potent antagonist was N⁶-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (ARC69931MX) (IC₅₀ = 4 nM), which behaved in a noncompetitive way. The active metabolite of clopidogrel was unable to displace bound 2MeSADP at concentrations up to 2 µM.

The recent molecular cloning of the platelet ADP receptor P2Y₁₂ (Hollopeter et al., 2001) revealed the existence of two structurally-distinct subfamilies of P2Y receptors. The first one is composed of P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors, all coupled to the phospholipase C pathway, except P2Y₁₁, which is also positively coupled to the cAMP pathway. The members of the second subfamily are P2Y₁₂, P2Y₁₃, and the UDP-glucose receptor recently renamed P2Y₁₄, which are all coupled to G_i. This group shows a low amino acid identity with the other P2Y receptors but shares some conserved positively charged amino acids in TM6 and TM7 (Erb et al., 1995). The P2Y₁₂ receptor is the target of antithrombotic agents: clopidogrel, which acts via the covalent binding of an active metabolite (Savi et al., 2001), and ATP derivatives (AR-C69931MX, etc.), which behave as competitive antagonists (Ingall et al., 1999).

The human P2Y₁₃ receptor was recently identified (Communi et al., 2001; Zhang et al., 2002). Its sequence displays 48% amino acid identity with the P2Y₁₂ receptor. It is coupled to G_i and responds to diphosphate adenine nucleotides (ADP, 2MeSADP, ADPS). The abundance of P2Y₁₃ messengers is highest in the brain and the spleen, suggesting roles in nervous and immune systems. As a preliminary step to the evaluation of its potential physiological roles, we have now performed a detailed pharmacological characterization of the P2Y₁₃ receptor.

	Materials and Methods

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Cell Culture and Transfection. CHO-K1 and AG32 cells were transfected with the recombinant P2Y₁₃-pEFIN5 plasmid or the plasmid alone using FuGENE6 transfection reagent (Roche Applied Science, Mannheim, Germany) as described previously (Communi et al., 2001). AG32 are 1321N1 cells transfected with the recombinant G₁₆-pERAEQ2 plasmid.

Materials. ADP, ADPS, 2MeSADP, ATP, 2MeSATP, ATPS, Ap₃A, Ap₄A, Ap₅A, Ap₆A, 2MeSAMP, poly[A], poly[A].[G], creatine phosphate (CP), creatine phosphokinase (CPK), apyrase (grade I), TBAP, iodoacetamide, and pertussis toxin were from Sigma Chemicals (St. Louis, MO). Suramin, reactive blue 2, PPADS and 8-p-SPT were from RBI/Sigma (Natick, MA). AR-C67085MX and ARC69931MX were generous gifts from Drs. J. D. Turner and D. Shah (AstraZeneca, Wilmington, DE). MRS-2179 was obtained from Tocris Chemicals (Ellisville, MO). Forskolin was purchased from Calbiochem (La Jolla, CA). Rolipram was a gift from the Laboratories Jacques Logeais (Trappes, France). Methanol SPECTRANAL was purchased from Riedel-de-Haen (Seelze, Germany). pEFIN5 and pERAEQ2 are expression vectors developed by Euroscreen (Brussels, Belgium). The radioactive products [myo-D-2-³H]inositol (17.7 Ci/mmol) and ¹²⁵I were from Amersham Biosciences (Piscataway, NJ). Dowex AG1 x 8 (formate form) was from Bio-Rad Laboratories (Hercules, CA). The radioactive ligands [-³³P]2MeSADP (2000 Ci/mmol) and [³H]ADP (33.9 Ci/mmol) were purchased from PerkinElmer Life Sciences Inc. (Boston, MA). The radioactive ligand [³H]2MeSADP (3 Ci/mmol) was purchased from Moravek Biochemicals Inc (Brea, CA). The active metabolite of clopidogrel was obtained as described previously (Savi et al., 2000).

Binding of [³³P]2MeSADP. Experiments on the specific binding of [³³P]2MeSADP to 1321N1 cells were performed with a filtration technique to separate the free from bound [³³P]2MeSADP, according to a previously published method (Savi et al., 1994). When the cultures were 80% confluent, the cells were harvested in phosphate-buffered saline-EDTA (5 mM), washed once, and resuspended in dialyzed fetal calf serum containing culture medium, supplemented with 1 mM EDTA, and used within 15 min. Incubations were carried out in 0.20 ml of culture medium that contained P2Y₁₃-expressing 1321N1 cells (5000 cells/well) and [³³P]2MeSADP (0 to 3 nM). Triplicate incubations were carried out at 20°C for 10 min and were stopped by the addition of 3 ml of ice-cold assay buffer followed by a rapid vacuum filtration over glass fiber filter (Filtermats 11734; Skatron Instruments Inc., Sterling, VA). The active metabolite of clopidogrel was incubated 1 h before radioligand addition.

