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Agonist Binding and Gq-Stimulating Activities of the Purified Human P2Y₁ Receptor

Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina

Received September 3, 2003; accepted November 5, 2003

	Abstract

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The human P2Y₁ receptor (P2Y₁-R) was purified after high-level expression from a recombinant baculovirus in Sf9 insect cells. Quantification by protein staining and with a radioligand binding assay using the high-affinity P2Y₁-R antagonist [³H]MRS2279 ([³H]2-chloro-N⁶-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bis-phosphate) indicated a nearly homogenous preparation of receptor protein. K_i values determined in [³H]MRS2279 binding assays for antagonists with the purified P2Y₁-R were in good agreement with the K_i and K_B values determined for these molecules in membrane binding and activity assays, respectively. Availability of P2Y₁-R in purified form allowed direct determination of nucleotide agonist affinities under conditions not compromised by nucleotide metabolism/interconversion, and an order of affinities of 2-methylthio-ADP (2MeSADP) > ADP = 2-methylthioATP = adenosine-5'-O-(3-thio)triphosphate = adenosine-5'-O(2-thiodiphosphate) >> ATP was obtained. The signaling activity of the purified P2Y₁-R was quantified after reconstitution in proteoliposomes with heterotrimeric G proteins. Steady-state GTP hydrolysis in vesicles reconstituted with P2Y₁-R and G_q12 was stimulated by the addition of either 2MeADP or RGS4 alone and was increased by up to 50-fold in their combined presence. EC₅₀ values of agonists for activation of the purified P2Y₁-R were similar to their respective K_i values determined in radioligand binding experiments with the purified receptor. Moreover, ATP exhibited 20-fold higher EC₅₀ and K_i values than did ADP and was a partial agonist relative to ADP and 2MeSADP under conditions in which no metabolism of the nucleotide occurred. Both RGS4 and PLC-1 were potent and efficacious GTPase-activating proteins for G_q and G₁₁ in P2Y₁-R–containing vesicles. These results illustrate that the binding and signaling properties of the human P2Y₁-R can be studied with purified proteins under conditions that circumvent the complications that occur in vivo.

Although a burgeoning interest in the biology and therapeutic potential of extracellular nucleotide signaling now exists, the complex regulatory pathways that underlie the action of extracellular nucleotides remain difficult to study. Few truly selective agonists and antagonists are available for the P2 receptors, and studies with intact tissues are compromised both by nucleotide release and by metabolism and interconversion of P2 receptor agonists. In the face of these difficulties in the study of nucleotide signaling, little molecular insight into the mechanisms of action of P2 receptors has accrued. This holds particularly true with the P2Y receptors, which, with the exception of the P2Y₁-R of turkey erythrocyte membranes (Boyer et al., 1989, 1996b) and the P2Y₁₂-R on platelet membranes (Cooper and Rodbell, 1979; Kunapuli, 1998), have been investigated only in intact cell studies. As with other G protein-coupled receptors, the P2Y receptors function in a multiprotein cohort of receptor, heterotrimeric G protein, and effector enzyme or ion channel that transduces nucleotide-promoted signals across the plasma membrane. The recent discovery of a large class of regulators of G protein signaling (RGS) proteins that promote GTPase activities of heterotrimeric G proteins (Dohlman and Thorner, 1997; Ross and Wilkie, 2000; Hollinger and Hepler, 2002) adds a fourth component of complexity. Observation of activities of RGS proteins in addition to the promotion of GTP hydrolysis (Siderovski et al., 1999; Sierra et al., 2000) as well as the interaction of G protein-coupled receptors with other proteins (Brzostowski and Kimmel, 2001) suggests that traditional models of G protein-coupled receptor signaling are insufficient to describe fully hormone action. Indeed, G protein-coupled receptors may interact directly with proteins to produce signals that largely or entirely circumvent involvement of heterotrimeric G proteins.

We reasoned that problems inherent in the mechanistic study of the P2Y receptors can be circumvented in part by purifying these proteins to homogeneity and reconstituting them with their functional signaling cohorts in model vesicles. This task is made problematic because of the anticipated selectivity of coupling of the P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors to members of the Gq family of G protein subunits, which are difficult to purify and study. However, the elegant studies of Ross and coworkers (Berstein et al., 1992; Biddlecome et al., 1996; Mukhopadhyay and Ross, 1999) with purified M1 muscarinic receptors functionally reconstituted with G_q provide an encouraging model. As such, we generated an appropriately tagged construct of the P2Y₁-R and purified this protein to homogeneity after expression from a recombinant baculovirus in Sf9 insect cells. A radioligand binding assay was developed for the soluble, purified P2Y₁-R using a high-affinity radiolabeled antagonist, [³H]MRS2279, and this assay was applied to determine directly the affinities of ligands, including nucleotide agonists, for the purified receptor. The purified P2Y₁-R also was reconstituted functionally in phospholipid vesicles with either G_q or G₁₁ in heterotrimeric form with G₁₂. Activation kinetics of the receptor were quantified with these model vesicles to establish unambiguously both agonist activity and agonist selectivity for the P2Y₁-R.

	Materials and Methods

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Materials. Phosphatidylserine and phosphatidylethanolamine were obtained from Avanti Polar Lipids (Birmingham, AL), and cholesterol hemisuccinate was from Sigma (St. Louis, MO). Superdex 200, chelating Sepharose FF, and Mono-Q and HiTrap SP FPLC columns were from Amersham Biosciences Inc. (Piscataway, NJ). Ni-NTA agarose and Penta-His antibody were from QIAGEN (Valencia, CA). Anti-FLAG M1 monoclonal antibody was from Sigma. High-purity digitonin was obtained from Calbiochem (San Diego, CA), and Centricon YM-30 concentrators were from Millipore Corporation (Bedford, MA). Other reagents were from sources cited previously (Waldo et al., 2002).