Binding of GTP[³⁵S]. The measurement of nucleotide-stimulated GTP[³⁵S] binding to membranes of cells expressing P2Y₁₃ was performed as described previously (Kotani et al., 2001). Briefly, membranes (15 µg) from AG32-P2Y₁₃ or 1321N1-P2Y₁₃ cells were incubated for 15 min at room temperature in GTP[³⁵S] binding buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 3 mM MgCl₂, 3 µM GDP, and 10 µg/ml saponin) containing different concentrations of ADP and 2MeSADP in 96-well microplates (Basic FlashPlates; PerkinElmer). GTP[³⁵S] (0.1 nM) (Amersham Biosciences) was added, microplates were shaken for 1 min and further incubated at 30°C for 30 min. The incubation was stopped by centrifugation of the microplate for 10 min at 800g and 4°C, and the supernatant was aspirated. Microplates were counted in a TopCount scintillation and luminescence counter (PerkinElmer) for 1 min per well.

For the kinetic studies, incubation was initiated with the addition of ligand simultaneously with GTP[³⁵S] and was terminated after 1, 3, 10, or 30 min by the filtration through GF/B filters using a multiple membrane filter (Linca Lamon Instrumentation, Tel Aviv, Israel). Filters were washed three times with 4 ml of ice-cold binding buffer, dried, and bound GTP[³⁵S] was measured by liquid scintillation counting. In some case, AG32-P2Y₁₃ cells were treated overnight with 100 ng/ml pertussis toxin added to the culture medium.

Inositol Phosphate Measurement. AG32 cells were seeded (200,000 cells per dish) on 35-mm (diameter) Petri dishes and labeled for 24 h with 5 µCi/ml [myo-D-2-³H]inositol in inositol-free Dulbecco's modified Eagle's medium containing 5% fetal calf serum, antibiotics, amphotericin, sodium pyruvate, and 400 µg/ml G418. Cells were incubated for 2 h in Krebs-Ringer-HEPES (KRH) buffer (124 mM NaCl, 5 mM KCl, 1.25 mM MgSO₄, 1.45 mM CaCl₂, 1.25 mM KH₂PO₄, 25 mM HEPES, pH 7.4, and 8 mM D-glucose). The cells were then incubated in presence of the tested compounds for 30 s up to 10 min. The incubation was stopped by the addition of 1 ml of an ice-cold 3% perchloric acid solution. Inositol phosphates were extracted and separated on Dowex columns as described previously (Communi et al., 1995).

Cyclic AMP Assays. CHO-K1 Chinese hamster ovary cells were seeded (150,000 cells per dish) on 35-mm (diameter) Petri dishes in Ham's F-12 medium containing 10% fetal calf serum, antibiotics, amphotericin, sodium pyruvate, and 400 µg/ml G418. Cells were incubated for 2 h in KRH (see below) containing rolipram (25 µM) and incubated in the presence of the tested compound for 10 min with or without forskolin (3 µM). The incubation was stopped by the addition of 1 ml of HCl (0.1 M). The cellular lysate was dried and resuspended in an appropriate volume of water. Cyclic AMP was quantified by ¹²⁵I radioimmunoassay as described previously (Brooker et al., 1979).

Evaluation of Nucleotide Degradation. AG32 cells were seeded (200,000 cells per dish) on 35-mm (diameter) Petri dishes in inositol-free Dulbecco's modified Eagle's medium containing 5% fetal calf serum, antibiotics, amphotericin, sodium pyruvate, and 400 µg/ml G418. Cells were incubated for 2 h in KRH buffer. Cells were then incubated in the presence of 100 nM [³H]ADP or 100 nM [³H]2MeSADP for various times. Cell supernatants were filtered through a 0.4-µm filter (Millipore, Brussels, Belgium) and stored at -20°C. Radiolabeled nucleotides were separated by HPLC on a µBondapack C18 reverse phase 3.9 x 300 mm column (Millipore) and eluted at 1.5 ml/min with a linear methanol gradient between solution A (5 mM TBAP, 60 mM KH₂PO₄, and 5% methanol) and solution B (5 mM TBAP, 60 mM KH₂PO₄, and 35% methanol) at 2% per min. HPLC fractions were counted after addition of scintillation liquid, Insta-Gel II Plus (PerkinElmer). The column was calibrated using unlabeled nucleotides (AMP, ADP, ATP, 2MeSAMP, 2MeSADP, and 2MeSAMP) and radiolabeled nucleotides ([³H]ADP and [³H]2MeSADP).

Nucleotide Pretreatment. To eliminate contaminating diphosphate nucleotides, triphosphate nucleotides were pretreated as described previously (Hechler et al., 1998). In brief, ATP and 2MeSATP (1 mM solutions) were incubated for 90 min at room temperature with 20 units/ml CPK and 10 mM CP. The reaction was stopped by addition of 10 mM iodoacetamide. Apyrase (grade I) was also used as another way to remove the contaminating diphosphate nucleotides. In brief, poly[A] and ADP (10 mM solutions) were incubated for 5 min at 30°C in HEPES buffer, pH = 6.5, with 20 units/ml apyrase (grade I). The apyrase activity was checked by HPLC using the same conditions as described in the previous section.

Data Analysis. Graphs and data analysis were performed with Prism from GraphPad Software (San Diego, CA).

	Results

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Assay of [³³P]2MeSADP binding to 1321N1 cells. The binding assay of [³³P]2MeSADP was performed on 1321N1 cells transfected with the P2Y₁₃-pEFIN5 vector. As shown in Fig. 1A, binding of [³³P]2MeSADP to 1321N1 cells was concentration dependent, and the nonspecific binding measured in presence of unlabeled ADP (10 mM) was very low and depended linearly on the [³³P]2MeSADP concentration. A very low binding of [³³P]2MeSADP was found in nontransfected 1321N1 cells (Fig. 1A). The Scatchard plot (Fig. 1B) revealed the presence of one class of binding sites that exhibit high affinity with an apparent equilibrium dissociation constant (K_d) of 0.28 ± 0.12 nM. The total number of binding sites (B_max) corresponds to 72,240 ± 18,540 receptors per cell.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Binding of [33P]2MeSADP to P2Y13-1321N1 cells. A, saturation. P2Y13-1321N1 cells () and 1321N1 cells () were incubated (5000 cells/sample) for 10 min with [33P]2MeSADP at concentrations ranging from 0.01 to 1.8 nM. Nonspecific binding was evaluated in P2Y13-1321N1 cells by adding 10 mM unlabeled ADP in the binding medium. B, Scatchard representation of the specific binding. C, effect of different compounds on the binding of [33P]2MeSADP to P2Y13-1321N1 cells. ADP, ADPS, ATP, ATPS, 2MeSADP, 2MeSATP, and AR-C67085MX were incubated for 10 min with P2Y13-1321N1 cells. The active metabolite of clopidogrel was incubated 1 h before radioligand addition.

As shown in Fig. 1C and Table 1, the order of affinity for the receptor was 2MeSADP >> 2MeSATP > ADP = ARC67085MX > ADPS = ATPS = ATP. The active metabolite of clopidogrel (Savi et al., 2000), a potent antagonist of the P2Y₁₂ receptor (Savi et al., 2001), did not inhibit the binding of [³³P]2MeSADP to P2Y₁₃-expressing 1321N1 cells.

Assay of GTP[³⁵S] Binding to Membranes. The effect of ADP and 2MeSADP on the binding of GTP[³⁵S] was assessed on membranes of both 1321N1 and AG32 cells transfected with P2Y₁₃-pEFIN5 vector. The same results were found with both cell lines. As shown in Fig. 2A, the binding of GTP[³⁵S] increased progressively after the addition of ADP or 2MeSADP. At all the times tested, the effect of 2MeSADP was greater than that of ADP. As shown in Fig. 2B, after 15 min of incubation, 2MeSADP was at least 30 times more potent than ADP. The binding of GTP[³⁵S] induced by 30 nM of ADP or 2MeSADP was abolished by a 20-h preincubation with pertussis toxin (100 ng/ml) (data not shown) or by coincubation with AR-C67085MX (5 µM) (Fig. 2C). In 1321N1 cells not transfected with the P2Y₁₃ receptor, neither ADP nor 2MeSADP had an effect on GTP[³⁵S] binding (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 2. Binding of GTP[35S] A, kinetic study of binding to 1321N1-P2Y13 cells. ADP and 2MeSADP were incubated for various times. The data represent the mean ± S.D. of triplicate experimental points obtained in one experiment that was representative of five. B, concentration-action curves for ADP and 2MeSADP incubated for 15 min with AG-32-P2Y13. The data represent the mean ± S.D. of triplicate experimental points obtained from one experiment that was representative of five. C, ADP (30 nM) and 2MeSADP (30 nM) stimulation is reversed by AR-C67085MX (5 µM). The data represent the mean ± S.D. of triplicate experimental points obtained in one experiment that was representative of three.