Preparation of Membranes from P2Y₁-R–Expressing Sf9 Insect Cells. A recombinant baculovirus was constructed for expression of the P2Y₁-R to high levels as was described previously (Waldo et al., 2002). The recombinant virus includes the coding sequence of the human P2Y₁-R with a FLAG tag incorporated at the amino terminus and a hexahistidine tag at the carboxyl terminus. Preliminary experiments optimized cell density, multiplicity of infection of virus particles/cell, and time of incubation for optimal P2Y₁-R expression. Eight 500-ml spinner cultures (4 liters total) typically were grown to a density of 1.4 x 10⁶ Sf9 cells/ml before infection (multiplicity of infection = 2 virus particles/cell) with recombinant P2Y₁-R baculovirus. After incubation at 27°C for 48 h, the infected cells were collected by centrifugation for 10 min at 500g in a J6 centrifuge (Beckman Coulter, Inc., Fullerton, CA). All subsequent steps in the membrane preparation were performed at 4°C. The cell pellet was resuspended in cavitation buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 2 mM 2-mercaptoethanol, 100 µg/ml PMSF, 100 µM benzamidine, and 50 µg/ml TPCK), transferred to a Parr cavitation apparatus, and maintained under 600 psi nitrogen for 1 h with constant stirring. The cells were disrupted by rapid return to atmospheric pressure, and disrupted cells were collected after centrifugation for 30 min at 30,000g in a JA-14 rotor and J2-21 preparative centrifuge (Beckman Coulter). The pellet was collected, and the supernatant was centrifuged for 30 min at 41,600g in a JA-20 rotor (Beckman Coulter). Both pellets were combined and homogenized with a Dounce homogenizer in 200 ml of cavitation buffer. Nuclei and intact cells were removed by centrifugation in conical bottles for 10 min at 200g in a J6 centrifuge. The plasma membrane-containing supernatant was collected, and the nuclear pellet was washed twice by Dounce homogenization in 100 ml of cavitation buffer followed by centrifugation for 10 min at 200g in the J6 centrifuge. The supernatants from the two nuclear fraction washes were combined with the plasma membrane fraction and centrifuged for 30 min at 41,600g in a JA-20 rotor. The resultant pellet was resuspended in 100 ml of freezing buffer (20 mM Tris, pH 8.0, 250 mM sucrose, 2 mM 2-mercaptoethanol, 100 µg/ml PMSF, 100 µM benzamidine, and 50 µg/ml TPCK) by Dounce homogenization and stored at –80°C. Approximately 140 mg of plasma membrane protein was obtained per liter of infected cells.

Purification of P2Y₁-R. Plasma membranes (1.1 g of protein) prepared from 8 liters of P2Y₁-R–expressing cells were thawed and combined. The combined membrane fraction was centrifuged for 30 min at 100,000g at 4°C in a type 35 rotor and L8-70 ultracentrifuge (Beckman Coulter). The resultant pellet was resuspended by Dounce homogenization in 370 ml (3 mg membranes/ml) of extraction buffer (70 mM Na₂HPO₄, pH 8.0, 10% glycerol, 5 mM 2-mercaptoethanol, 1% digitonin, 20 mM imidazole, 500 mM NaCl, 100 µg/ml PMSF, 50 µg/ml TPCK, 100 µM benzamidine, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and stirred for 1 h at room temperature. The digitonin-extracted membranes were centrifuged for 1 h at 100,000g in a type 35 rotor (Beckman Coulter). The soluble P2Y₁-R fraction was incubated for 4 h at room temperature with 12 ml of chelating Sepharose FF charged previously with NiSO₄ according to the manufacturer's instructions. The P2Y₁-R–bound resin was transferred to a 2.5-cm diameter chromatography column and washed with 4 bed volumes of extraction buffer followed by 12 bed volumes of extraction buffer containing a lower concentration (0.1%) of digitonin. The recombinant P2Y₁-R was eluted with 36 ml of buffer containing 50 mM Na₂HPO₄, pH 7.4, 150 mM NaCl, 0.1% digitonin, 10% glycerol, 250 mM imidazole, 100 µg/ml PMSF, 50 µg/ml TPCK, 100 µM benzamidine, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. The P2Y₁-R–containing eluant was desalted on a 100-ml column of Sephadex G-50 equilibrated with buffer A (50 mM Na₂HPO₄, pH 7.4, 10% glycerol, 0.1% digitonin, 100 µg/ml PMSF, 50 µg/ml TPCK, 100 µM benzamidine, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The desalted P2Y₁-R–containing fraction was loaded onto a 1-ml HiTrap SP FPLC column and eluted with a 30-ml gradient from 0 to 400 mM NaCl in buffer A. P2Y₁-R–containing fractions eluting from the Hi-Trap SP column were concentrated to 2 ml by passage through a Centricon YM-30 membrane (Millipore) and applied to a Superdex 200 (1.6 x 66 cm, 133 ml) gel-filtration column equilibrated with 50 mM Na₂HPO₄, pH 7.4, 10 mM glycerol, 100 mM NaCl, and 0.1% digitonin. The P2Y₁-R–containing fraction eluting from the Superdex 200 column was concentrated to 0.6 ml by passage through a Centricon YM-30 membrane. The final yield was approximately 2 nmol of P2Y₁-R.

Purification of G_q,G₁₁,G₁₂, RGS4, and PLC-1. G_q and G₁₁ were purified according to the method described by Kozasa and Gilman (1996). Briefly, membranes expressing recombinant G₁, hexahistidine-tagged G₂, and either G_q or G₁₁ were prepared from Sf9 insect cells 48 h after infection with the corresponding recombinant baculoviruses. Heterotrimeric G protein was extracted from the Sf9 membranes with a buffer containing 1% sodium cholate. The soluble extract was diluted 4-fold with buffer containing 0.5% C₁₂E₁₀ before binding the soluble G protein heterotrimer to nickel nitrilotriacetic acid affinity resin via the hexahistidine-tagged G₂ subunit. The G subunit was eluted from the affinity matrix with a buffer containing 50 mM MgCl₂, 10 mM NaF, and 0.03 mM AlCl₃. The G subunit was further purified by Mono-Q FPLC ion-exchange chromatography as described elsewhere.

G₁₂ purification (Kozasa and Gilman, 1996) was performed with use of a strategy similar to that used for the purification of G_q and G₁₁, except that G₁ and G₂ were coexpressed in Sf9 cells with hexahistidine-tagged G_i1. RGS4 was purified after bacterial expression as described by Hepler et al. (1997). PLC-1 was purified from the cytosol of Sf9 insect cells after infection with a recombinant baculovirus as described previously (Paterson and Harden, 1996).

N-Glycosidase F Treatment of Purified P2Y₁-R and SDS-PAGE Analysis. Approximately 6 pmol of P2Y₁-R was incubated overnight at room temperature with or without 1000 U of peptide N-glycosidase (PNGase) F (New England BioLabs, Beverly, MA). After the overnight incubation SDS-PAGE sample buffer was added to each reaction and the proteins were separated on 12.5% SDS-PAGE gels. The proteins were either stained with Coomassie blue or transferred to nitrocellulose for immunoblot analysis with anti-FLAG M1 or anti-Penta-His monoclonal antibodies.

Radioligand Binding Assay of Soluble P2Y₁-R. Approximately 18 ng of purified P2Y₁-R was incubated with [³H]MRS2279 (Waldo et al., 2002) and buffer or the indicated concentration of competing drug for 60 min in an ice-water bath in a total assay volume of 50 µl. All subsequent steps were performed at 4°C. Saturation-binding isotherms were generated by incubation of soluble receptor with [³H]MRS2279 at concentrations of radioligand ranging from 0.5 to 60 nM. Specific binding was defined as total [³H]MRS2279 binding minus binding occurring in the presence of 10 µM MRS2179. Agonist and antagonist competition curves were generated by incubation of approximately 20,000 cpm (5 nM) of [³H]MRS2279 with increasing concentrations of competing drug. After the 60-min incubation, bound radioligand was separated from free radioligand by Sephadex G-50 spin columns. Briefly, 4 ml of Sephadex G-50 in Poly-Prep chromatography columns (0.8 x 4 cm) (Bio-Rad, Hercules, CA) was washed with 20 ml of wash buffer (20 mM Tris, pH 7.5, 145 mM NaCl, 1 mM EDTA, and 5 mM MgCl₂) and then was equilibrated with 4 ml of wash buffer containing 0.1% digitonin. The columns were centrifuged for 10 to 15 s at 750g in a JS-3.0 rotor (Beckman Coulter) before application of samples. Bound radioligand was isolated sequentially from each set of triplicate assays by transferring 40 µl of each reaction to a prespun G-50 column and immediately centrifuging the three columns for 5 min at 1775g in a JS-4.2 rotor (Beckman Coulter). Bound radioligand was collected directly into miniscintillation vials during the centrifugation. Scintillant was added, and radioactivity was quantified by liquid scintillation spectrometry. K_i values were calculated for the agonists and antagonists from the EC₅₀ values obtained in competition experiments using the average K_d value (3.8 nM) from three saturation-binding isotherms for [³H]MRS2279.