Kinetics of IP₃ Formation in AG32 Cells. We studied the time course of IP₃ accumulation in AG32 cells stably expressing the P2Y₁₃ receptor using two distinct P2Y₁₃ agonists: ADP and 2MeSADP. As shown in Fig. 3A, the 2MeSADP-induced IP₃ accumulation had a longer duration than the response to ADP. The peak of IP₃ accumulation was at 1 min and 30 s, respectively, for 2MeSADP and ADP. The concentration-action curves characterizing the effects of ADP, 2MeSADP, and ADPS on IP₃ formation after 30 s have been reported previously (Communi et al., 2001): they were almost superimposable for ADP and 2MeSADP; ADPS was slightly less potent. We have now obtained the corresponding concentration-action curves after 10 min, in experiments performed in the presence of LiCl (10 mM) and 8-p-SPT (100 µM), to prevent an effect of adenosine formed by ADP degradation on the A₂ receptor endogenously expressed by 1321N1 cells. As shown in Fig. 3B, after 10 min of stimulation, the 2MeSADP concentration-action curve was shifted to the left compared with the 30-s stimulation, whereas the ADP and ADPS curves were shifted to the right. In these 10-min experiments, the following EC₅₀ values were computed for ADP, 2MeSADP, and ADPS, respectively: 194 ± 26, 1.2 ± 0.3, and 280 ± 63 nM (mean ± S.D. of three independent experiments). For 30-s experiments, the following EC₅₀ values were computed for ADP, 2MeSADP, and ADPS, respectively: 45 ± 4.7, 57 ± 19, and 82 ± 13 nM (mean ± S.D. of three independent experiments). The time-dependent variation of the relative potencies of ADP and 2MeSADP cannot be explained by differences in the degradation rate, which was very similar for the two nucleotides (Fig. 4).

View larger version (23K): [in this window] [in a new window]   Fig. 3. A, time course of the IP3 response of hP2Y13-AG32 cells to ADP and 2MeSADP. 1321N1 cells expressing both hP2Y13 receptor and G16 protein (AG32) were incubated with 100 nM of ADP or 2MeSADP for different times. The data represent the mean ± S.D. of triplicate experimental points obtained in one experiment that was representative of three. B, concentration-action curves of ADP, 2MeSADP, and ADPS on IP3 accumulation in AG32 cells. AG32 cells were incubated with different agonist concentrations for 10 min. A preincubation with LiCl (10 mM) and 8-p-SPT (100 µM) was performed 10 min before stimulation with agonist. The data represent the mean ± S.D. of triplicate experimental points obtained in one experiment that was representative of three.

View larger version (59K): [in this window] [in a new window]   Fig. 4. Estimation of extracellular ADP and 2MeSADP degradation by AG32-P2Y13. Cells were incubated with [3H]ADP and [3H]2MeSADP for various times. Cell supernatants were fractionated by HPLC and fractions were then counted for [3H] radioactivity.

ATP and 2MeSATP Effect on IP₃ in AG32 Cells. ATP and 2MeSATP were tested on the hP2Y₁₃-expressing AG32 cells. Commercial powders are contaminated by degradation products: contamination by the respective diphosphate derivative is around 1% for ATP and 10% for 2MeSATP (Hechler et al., 1998). Therefore, the diphosphate contaminants were enzymatically removed using CP/CPK as described under Materials and Methods. As shown in Fig. 5, after such treatment, ATP and 2MeSATP behaved as partial agonists with EC₅₀ values of 4.2 ± 0.8 and 1.5 ± 0.2 µM, respectively (mean ± S.D. of three independent experiments).

View larger version (21K): [in this window] [in a new window]   Fig. 5. Partial agonist action of ATP and 2MeSATP on human P2Y13 receptor. 1321N1 cells transfected with both hP2Y13 receptor and G16 protein were incubated with various concentrations of ADP, ATP, and 2MeSATP for 30 s. ATP and 2MeSATP were treated with 10 mM CP and 20 units/ml CPK, as described under Materials and Methods, before the stimulation. The data represent the mean ± S.D. of triplicate experimental points obtained in one experiment that was representative of three.