Reconstitution of P2Y₁-R into Proteoliposomes. Purified P2Y₁-R was co-reconstituted with either G_q or G₁₁ and G₁₂ into proteoliposomes by a modification of the method used by Brandt et al. (1983) and as described in detail by Cunningham et al. (2001). Briefly, 15 pmol of purified P2Y₁-R, 50 pmol of G, and 150 pmol of G₁₂ were reconstituted by Sephadex G-50 chromatography into phospholipid vesicles containing L--phosphatidylserine from brain, L--phosphatidylethanolamine from liver, and cholesteryl hemisuccinate.

Quantification of Steady-State GTP Hydrolysis. Steady-state GTPase activity of the reconstituted proteoliposomes was determined in the absence or presence of the indicated concentrations of RGS4 or PLC-1 and with or without the indicated concentrations of P2Y₁-R agonist or antagonist. Unless otherwise indicated, the assays (final volume, 50 µl) were incubated for 30 min at 30°C and contained 20 mM HEPES, pH 8.0, 50 mM NaCl, 2 mM MgCl₂, 1 mM EDTA, and 2 µM [-³²P]GTP (4500 cpm/pmol). The assays were terminated by the addition of 950 µlofa4°C solution of 5% activated charcoal in 20 mM H₃PO₄. After centrifugation, liberated [³²P]P_i in the supernatant was quantified by liquid scintillation spectrometry.

	Results

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With the goal of purifying the P2Y₁-R to homogeneity in a fully functional form, a recombinant baculovirus was constructed for high-density expression of the human P2Y₁-R in Sf9 insect cells. Conditions, including multiplicity of virus infection, time of incubation, and cell density, were optimized for the expression of immunoreactivity of the recombinant receptor (see Materials and Methods). Although both dodecylmaltoside and digitonin were effective in solubilizing a large percentage of the immunoreactivity from a membrane preparation of P2Y₁-R–expressing Sf9 cells, receptor signaling activity only was retained in the presence of digitonin. Therefore, the purification and all of the analyses of the human P2Y₁-R described below were carried out with receptor purified in the presence of digitonin.

We recently reported that [³H]MRS2279 is a reliable radioligand for quantification of both recombinant and natively expressed P2Y₁-R (Waldo et al., 2002). Much of our initial analyses of this radioligand were accomplished with membranes prepared from Sf9 cells expressing the recombinant human P2Y₁-R to high levels. Given the relatively high hydrophilicity of [³H]MRS2279 and the reliability of this radioligand for quantification of membrane P2Y₁-R, we examined whether [³H]MRS2279 binding might also be a useful means to directly assess the receptor in a soluble form. As was reported previously (Waldo et al., 2002), [³H]MRS2279 bound to the solubilized receptor with an affinity similar to that of the membrane receptor. The relative affinities of competing P2Y₁-R ligands determined with the soluble receptor (see below) also were similar to those obtained with the P2Y₁-R in its native membrane form. Thus, [³H]MRS2279 was used as a means to quantify active P2Y₁-R during purification.

The P2Y₁-R was expressed to high levels in Sf9 cells, membranes were prepared, and the receptor was purified as described in detail under Materials and Methods. The [³H]MRS2279 binding assay was applied to quantify soluble P2Y₁-R (Table 1), and approximately 45% of receptor binding activity solubilized from membranes was recovered in the final purification step. Approximately 2 nmol of [³H]MRS2279 binding sites was purified in a typical preparation from 8 liters of infected Sf9 cells, and approximately 1000-fold purification of [³H]MRS2279 binding activity was observed. Saturation-binding studies of the purified P2Y₁-R are considered below. Analyses of the purified receptor fraction by SDS-PAGE revealed a diffusely migrating protein species of approximately 44 kDa identified either by protein staining or by immunoblots with anti-FLAG or anti-His antiserum (Fig. 1). A much less abundant, slower migrating species also was observed at approximately 95 kDa with most receptor preparations. The amount of this 95-kDa species markedly increased after incubation of the purified receptor at room temperature or 37°C; incubation also increased the occurrence of both an immunoreactive species of approximately 130 kDa and an aggregated protein that did not enter the gel (Fig. 1). Thus, multimeric forms of the P2Y₁-R were barely detectable with the freshly purified receptor but could be induced to occur by maintaining the receptor preparation at elevated temperature.

View larger version (75K): [in this window] [in a new window]   Fig. 1. SDS-PAGE and immunoblot analysis of purified recombinant human P2Y1-R. Recombinant P2Y1-R tagged with His-6 and FLAG epitopes was purified from a digitonin extract of Sf9 insect cell plasma membranes, and N-glycan moieties were removed from the glycoprotein as described under Materials and Methods. Purified P2Y1-R incubated with PNGase F (lane 3), without PNGase F (lane 2), or untreated P2Y1-R (lane 1) were subjected to SDS-PAGE analysis. The resulting gels were stained with Coomassie blue (A) or transferred to nitrocellulose and immunoblotted with anti-FLAG antibody (B) or anti-Penta-His antibody (C). The PNGase enzyme is identified (P) in the Coomassie-stained gel.

Treatment of the purified P2Y₁-R preparation with N-glycosidase F resulted in a faster migrating receptor species presumably because of the removal of N-linked carbohydrate moieties (Fig. 1). Although the faster migrating species was somewhat less diffuse than the native P2Y₁-R, the appearance of heterogeneity remained. Variation in the temperature and time of incubation with glycosidase treatment did not produce a sharper P2Y₁-R species. Similarly, denaturation of the preparation with SDS and treatment with -mercaptoethanol before incubation with N-glycosidase F also failed to decrease the apparent heterogeneity. We are uncertain whether these observations follow from incomplete deglycosylation or the occurrence of additional covalent modifications (e.g., phosphorylation, palmitoylation) of the P2Y₁-R.