Effect of Other Nucleotide Derivatives on IP₃ in AG32 Cells. The effect of diadenosine polyphosphates was also tested on AG32 cells stably expressing the P2Y₁₃ receptor (Fig. 6). After 30 s of incubation, the effect of Ap₃A on IP₃ accumulation was almost equal to that of ADP. In contrast, Ap₄A, Ap₅A, and Ap₆A were inactive. In view of a recent report showing that extracellular mRNA can activate a P2Y receptor via its poly[A] tail in a pertussis toxin-sensitive manner (Ni et al., 2002), we tested the effect of poly[A] on the P2Y₁₃ receptor in AG32 cells. As shown in Fig. 7A, poly[A] and poly[A].[G] also induce an increase of IP₃ in AG32 transfected cells. As previously discussed, contamination with ADP derivatives can be avoided by CP/CPK treatment. This treatment totally abolished poly[A].[G] activity and partially abolished that of poly[A]. After such treatment, the EC₅₀ value expressed in AMP equivalents, was 205 ± 60 µM. However pretreatment with apyrase, which degrades ADP into AMP instead of converting it into ATP, a partial agonist of P2Y₁₃, totally abolished the effect of poly[A] (100 µM) (Fig. 7B).

View larger version (30K): [in this window] [in a new window]   Fig. 6. Effect of different diadenosine polyphosphates on hP2Y13. 1321N1 cells transfected with both hP2Y13 receptor and G16 protein were challenged for 30 s with two different concentrations (100 nM and 10 µM) of ADP or ApnA (n = 3 to 6). The data represent the mean ± S.D. of triplicate experimental points obtained from one experiment that was representative of three.

View larger version (35K): [in this window] [in a new window]   Fig. 7. Concentration-response curves of different nucleotide derivatives at hP2Y13 receptor. A, poly[A] and poly[A].[G] were treated with 10 mM CP and 20 units/ml CPK, as described under Materials and Methods, before the stimulation. Both treated and untreated poly[A] and poly[A].[G] were added for 30 s on 1321N1 cells transfected with both hP2Y13 receptor and G16 protein. ADP was used as reference compound. B, poly[A] was treated with 20 units/ml apyrase, as described under Materials and Methods, before the stimulation. Poly[A] was added for 30 s on 1321N1 cells transfected with both hP2Y13 and G16 protein. ADP was used as reference compound. For A and B, the data represent the mean ± S.D. of triplicate experimental points obtained from one experiment that was representative of three.

Effect of Selective and Nonselective Antagonists on IP₃ in AG32 Cells. The classic nonselective antagonists of P2 receptors, RB-2, suramin, and PPADS (von Kugelgen and Wetter, 2000), were tested on the hP2Y₁₃ receptor in the presence of 100 nM of ADP. As shown in Table 2 and Fig. 8A, all three agents were inhibitory: RB-2 was slightly more potent than suramin, whereas PPADS was significantly less potent. We also tested three selective P2Y antagonists currently available: AR-C67085MX and AR-C69931MX, reported to be selective for P2Y₁₂ (Ingall et al., 1999), and MRS-2179, selective for P2Y₁ (Boyer et al., 1998). As shown in Fig. 8B, AR-C67085MX and AR-C69931MX were able to completely inhibit the response to 100 nM ADP with IC₅₀ values of 0.63 and 4.6 nM, respectively. The action of ARC69931MX was noncompetitive (Fig. 8C). At 100 µM, MRS-2179 had almost no effect on IP₃ accumulation induced by 100 nM ADP (data not shown). As shown in Fig. 8D, MRS-2179 and PPADS were unable to displace [³³P]2MeSADP bound to 1321N1 cells expressing P2Y₁₃, whereas suramin and RB-2 could partially displace [³³P]2MeSADP at high concentration. These results suggest that PPADS, suramin, and RB-2 act, at least in part, by a noncompetitive mechanism. Furthermore, RB-2, suramin, and PPADS (100 µM) had no specificity for the P2Y₁₃ receptor, because they also inhibited the IP₃ response to adenosine that in AG32 cells results from the coupling to G₁₆ of an endogenous adenosine receptor normally coupled to G_s (Communi et al., 1999) (Fig. 8E). As shown in Fig. 8F, two other nucleotides that are antagonists of P2Y₁₂ receptor were tested: Ap₄A (Zamecnik et al., 1992) and 2MeSAMP (Hollopeter et al., 2001). Ap₄A was a complete antagonist of hP2Y₁₃ with an IC₅₀ of 216 nM. In contrast, 2MeSAMP behaved as a partial agonist, producing a maximal effect that was 38% of that of ADP. Consistent with that partial agonist property, increasing amounts of 2MeSAMP decreased ADP-promoted IP₃ level by 62%, with an IC₅₀ of 2.4 µM.