The purified P2Y₁-R bound [³H]MRS2279 with high affinity (K_d = 3.8 ± 0.3 nM) and by law of mass action-binding kinetics for ligand interaction with a single site (Fig. 2). The density of binding sites (approximately 3 nmol/mg protein) represents a stoichiometry of binding that is only 10 to 15% of theoretical binding to a single site of a protein with a molecular size of approximately 44,000. This observation was surprising given the apparent purity of the protein determined in protein-staining analyses (Fig. 1). Several factors probably contribute to the somewhat low binding stoichiometry, including the difficulty of accurately quantifying protein in relatively small samples and the somewhat cumbersome G-50 Sephadex centrifugation technique used to separate bound from free [³H]MRS2279, which allows the dissociation of significant amounts of specific radioligand binding (k₂ = 0.721 min^–1 in membrane filtration assay) (Waldo et al., 2002). Thus, the values obtained in the soluble binding assay underestimate the true B_max values. Extrapolation of a first-order rate plot (data not shown) of the dissociation of radioligand– P2Y₁-R complexes that occurs after the addition of reactants to the G-50 column predicts that the specific activity of [³H]MRS2279 binding to the purified P2Y₁-R is at least 5.5 nmol/mg (approximately 25% of theoretical). Moreover, because the purification scheme used here resulted in the recovery of up to 50% of the P2Y₁-R present in the detergent extract (Table 1), we believe that a significant amount of the P2Y₁-R present in the starting membrane preparation represents P2Y₁-R protein that lacks functional binding activity. That is, significant loss of binding activity does not occur during purification, and protein-stained gels indicate >90% purity of the P2Y₁-R protein.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Saturation-binding isotherm for [3H]MRS2279 binding to purified P2Y1-R. P2Y1-Rs were purified from a digitonin extract of P2Y1 Sf9 membranes as described under Materials and Methods. Approximately 18 ng of purified P2Y1-R was incubated with the indicated concentrations (0.5–60 nM) of [3H]MRS2279 for 60 min at 4°C. Bound radioligand was separated from free by Sephadex G-50 chromatography. Data are plotted as specific binding and are representative of three similar experiments. A Scatchard transformation of the data is presented in the inset.

The pharmacological properties of the P2Y₁-R have been widely studied. However, consensus conclusions on the relative potencies and efficacies of agonists at the P2Y₁-R have not been attained despite rigorous attempts to do so by many laboratories. This deficit has followed from the propensity of most of these nucleotide agonists to undergo breakdown and/or interconversion. The recent elucidation of a direct binding assay using a high-affinity non-nucleotide antagonist of the P2Y₁-R has for the first time provided a means to directly determine ligand affinities for the P2Y₁-R (Waldo et al., 2002). However, assessment with [³H]MRS2279 of binding affinities of nucleotide agonists using membranes expressing recombinant P2Y₁-R to high levels is still compromised by nucleotide metabolism, as we reported previously (Waldo et al., 2002). In contrast, application of the [³H]MRS2279 binding assay to P2Y₁-R purified to homogeneity by definition circumvents any problems contributed by enzymatic metabolism of nucleotides. Therefore, we carried out detailed radioligand binding analyses with the purified human P2Y₁-R. Competition binding isotherms were generated for nucleotides and nucleotide analogs known to interact with the P2Y₁-R (Fig. 3), and K_i values of these analogs for the P2Y₁-R were determined. It should be pointed out that, because these binding affinities were determined with purified receptor in the absence of heterotrimeric G_q, they represent affinities for the uncoupled low-affinity agonist binding state of the P2Y₁-R. The Hill slopes of all competition curves were near unity (data not shown). The nucleotide analog 2MeADP exhibited the lowest K_i, and ADP, the cognate agonist of the receptor, exhibited approximately 10-fold lower affinity than did 2MeSADP. ATP, which has been concluded to be an agonist (Webb et al., 1993; Filtz et al., 1994; Henderson et al., 1995; Schachter et al., 1996), partial agonist (Palmer et al., 1998), or antagonist (Leon et al., 1997; Hechler et al., 1998) of the P2Y₁-R, exhibited a K_i value (18 µM) approximately 20-fold higher than that of ADP. K_i values of 1 to 2 µM were observed for ADPS, 2MeSATP, and ATPS (Table 2). The molecule adenosine 3',5'-bisphosphate, originally identified (Boyer et al., 1996a) as a selective bisphosphate antagonist of the P2Y₁-R, exhibited a K_i value of approximately 1 µM (Table 1), whereas the K_i values of MRS2179 and MRS2279, which are bisphosphate analogs synthesized (Boyer et al., 1998, 2002; Camaioni et al., 1998; Nandanan et al., 2000) to enhance affinity for the P2Y₁-R, were 15 and 11 nM, respectively. These K_i values determined with the purified P2Y₁-R are in good agreement with the respective K_i values determined in binding assays with membranes and with respective K_B values determined in studies of antagonism of 2MeSADP-stimulated responses in intact cells.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Pharmacological selectivity of 2MeSADP, ADP, and ATP for the inhibition of [3H]MRS2279 binding to purified P2Y1-R. Approximately 18 ng of purified P2Y1-R was incubated for 60 min at 4°C with 5.4 nM [3H]MRS2279 and the indicated concentrations of the P2Y1-R nucleotide ligands 2MeSADP (), ADP (), and ATP (). Bound radioligand was separated from free by Sephadex G-50 chromatography. Data are representative of five similar experiments. Ki values are indicated.

These direct binding studies with the purified human P2Y₁-R provide the first unequivocal determination of binding affinities of ligands for this receptor. They do not, however, indicate whether the purified P2Y₁-R retains signaling activity, and they also do not provide assessment of the relative agonist activities of nucleotides and nucleotide analogs at the purified receptor. As such, a model phospholipid-vesicle system was developed in which the purified P2Y₁-R was reconstituted with various purified G subunits and purified G₁₂, as described under Materials and Methods. GTPase activity of the reconstituted G subunits was quantified by measuring the release of [³²P]P_i from [-³²P]GTP, and potential agonist activity of P2Y₁-R ligands was assessed by measuring the concentration-dependent capacity of these molecules to increase GTPase activity of the reconstituted G subunits. Addition of the P2Y₁-R agonist 2MeSADP to proteoliposomes reconstituted with purified P2Y₁-R, G₁₁, and G₁₂ resulted in a small but reproducible increase in GTPase activity (Fig. 4). Addition of the RGS protein, RGS4, to these vesicles in the absence of a P2Y₁-R agonist also elicited a small increase in GTPase activity. Guanine nucleotide exchange is rate-limiting in the catalytic cycle of heterotrimeric G proteins. Therefore, coaddition of 2MeSADP to promote guanine nucleotide exchange and RGS4 to activate the GTPase of G₁₁ resulted in GTP hydrolysis at rates that were up to 50-fold greater than those observed with either agonist or RGS protein alone (Fig. 4). GTP hydrolysis was linear for up to 45 min, and therefore, steady-state GTPase activity was measured under the conditions of these assays. The rate of GTP hydrolysis also was linearly dependent on the amount of proteoliposomes in the assay (data not shown). The purified P2Y₁-R also functionally coupled to G_q12, as is illustrated below. In contrast, relatively small effects of 2MeSADP and RGS4 were observed in vesicles reconstituted with purified P2Y₁-R and G₀, G_i1, G_i2, or G_i3 in heterotrimeric form with G₁₂ (data not shown). Under these same conditions, marked agonist and RGS4-stimulated GTP hydrolysis was observed in proteoliposomes reconstituted with M2 muscarinic receptors and G₀₁₂, G_i112, G_i212, and G_i312 (data not shown). Thus, as is suggested from studies with intact cell systems, the P2Y₁-R preferentially couples to G_q rather than to G subunits of the G_i family.

View larger version (21K): [in this window] [in a new window]   Fig. 4. 2MeSADP-promoted steady state GTP hydrolysis by P2Y1-R/G11 proteoliposomes. Purified human P2Y1-R, G11, and G12 were reconstituted in phospholipid vesicles. GTP hydrolysis was measured at 30°C for the indicated times in the presence of 100 nM RGS4 without proteoliposomes (), in the presence of proteoliposomes without RGS4 (), or in the presence of 100 nM RGS4 and proteoliposomes in the absence () or presence () of 1 µM 2MeSADP.

The responses illustrated in Fig. 4 suggested that this reconstitution system can be used to determine pharmacological properties of the P2Y₁-R by varying agonist and antagonist concentrations in the presence of a fixed concentration of RGS protein. Pharmacological studies are demonstrated with P2Y₁-R/G₁₁/G₁₂ vesicles in Fig. 5. In the absence of added GTPase-activating protein, 2MeSADP elicited a small but significant concentration-dependent increase in steady-state GTPase activity (Fig. 5A). However, in the presence of 100 nM RGS4, a marked concentration of 2MeSADP-dependent stimulation of GTP hydrolysis was observed. As illustrated in Fig. 5B, the P2Y₁-R antagonist MRS2279 inhibited the capacity of 1 µM 2MeSADP to stimulate GTPase with an IC₅₀ value consistent with the affinity of MRS2279 for binding to the human P2Y₁-R.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Concentration-dependence of agonist and antagonist activities measured with purified P2Y1-R reconstituted in proteoliposomes. Purified P2Y1-R, G11, and G12 were reconstituted in proteoliposomes. A, steady-state GTP hydrolysis was measured in proteoliposomes incubated in the absence () or presence () of 100 nM RGS4 and the indicated concentration of the P2Y1-R agonist 2MeSADP. B, steady-state GTP hydrolysis was measured in proteoliposomes incubated with 100 nM RGS4 and the indicated concentrations of the P2Y1-R antagonist MRS2279 with () or without () 1 µM 2MeSADP.

This reconstitution system using purified proteins provides a well-controlled system that circumvents many of the difficulties inherent in studying activities of P2Y receptors under conditions in which significant hydrolysis and intercoversion of added nucleotides and/or release of cellular nucleotides occurs. Given that the purified receptor exhibited signature responses to an agonist and antagonist of the P2Y₁-R (Fig. 5, A and B), we generated a series of concentration-effect curves for activation of the purified receptor (Fig. 6). These studies were carried out with vesicles reconstituted with purified recombinant G_q rather than with the very similar G subunit G₁₁, which was used in the experiments presented in Figs. 4 and 5. The maximal effects of 2MeSADP and 2Me-SATP on GTPase activity were equivalent, and the potency of 2MeSADP (EC₅₀ = 150 nM) was approximately 20-fold greater than that of 2MeSATP (EC₅₀ = 3 µM). The presence of 2MeSADP in the preparation of 2MeSATP apparently did not contribute to its activity because HPLC purification of commercial 2MeSATP just before its use did not result in a significant change in its activity (Fig. 6A). The maximal effect observed with ADP was similar to that observed with 2MeSADP, and ADP exhibited an EC₅₀ value of approximately 1 µM (Fig. 6B). Thus, 2MeSADP, 2MeSATP, and ADP are all full agonists at the purified P2Y₁-R. ADPS and ATPS were less effective agonists than 2MeSADP and ADP for activation of the purified P2Y₁-R, and both ADPS and ATPS seemed to be partial agonists. UTP, UDP, MeATP, and MeATP all were inactive at the purified P2Y₁-R (Fig. 6C).

View larger version (29K): [in this window] [in a new window]   Fig. 6. Agonist selectivity of the purified P2Y1-R. Purified P2Y1-R, Gq, and G12 were reconstituted in proteoliposomes. A and B, Steady-state GTP hydrolysis was measured in proteoliposomes incubated with 100 nM RGS4 and the indicated concentration of P2Y1-R agonist: A, commercial 2MeSATP (), HPLC-purified 2MeSATP (), and 2MeSADP (); B, 2MeSADP (), ADP (), ADPS (), ATPS (), and ATP (). C, Steady-state GTP hydrolysis was measured in proteoliposomes incubated with no drug, with 10 µM 2MeSADP, or with 100 µM of the indicated drugs.

Whether ATP is an agonist or an antagonist at the P2Y₁-R is an important question in understanding the biological regulation of this receptor. This issue has not been entirely resolved, despite rigorous studies from a number of laboratories (Hechler et al., 1998, Palmer et al., 1998). Availability of purified P2Y₁-R reconstituted with purified G_q allowed us to assess the action of ATP under conditions in which no conversion of ATP to ADP occurs. ATP exhibited an efficacy for stimulation of GTPase activity that was approximately 30% of that of the full P2Y₁-R agonists 2MeSADP, 2MeSATP, and ADP (Figs. 6B and 7A). The partial agonist activity of ATP was further confirmed by illustrating that over the range of ATP concentrations that stimulated GTPase activity, inhibition of the stimulatory activity of nearly maximally effective concentrations of 2MeSADP also occurred (Fig. 7B). Metabolism of [-³²P]ATP was simultaneously measured under the conditions of the assays presented in Fig. 7, A and B, to ascertain whether the formation of ADP from ATP accounted for any of the activity of ATP. Only 0.07% of the ATP was hydrolyzed in a 30-min incubation under these conditions (Table 3). This level of metabolism would not result in a P2Y₁-R–activating concentration of ADP, even assuming that all of the conversion was to ADP alone and that this ADP was present during an entire 30-min test of agonist activity. Thus, we conclude that ATP is a relatively weak partial agonist at the P2Y₁-R.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Concentration-dependence of ATP as a partial agonist at the P2Y1-R. Purified P2Y1-R, Gq, and G12 were reconstituted in proteoliposomes. A, steady-state GTP hydrolysis was measured in proteoliposomes incubated with (, ) or without (, ) 100 nM RGS4 and with the indicated concentrations of 2Me-SADP (, ) or ATP (,). B, Steady-state GTP hydrolysis was measured in proteoliposomes incubated with the indicated concentrations of ATP in the presence (, ) or absence (, ) of 100 nM RGS4 and with (, ) or without (, ) 1 µM 2MeSADP.

View this table: [in this window] [in a new window]   TABLE 3 Metabolism of [32P]ATP under GTPase assay conditions ATP hydrolysis was determined by measuring the release of 32P from [-32P]ATP after incubation at 30° C for the indicated times in the presence or absence of P2Y1/Gq/12 proteoliposomes, 100 nM RGS4, and 10 µM 2MeSADP as indicated. The assay conditions were identical with those used in Fig. 7 except that [-32P]ATP was substituted for [-32P]GTP.