View larger version (42K): [in this window] [in a new window]   Fig. 8. A, effect of nonselective P2 antagonists on hP2Y13 receptor. 1321N1 cells transfected with both hP2Y13 receptor and G16 protein (AG32) were preincubated with different concentrations of RB-2, suramin, or PPADS for 10 min. After 10 min, the cells were stimulated with 100 nM ADP for 30 s. B, effect of selective P2 antagonists on hP2Y13 receptor. The same protocol was applied for AR-C67085MX and AR-C69931MX. C, characterization of the antagonism by AR-C69931MX. 1321N1 cells transfected with both hP2Y13 receptor and G16 protein were preincubated with different concentrations of AR-C69931MX (, Ctrl, , 0.1 nM; , 1 nM; , 10 nM) for 10 min. After 10 min, cells were stimulated with different concentration of ADP for 30 s. D, binding of [33P]2MeSADP to 1321N1 cells. Displacement of [33P]2MeSADP by different antagonists of P2Y13 (suramin, RB-2, PPADS) and a P2Y1 antagonist, MRS-2179. E, nonselective effect of suramin, RB-2, and PPADS. AG32 cells were preincubated with 100 µM RB-2, suramin, or PPADS for 10 min. After 10 min, the cells were stimulated with 100 nM ADP or 30 µM of adenosine for 30 s. F, effect of two selective P2Y12 antagonists: Ap4A () and 2MeSAMP (). The protocol is identical to the one followed for other antagonists. and , cells preincubated 10 min with Ap4A and 2MeSAMP, respectively, without addition of ADP (100 nM). , control cells. For A to F, the data represent the mean ± S.D. of triplicate experimental points obtained in one experiment that was representative of three.

Effect on cAMP in CHO-K1 Cells. As shown in Fig. 9A, ADP and 2MeSADP produced a comparable inhibition of forskolin (3 µM)-induced accumulation of cAMP in CHO-K1 cells stably expressing the P2Y₁₃ receptor. The IC₅₀ values of ADP and 2MeSADP were 0.07 ± 0.14 and 0.1 ± 0.3 nM, respectively (mean ± S.D. of three independent experiments). The maximal inhibition was of 49 ± 7.6% at 10 nM for ADP and of 54 ± 9.7% at 1 nM for 2MeSADP (mean ± S.D. of three independent experiments). At higher concentrations, the inhibition was reversed. The EC₅₀ values characterizing that reversal were strikingly different for 2MeSADP and ADP: 6.3 ± 2 and 397 ± 130 nM, respectively (mean ± S.D. of three independent experiments). After an 18-h pretreatment with 100 ng/ml pertussis toxin, ADP and 2MeSADP produced only enhancement of cAMP accumulation in response to forskolin (3 µM) (Fig. 9B). The EC₅₀ values of 2MeSADP and ADP were 4.2 ± 1.6 and 158 ± 15 nM, respectively (mean ± S.D. of three independent experiments).

View larger version (23K): [in this window] [in a new window]   Fig. 9. A, CHO-K1 transfected cells were incubated with ADP or 2MeSADP for 10 min in presence of 3 µM forskolin. The data represent the mean ± S.D. of triplicate experimental points obtained from one experiment that was representative of three. B, CHO-K1 transfected cells were preincubated with 100 ng/ml pertussis toxin for 18 h and then incubated with ADP or 2MeSADP for 10 min in presence of 3 µM forskolin. The data represent the mean ± S.D. of triplicate experimental points obtained from one experiment that was representative of three.

	Discussion

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The human P2Y₁₃ receptor was initially characterized after stable expression in 1321N1 cells coexpressing G₁₆ (AG32 cells) and CHO-K1 cells (Communi et al., 2001). In AG32 cells, but not in 1321N1 cells, ADP, 2MeSADP, and ADPS stimulated inositol phosphate formation in a pertussis toxin-sensitive manner. In CHO-K1 cells, ADP inhibited forskolin-induced cAMP accumulation, an effect that was abolished by pertussis toxin. Therefore, the P2Y₁₃ receptor was defined as a receptor for diphosphate adenine nucleotides coupled to G_i (Communi et al., 2001). Using different expression systems, Zhang et al. (2002) reached essentially similar conclusions. In the present study, we have further characterized the pharmacology of the P2Y₁₃ receptor and its differences with that of the P2Y₁₂ receptor, with the use of additional assays (binding of [³³P]2MeSADP and GTP[³⁵S]) and a special emphasis on the following issues: activity of triphosphate adenine nucleotides, relative potency of ADP and 2MeSADP, sensitivity to P2Y₁₂ antagonists (clopidogrel active metabolite, AR-C67085MX, and AR-C69931MX).