In the presence of a P2Y₁-R agonist, guanine nucleotide exchange is no longer rate-limiting, and the capacity of various GTPase activating proteins to stimulate GTP hydrolysis by G_q can be assessed. Thus, we also applied vesicles reconstituted with purified P2Y₁-R and heterotrimeric G_q or G₁₁ to assess the action of proteins known to be GAPs of G_q family G subunits. As illustrated in Fig. 8, both RGS4 and PLC-1 caused a concentration-dependent stimulation of the GTPase activity of G_q and G₁₁. The maximal activity observed with RGS4 was slightly greater than that of PLC-1, whereas PLC-1 was consistently slightly more potent than RGS4 under the conditions of these assays. G_q and G₁₁ exhibit approximately 90% sequence identity, and these ubiquitously expressed G subunits exhibit completely interchangeable activities in a variety of biological test conditions. As illustrated in Fig. 8, the concentration-dependence for stimulation of GTPase activities by RGS4 and of PLC-1 also were essentially identical whether G_q- or G₁₁-containing vesicles were studied.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Concentration-dependence of RGS4 and phospholipase C-1 for promoting 2MeSADP-stimulated GTP hydrolysis in Gq and G11 proteoliposomes. P2Y1-Rs were reconstituted with G12 and Gq (left) or G11 (right). Steady-state GTP hydrolysis was measured in the presence of 10 µM 2MeSADP and the indicated concentrations of PLC-1 () or RGS4 ().

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
High-level expression of a hexahistidine-tagged P2Y₁-R in Sf9 insect cells provided technological advantages for purifying the human P2Y₁-R. When reconstituted with its protein-signaling cohorts in model phospholipid vesicles, the purified receptor exhibited signaling and pharmacological characteristics reminiscent of those predicted from studies of the native P2Y₁-R observed in a variety of target tissues. Thus, recombinant P2Y₁-R can be obtained in a nearly homogeneous form that retains activity in its agonist-binding site and in the domain(s) that functionally interacts with G_q. Approximately 25% of the purified P2Y₁-R is active as assessed by radioligand binding, which is similar to that reported for epitope-tagged ₂ adrenergic receptor (30% of theoretical) after purification to homogeneity and before a final ligand affinity-purification step (Kobilka, 1995). This model system allowed the unambiguous illustration of the overall agonist selectivity of the P2Y₁-R and the partial agonist activity of ATP at the P2Y₁-R. P2Y₁-R–containing proteoliposomes also were used to illustrate that the interaction of the purified receptor with downstream regulatory proteins RGS4 and PLC-1 could be studied and to establish that neither RGS4 nor PLC-1 exhibits selectivity as GTPase-activating proteins for G_q versus G₁₁.

The affinities of agonists for the P2Y₁-R have not been known because reliable means for their determination have not been available. Tissues contain high levels of nucleotidemetabolizing enzymes, and we found that expression of recombinant P2Y₁-R to very high levels still does not allow sufficiently low membrane concentrations in biochemical assays to overcome nucleotide metabolism (Waldo et al., 2002). Drugs of sufficient selectivity and affinity also are not available to effect pharmacological blockade of endogenous nucleotide metabolism. The development of [³H]MRS2279 as an antagonist radioligand for the P2Y₁-R provided the first reliable means to quantify directly the interaction of ligands with membrane P2Y₁-R. As we described recently in detail, this methodology alone did not circumvent the aforementioned problems encountered in determining agonist affinity at membrane P2Y₁-R (Waldo et al., 2002). However, the [³H]MRS2279 binding assay now applied to studies of the human P2Y₁-R purified to homogeneity have circumvented these concerns. Thus, we directly measured the binding of molecules to the P2Y₁-R under conditions in which molecules are not metabolized or interconverted to other nucleotides. Although the general consensus has been that the P2Y₁-R is preferentially activated by ADP, both the affinity and agonist activity of ATP relative to ADP have been questions of considerable disagreement. We show here by direct and unambiguous means that the affinity of ADP for the human P2Y₁-R is approximately 20-fold greater than that of ATP.

Cell signaling through heterotrimeric G proteins is ultimately governed by the rate of exchange of guanine nucleotides and the rate of hydrolysis of GTP by the involved G protein subunit (Gilman, 1987). Thus, in conditions in which multiple rounds of nucleotide hydrolysis occur, GT-Pase activity is regulated through the capacity of receptors to act as guanine nucleotide exchange factors and through the activity of various classes of proteins to increase the k_catalysis of the GTPase activity of the involved G protein subunit. We took advantage of the rate-limiting nature of both of these steps in the catalytic cycle of G proteins to analyze the concentration-dependence of agonists at the P2Y₁-R for enhancing GTPase activity in the presence of a saturating concentration of a GAP, and the concentration-dependence of GAPs, e.g., PLC-1 or RGS4, for enhancing GTPase activity in the presence of a maximally effective concentration of a P2Y₁-R agonist. Our results indicate that under the conditions of these assays, the level of agonist-stimulated GTPase activity observed is proportional to the amount of reconstituted P2Y₁-R. Thus, the relative potencies and maximal effects of agonists observed under these conditions are related to their relative affinities and intrinsic efficacies for the P2Y₁-R. Similarly, in the presence of a maximally effective concentration of agonist, relative affinities and maximal activities for GAPs can be compared for P2Y₁-R–activated G protein subunits.

Studies designed to minimize the confounding contributions of nucleotide release, metabolism, and interconversion have led to the view that ADP is the preferred natural agonist for the P2Y₁-R, with ATP acting either as a partial agonist or antagonist, possibly depending on the level of expression of P2Y₁-R in the test system (Hechler et al., 1998; Palmer et al., 1998; Simon et al., 2001). This view is strongly supported by the results of the current study with a functional system reconstituted from purified components. Negligible hydrolysis of ATP to ADP occurred under conditions in which ATP was illustrated to be a partial agonist exhibiting an EC₅₀ approximately 20-fold greater than that of ADP. In contrast, 2MeSATP was a full agonist exhibiting a potency approximately 20-fold less than 2MeSADP and similar to that of ADP. Thus, we conclude that ADP is the cognate agonist for the P2Y₁-R and that the much lower efficacy and apparent affinity of ATP are both consistent with the idea that this nucleotide is not of major importance as a physiological regulator of the P2Y₁-R.