When contaminating diphosphates were enzymatically removed and testing was performed over a short period (30 s), ATP and 2MeSATP seemed to be partial agonists of the P2Y₁₃ receptor with a weak potency. In the radioligand binding assay, the IC₅₀ of 2MeSATP was 20-fold greater than that of 2MeSADP, but this difference is likely to have been underestimated because of ectonucleotidase-catalyzed conversion of 2MeSATP into 2MeSADP during the 10-min course of the assay. In contrast, 2MeSATP has been consistently reported by several groups to be almost equipotent to 2MeSADP at the P2Y₁₂ receptor (Savi et al., 2001; Takasaki et al., 2001; Zhang et al., 2001), although it is known that 2MeSATP behaves as an antagonist rather than an agonist of the P2Y₁₂ receptor natively expressed on platelets (Park and Hourani, 1999). Zhang et al. (2002) have reported that after CP/CPK treatment, 2MeSATP retains a high potency on the P2Y₁₃ receptor (EC₅₀ = 83 nM): the reason for this discrepancy with our data are unclear but might be related to different levels of receptor expression. Palmer et al. (1998) have indeed shown that the capacity of ATP to activate the P2Y₁ receptor depends on the degree of receptor reserve.

2MeSADP is 1 to 2 orders of magnitude more potent than ADP at the human P2Y₁₂ receptor (Hollopeter et al., 2001; Savi et al., 2001; Takasaki et al., 2001; Zhang et al., 2001). The situation seems more complex in the case of the P2Y₁₃ receptor. Binding of [³³P]2MeSADP to intact 1321N1 cells revealed a greater affinity of 2MeSADP compared with ADP. 2MeSADP was also more potent than ADP in stimulating the binding of GTP[³⁵S] to 1321N1 or AG32 membranes, and this difference was observed at all the times tested. However, in AG32 cells, the EC₅₀ values characterizing the effects of 2MeSADP and ADP on IP₃ measured after 30 s have been reported to be almost equal (Communi et al., 2001). Using a different expression system, Zhang et al. (2002) reported EC₅₀ values of 19 and 60 nM, respectively. We have now shown that the relative potencies of 2MeSADP and ADP are critically dependent on the experimental conditions. In AG32 cells, the ADP and 2MeSADP EC₅₀ values for IP₃ accumulation were almost equal after 30 s. However, a 200-fold difference was noticed after 10 min, because of decreased potency of ADP and increased potency of 2MeSADP. Degradation by ectonucleotidases cannot explain these discrepancies because the degradation rates of ADP and 2MeSADP were comparable and relatively slow. Neither can they be considered an artifact resulting from the use of G₁₆-mediated activation of phospholipase C as a readout of receptor activation. Indeed, using the GTP[³⁵S] binding assay, 2MeSADP was more potent than ADP in both plain 1321N1 cells and 1321N1 cells cotransfected with G₁₆(AG32 cells). Furthermore, discrepancies were also found in CHO-K1 cells where G₁₆ was not coexpressed. In CHO-K1 cells incubated for 10 min, ADP and 2MeSADP produced an equipotent inhibition of cAMP. However, the reversal of that inhibition at higher concentration, as well as the enhancement of cAMP accumulation observed after pertussis toxin pretreatment, were characterized by a greater potency of 2MeSADP. These effects are explained by a promiscuous coupling to G_s, which has been observed with several other G_i-coupled receptors (Kukkonen et al., 2001). Although the binding assays indicate that 2MeSADP is intrinsically more potent than ADP at the P2Y₁₃ receptor, as it is on the P2Y₁₂ receptor, the other results, taken together, suggest that the P2Y₁₃ receptor could exist in multiple active conformations, characterized by differences in affinity for 2MeSADP versus ADP, kinetics, and preference for G proteins (Leff et al., 1997).

The antiplatelet and antithrombotic action of clopidogrel is mediated by an active metabolite (Savi et al., 2000). That active metabolite has been shown to prevent the binding of [³³P]2MeSADP to the P2Y₁₂ receptor with an IC₅₀ of 100 nM (Savi et al., 2001). When the same radioligand binding assay was performed on the P2Y₁₃ receptor, no displacement of the ligand was observed at concentrations up to 2 µM. On the other hand, AR-C67085MX and AR-C69931MX belong to a group of ATP derivatives that are specific antagonists of the P2Y₁₂ receptor and inhibit platelet aggregation at nanomolar concentrations (Ingall et al., 1999). At the recombinant P2Y₁₂ receptor, AR-C69931MX had an IC₅₀ of 2.4 nM (Takasaki et al., 2001). In AG32 cells expressing the P2Y₁₃ receptor, the IC₅₀ of AR-C67085MX was 0.6 µM. This lower potency on the P2Y₁₃ receptor is consistent with the low affinity of triphosphates (ATP, 2MeSATP) mentioned earlier. However, ARC69931MX was a very potent antagonist of the P2Y₁₃ receptor, with an IC₅₀ comparable with that obtained at the P2Y₁₂ receptor. Interestingly, this action of AR-C69931MX seems to be noncompetitive. MRS-2179 had no significant inhibitory effect on the P2Y₁₃ receptor at concentrations up to 100 µM. This observation further strengthens the claim that it is a selective antagonist of the P2Y₁ receptor (Boyer et al., 1998).

We have confirmed the observation by Zhang et al. that Ap₃A is a potent agonist of the P2Y₁₃ receptor. Furthermore, we have shown that Ap₄A, Ap₅A, and Ap₆A are completely inactive. The same profile has been observed with the P2Y₁ receptor (Patel et al., 2001), suggesting that a selective sensitivity to Ap₃A is a common feature of ADP receptors. Ap₄A, previously shown to inhibit platelet aggregation (Zamecnik et al., 1992), was an antagonist of the P2Y₁₃ receptor. Recently Ni et al. (2002) have suggested that extracellular mRNA can activate dendritic cells via an interaction between the poly[A] tail and an unidentified P2Y receptor. This conclusion was based on the observation that commercial poly[A] increased cytosolic calcium in dendritic cells, a response blocked by suramin and pertussis toxin. We have now shown that poly[A] activates the P2Y₁₃ receptor. Because commercial poly[A] is prepared using ADP as starting material, contamination by residual ADP may complicate the interpretation of these results. Indeed pretreatment with CP/CPK abolished the effect of poly[A].[G] and shifted the poly[A] concentration-action curve to the right, whereas apyrase abolished its effect completely. Therefore, it is unlikely that the P2Y₁₃ receptor plays a role in the activation of dendritic cells by extracellular mRNA.

In conclusion, we have identified three properties that distinguish the recombinant human P2Y₁₃ receptor from the P2Y₁₂ receptor: it is less sensitive than the P2Y₁₂ receptor to activation by triphosphate nucleotides; the potency of 2MeSADP is superior or equal to that of ADP, depending on the measured endpoint; and it is antagonized less potently by AR-C67085MX and not at all by the active metabolite of clopidogrel. However, these studies have also revealed that in several respects, the P2Y₁₃ receptor is even more similar to the P2Y₁₂ than initially believed: greater potency of 2MeSADP compared with ADP in radioligand binding assays ([³³P]2MeSADP, GTP[³⁵S]) that assess binding to the receptor or its interaction with G_i, and antagonism by 2MeSAMP, Ap₄A, and especially AR-C69931MX. These similarities between the pharmacological profiles of the hP2Y₁₂ and hP2Y₁₃ receptors seems logical when considering the high degree of amino acid identity (47.7%) and the short distance (10 kilobases) between these two syntenic genes located in tandem on 3q25 (Wittenberger et al., 2001).

	Acknowledgements

 
We thank Professors J. E. Dumont and G. Vassart for helpful advice and discussions.

	Footnotes

 
This work was supported by an Action de Recherche Concertée of the Communauté Française de Belgique, by the Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Federal Service for Science, Technology and Culture, by grants of the Fonds de la Recherche Scientifique Médicale, the Fonds Médical Reine Elisabeth, the Fonds Emile DEFAY, and Boehringer Ingelheim. F.M. is supported by the Fonds National de la Recherche Scientifique/FRIA, Belgium. D.C. and D.C. are associated researchers of the FNRS, Belgium. N.S.G. is supported by Euroscreen.

ABBREVIATIONS: AR-C67085MX, 2-(propylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid); 2MeS, 2-methylthio-; ADPS, adenosine 5'-O-(2-thiodiphosphate); CHO, Chinese hamster ovary; ATPS, adenosine 5'-O-(3-thiotriphosphate); Ap₃A, diadenosine triphosphate; Ap₄A, diadenosine tetraphosphate; Ap₅A, diadenosine pentaphosphate; Ap₆A, diadenosine hexaphosphate; CP, creatine phosphate; CPK, creatine phosphokinase; TBAP, tetrabutylammonium dihydrogen phosphate; PPADS, pyridoxal-phosphate-6-azophenyl-2',4'disulfonic acid; 8-p-SPT, 8-(p-sulfophenyl)theophylline; AR-C69931MX, N⁶-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid); MRS-2179, N⁶-methyl 2'-deoxyadenosine 3',5'-bisphosphate; KRH, Krebs-Ringer-HEPES; HPLC, high-performance liquid chromatography; IP₃, inositol trisphosphate; RB-2, reactive blue-2.

Address correspondence to: Frederic Marteau, Institute for Interdisciplinary Research, School of Medicine, 808, Route de Lennik, 1070 Brussels, Belgium. E-mail: fmarteau{at}ulb.ac.be

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