A number of studies using transient and stable heterologous expression of the recombinant human P2Y₁-R in mammalian cell lines have concluded that the P2Y₁-R is predominantly G_q-coupled (Filtz et al., 1994; Schachter et al., 1996, 1997). However, for many years, the unrealized presence of two different ADP-activated receptors in platelets resulted in uncertainty over the nature of the involved receptor(s) and signaling responses that accounted for the aggregation response to ADP. Furthermore, the identification of the P2Y₁-R in platelets before the unambiguous identification of the G_i/adenylyl cyclase-linked P2Y₁₂-R led to uncertainties over whether the P2Y₁-R coupled to both G_q/phospholipase C and G_i/adenylyl cyclase. Indeed, the eventual identification of the P2Y₁₂-R receptor gene (Zhang et al., 2000; Hollopeter et al., 2001) and the G_i-activating/adenylyl cyclase-inhibiting activity of this receptor does not formally prove that the P2Y₁-R couples only to G_q rather than to both G_q and G_i in the platelet or other tissues. However, the purified P2Y₁₂-R apparently couples to three different G_i subunits but not to G_q (Bodor et al., 2003), and the purified P2Y₁-R studied here primarily couples to G_q, supporting the view that the two ADP-activated receptors of platelets signal in a relatively linear manner down a G_q-mediated pathway, i.e., the P2Y₁-R, or G_i-mediated pathway, i.e., the P2Y₁₂-R.

G_q and G₁₁ exhibit approximately 90% amino acid identity and, with the exception of the mouse platelet (Offermanns et al., 1997), seem to be redundantly expressed in all tissues studied. These two ubiquitously expressed signaling proteins also have exhibited essentially identical signaling functions in all processes studied to date. Thus, a generally consensus view has been that these G subunits are interchangeable in most if not all of their actions. Ross and coworkers first observed GAP activity of an effector protein for heterotrimeric G proteins in their studies with phospholipase C-1 (Berstein et al., 1992). Thus, the capacity of a purified fraction containing both G_q and G₁₁ to hydrolyze GTP was markedly increased by PLC-1 in a concentration-of-enzyme–dependent manner. Subsequent work elegantly elucidated the kinetics of agonist-promoted guanine nucleotide exchange and enhancement of GTPase activity by PLC-1 in proteoliposomes reconstituted with M1 muscarinic receptors and heterotrimeric G_q (Biddlecome et al., 1996, Mukhopadhyay and Ross, 1999). As was illustrated here, the interaction of the purified P2Y₁-R with downstream signaling components can be studied in proteoliposomes with purified proteins, and both RGS4 and PLC-1 stimulated GTPase activities of G_q versus G₁₁ with equal effectiveness.

In summary, the data presented in this study illustrate that the human P2Y₁-R can be purified to near homogeneity in active form and that downstream signaling components can be reconstituted in a functional proteoliposome system with the purified P2Y₁-R. This model system should be useful in obtaining further molecular insight into this important signaling protein.

	Acknowledgements

 
We acknowledge the contribution of Robert Nicholas and R. Kyle Palmer in the construction of the P2Y₁-R baculovirus, Vijay Krishnamurthy for purification of G_q, Jose Boyer and Jay Douglass for HPLC-purified 2MeSATP, and David Rinker for assisting with the manuscript.

	Footnotes

 
This work was supported by National Institutes of Health grants GM29536, GM38213, and HL34322.

ABBREVIATIONS: P2Y₁-R, P2Y₁ receptor; P2Y₁₂-R, P2Y₁₂ receptor; PLC, phospholipase C; GAP, GTPase activating protein; MRS2279, 2-chloro-N⁶-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bis-phosphate; RGS, regulator of G-protein signaling; 2MeSADP, 2-methylthioADP; 2MeSATP, 2-methylthio-ATP; ADPS, adenosine-5'-O-(2-thiodiphosphate); ATPS, adenosine-5'-O-(3-thio)triphosphate; FPLC, fast-performance liquid chromatography; HPLC, high-performance liquid chromatography; PMSF, phenylmethylsulfonyl fluoride; TPCK, N-tosyl-Lphenylalanine chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis; PNGase, peptide N-glycosidase; MRS2179, N⁶-methyl-2'deoxyadenosine 3',5'-bisphosphate.

Address correspondence to: Dr. T. Kendall Harden, Department of Pharmacology, CB#7365, University of North Carolina, Chapel Hill, Chapel Hill, NC 27599-7365. E-mail: tkh{at}med.unc.edu

	References

Top Abstract Materials and Methods Results Discussion References

 
Berstein G, Blank JL, Jhon D-Y, Exton JH, Rhee SG, and Ross EM (1992) Phospholipase C-1 is a GTPase-activating protein for G_q/11, its physiologic regulator. Cell 70: 411–418.[CrossRef][Medline]

Biddlecome GH, Bernstein G, and Ross EM (1996) Regulation of phospholipase C-1 by Gq and m1 muscarinic cholinergic receptor. Steady-state balance of receptor-mediated activation and GTPase-activating protein-promoted deactivation. J Biol Chem 271: 7999–8007.[Abstract/Free Full Text]

Bodor ET, Waldo GL, Hooks SB, Corbitt J, Boyer JL, and Harden TK (2003) Purification and functional reconstitution of the human P2Y12 receptor. Mol Pharmacol 64: 1210–1216.[Abstract/Free Full Text]

Boyer JL, Adams M, Ravi RG, Jacobson KA, and Harden TK (2002) 2-Chloro-N⁶-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate is a selective high affinity P2Y₁ receptor antagonist. Br J Pharmacol 135: 2004–2010.[CrossRef][Medline]

Boyer JL, Downes CP, and Harden TK (1989) Kinetics of activation of phospholipase C by P₂ purinergic receptor agonists and guanine nucleotides. J Biol Chem 264: 884–890.[Abstract/Free Full Text]

Boyer JL, Mohanram A, Camaioni E, Jacobson KA, and Harden TK (1998) Competitive and selective antagonism of P2Y₁ receptors by N⁶-methyl 2'-deoxyadenosine 3',5'-bisphosphate. Br J Pharmacol 124: 1–3.[CrossRef][Medline]

Boyer JL, Romero T, Schachter JB, and Harden TK (1996a) Identification of competitive antagonists of the P2Y₁ receptor. Mol Pharmacol 50: 1323–1329.[Abstract]

Boyer JL, Schachter JB, Sromek SM, Palmer RK, Jacobson KA, Nicholas RA, and Harden TK (1996b) Avian and human homologues of the P2Y₁ receptor: pharmacological, signaling and molecular properties. Drug Dev Res 39: 253–261.[CrossRef]

Brandt DR, Asano T, Pedersen SE, and Ross EM (1983) Reconstitution of catecholamine-stimulated guanosinetriphosphatase activity. Biochemistry 22: 4357–4362.[CrossRef][Medline]

Brzostowski JA and Kimmel AR (2001) Signaling at zero G: G-protein-independent functions for 7-TM receptors. Trends Biochem Sci 26: 291–297.[CrossRef][Medline]

Burnstock G (1972) Purinergic nerves. Pharmacol Rev 24: 509–581.[Free Full Text]

Camaioni E, Boyer JL, Mohanram A, Harden TK, and Jacobson KA (1998) Deoxyadenosine-bisphosphate derivatives as potent antagonists at P2Y₁ receptors. J Med Chem 41: 183–190.[CrossRef][Medline]

Cooper DMF and Rodbell M (1979) ADP is a potent inhibitor of human platelet plasma membrane adenylate cyclase. Nature (Lond) 282: 517–518.[CrossRef][Medline]

Cunningham ML, Waldo GL, Hollinger S, Hepler JR, and Harden TK (2001) Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regulation of G11 signaling. J Biol Chem 276: 5438–5444.[Abstract/Free Full Text]

Dohlman HG and Thorner J (1997) RGS proteins and signaling by heterotrimeric G proteins. J Biol Chem 272: 3871–3874.[Free Full Text]

Dubyak GR and El-Moatassim C (1993) Signal transduction via P₂-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol 265: C577–C606.

Filtz TM, Li Q, Boyer JL, Nicholas RA, and Harden TK (1994) Expression of a cloned P_2Y-purinergic receptor that couples to phospholipase C. Mol Pharmacol 46: 8–14.[Abstract]

Gilman AG (1987) G proteins: transducers of receptor-generated signals. Annu Rev Biochem 56: 615–649.[CrossRef][Medline]

Harden TK, Barnard EA, Boeynaems JM, Burnstock G, Dubyak GR, Hourani SMO, and Insel PA (1998) P2Y receptors, in The IUPHAR Compendium of Receptor Characterization and Classification (Girdlestone D ed) pp 209–217, IUPHAR Media, London.

Harden TK, Boyer JL, and Nicholas RA (1995) P₂-purinergic receptors: subtype-associated signaling responses and structure. Annu Rev Pharmacol Toxicol 35: 541–579.[CrossRef][Medline]

Hechler B, Vigne P, Leon C, Breittmayer JP, and Gachet C (1998) ATP derivatives are antagonists of the P2Y1 receptor: similarities to the platelet ADP receptor. Mol Pharmacol 53: 727–733.[Abstract/Free Full Text]

Henderson DJ, Elliot DG, Smith GM, Webb TE, and Dainty IA (1995) Cloning and characterization of a bovine P_2Y receptor. Biochem Biophys Res Commun 212: 648–656.[CrossRef][Medline]

Hepler JR, Berman DM, Gilman AG, and Kozasa TR (1997) RGS4 and GAIP are GTPase-activating proteins for G_q and block activation of phospholipase C by -thio-GTP-G_q. Proc Natl Acad Sci USA 94: 428–432.[Abstract/Free Full Text]

Hollinger S and Hepler JR (2002) Cellular regulation of RGS proteins: modulators and integrators of G protein signaling. Pharmacol Rev 54: 527–529.[Abstract/Free Full Text]

Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden P, Nurden A, Julius D, et al. (2001) Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature (Lond) 409: 202–207.[CrossRef][Medline]

Khakh BS, Barnard EA, Burnstock G, Kennedy C, King BF, North RA, Seguela P, Voigt M, and Humphrey PPA (2000) P2X receptors, in The IUPHAR Compendium of Receptor Characterization and Classification (Girdlestone D ed) pp 291–305, IUPHAR Media, London.

Kobilka BK (1995) Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal Biochem 231: 269–271.[CrossRef][Medline]

Kozasa T and Gilman AG (1996) Protein kinase C phosphorylates G₁₂ and inhibits its interaction with G. J Biol Chem 271: 12562–12567.[Abstract/Free Full Text]

Kunapuli SP (1998) Multiple P2 receptor subtypes on platelets: a new interpretation of their function. Trends Pharmacol Sci 19: 391–394.[CrossRef][Medline]

Lazarowski E, Boucher RC, and Harden TK (2003) Mechanisms of release of nucleotides and integration of their action as P2X- and P2Y-receptor activating molecules. Mol Pharmacol 64: 785–795.[Free Full Text]

Leon C, Hechler B, Vial C, Leray C, Cazenave J-P, and Gachet C (1997) The P2Y₁ receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells. FEBS Lett 403: 26–30.[CrossRef][Medline]

Mukhopadhyay S and Ross EM (1999) Rapid GTP binding and hydrolysis by G(q) promoted by receptor and GTPase-activating proteins. Proc Natl Acad Sci USA 96: 9539–9544.[Abstract/Free Full Text]

Nandanan E, Jang SY, Moro S, Kim HO, Siddiqui MA, Russ P, Marquez VE, Busson R, Herdewijn P, Harden TK, et al. (2000) Synthesis, biological activity and molecular modeling of ribose-modified deoxyadenosine bisphosphate analogues as P2Y₁ receptor ligands. J Med Chem 43: 829–842.[CrossRef][Medline]

Offermanns S, Toombs CF, Hu YH, and Simon MI (1997) Defective platelet activation in G alpha(q)-deficient mice. Nature (Lond) 389: 183–186.[CrossRef][Medline]

Palmer RK, Boyer JL, Schachter JB, Nicholas RA, and Harden TK (1998) Agonist action of adenosine triphosphates at the human P2Y₁ receptor. Mol Pharmacol 54: 1118–1123.[Abstract/Free Full Text]

Paterson A and Harden TK (1996) Expression, purification and reconstitution of recombinant phospholipase C- isoenzymes. Methods Neurosci 29: 246–263.

Ralevic V and Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50: 413–492.[Abstract/Free Full Text]

Ross EM and Wilkie TM (2000) GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu Rev Biochem 69: 795–827.[CrossRef][Medline]

Schachter JB, Boyer JL, Li Q, Nicholas RA, and Harden TK (1997) Fidelity in functional coupling of the rat P2Y₁ receptor to phospholipase C. Br J Pharmacol 122: 1021–1024.[CrossRef][Medline]

Schachter JB, Li Q, Boyer JL, Nicholas RA, and Harden TK (1996) Second messenger cascade specificity and pharmacological selectivity of the human P_2Y1 receptor. Br J Pharmacol 118: 167–173.[Medline]

Siderovski DP, Strockbine B, and Behe CI (1999) Whither goest the RGS proteins? Crit Rev Biochem Mol Biol 34: 215–251.[CrossRef][Medline]

Sierra DA, Popov S, and Wilkie TM (2000) Regulators of G-protein signaling in receptor complexes. Trends Cardiovasc Med 10: 263–268.[CrossRef][Medline]

Simon J, Vigne P, Eklund KM, Michel AD, Carruthers AM, Humphrey PP, Frelin C, and Barnard EA (2001) Activity of adenosine diphosphates and triphosphates on a P2Y(T)-type receptor in brain capillary endothelial cells. Br J Pharmacol 132: 173–182.[CrossRef][Medline]

Waldo GL, Corbitt J, Boyer JL, Ravi G, Kim HS, Ji X, Lacy J, Jacobson KA, and Harden TK (2002) Quantitation of the P2Y1 receptor with a high affinity radiolabeled antagonist. Mol Pharmacol 62: 1249–1257.[Abstract/Free Full Text]

Webb TE, Simon J, Krishek BJ, Bateson AN, Smart TG, King BF, Burnstock G, and Barnard EA (1993) Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Lett 324: 219–225.[CrossRef][Medline]

Zhang FL, Luo L, Gustafson E, Lachowicz J, Smith M, Qiao X, Liu YH, Chen G, Pramanik B, Laz TM, et al. (2000) ADP is the cognate ligand for the orphan G-protein coupled receptor SP1999. J Biol Chem 276: 8608–8615.

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