1Department of Physiology, 2Department of Pharmacology, and 3The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania
Submitted 16 August 2004 ; accepted in final form 20 October 2004
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ABSTRACT |
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carboxyl terminus; adenosine diphosphate; truncation; inositol phosphate
The human P2Y1 receptor was originally cloned from human erythroleukemia cells (4) and endothelial cells (37). The human P2Y1 receptor is 373 amino acids long with 7 putative hydrophobic transmembrane regions and is encoded by a single exon (3). The gene for the human P2Y1 receptor has been localized to chromosome 3q25 (3). When heterologously expressed in astrocytoma cells, the P2Y1 receptor has been shown to activate PLC through pertussis toxin-insensitive G proteins of the Gq/11 class, resulting in inositol phosphate formation (44). Studies with mice lacking Gq revealed that signaling through Gq is essential for ADP-induced functional responses in platelets (40).
ADP and 2-methylthio-ADP (2-MeSADP) are potent agonists at the P2Y1 receptor, whereas adenosine 3'-phosphate 5'-phosphosulfate, adenosine 3'-phosphate 5'-phosphate, and adenosine 2'-phosphate 5'-phosphate are selective antagonists (6). Mutational analysis studies have been performed to locate the essential residues for ligand recognition, identifying several amino acids in the extracellular loops and transmembrane domains that were essential for ligand recognition (25, 28).
P2Y1 was the first P2Y receptor to be cloned and has a wide distribution, having been described in heart, vascular, connective, immune, and neural tissues (43). P2Y1 receptors may be involved in the modulation of neurotransmission (48). However, the function of the P2Y1 receptor has been studied most extensively in platelets (33). The P2Y1 receptor plays an important role in ADP-induced shape change (29), aggregation (30), and thromboxane A2 (TXA2) generation (31). P2Y1 receptor-deficient mice have increased bleeding times and are protected from thromboembolism (19, 36). These results suggest that the P2Y1 signaling blockade, at the receptor-G protein interaction level, might be a potential antithrombotic drug target. With this goal in mind, we initiated studies to elucidate the Gq coupling domains in the P2Y1 receptor.
In this study, we first sought to explore the role of the COOH-terminal region of human P2Y1 receptor in receptor-mediated Gq activation and found that the COOH terminus is critical for the Gq coupling ability of the receptor. Furthermore, we also identified two arginine residues (R333R334) in the COOH-terminal region that are essential for Gq coupling.
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MATERIALS AND METHODS |
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Construction of human P2Y1 truncation mutant plasmids.
Human platelet P2Y1 receptor (GenBank accession no. U42029) (3) was cloned into the pcDNA3 vector with a HA tag (YPYDVPDYA) inserted at the beginning of the translation initiation by polymerase chain reaction (PCR). Forward primer containing a BamHI restriction site (underlined) and a HA tag sequence was 5'-GCGGATCCACCATGTACCCATACGATGTTCCAGATTACGCTACCGAGGTGCTGTGGCCGGCT-3'. Three COOH-terminally truncated P2Y1 receptors (P2Y1-T330-L373, P2Y1-
R340-L373, and P2Y1-
D356-L373) were constructed using PCR with the same forward primer (as for wild-type P2Y1), a reverse primer with a stop codon at the desired location (Fig. 1 and Table 1), and an XhoI restriction site to aid in subcloning. All truncated receptors were subcloned into pcDNA3 and verified using DNA sequence analysis.
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Construction of P2Y1-R333A/R334A expression plasmid. Human P2Y1-R333A/R334A expression plasmid was constructed using wild-type human P2Y1 receptor expression plasmid as template by performing site-directed mutagenesis as described previously (13), using forward primer 5'-GGGAGATACTTTCAGAGCGGCACTCTCCCGAGCCACAAGG-3' and reverse primer 5'-CCTTGTGGCTCGGGAGAGTGCCGCTCTGAAAGTATCTCCC-3'. The nucleotide sequence of the P2Y1-R333A/R334A encoding sequence in the expression plasmid [P2Y1-R333A/R334A-pcDNA3/G418(+)] was confirmed using DNA sequence analysis.
Cell culture. Chinese hamster ovary (CHO-K1) cells were grown in Ham's F-12 medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum and 1% penicillin, streptomycin, and amphotericin B at 37°C with 5% CO2. CHO-K1 cells transfected with wild-type or mutant P2Y1 receptors were grown in the same medium supplemented with 500 µg/ml G418.
Stable expression of human P2Y1 receptor in CHO-K1 cells. The expression construct for the wild-type P2Y1 receptor or for each of the P2Y1 mutants (1 µg) was used to transfect CHO-K1 cells by using Lipofectamine as described previously (2). After 6 h, the growth medium was replaced with fresh medium. Stable transfectants were selected on medium containing 500 µg/ml G418 and were screened for the expression of wild-type or mutant P2Y1 receptor by HA tag detection using flow cytometry.
HA tag detection using flow cytometry. CHO-K1 cells, naive, vector transfected, or stably transfected with P2Y1 receptors, were cultured in 100-mm dishes, washed twice with PBS (137 mM NaCl, 2.68 mM KCl, 4.29 mM Na2HPO4, and 1.47 mM KH2PO4) and detached with Versene (0.5 mM Na4EDTA, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 15 mM KH2PO4, and 1 mM glucose). After spinning at 700 rpm for 3 min, the pellets were resuspended in Tyrode solution [137 mM NaCl, 2.67 mM KCl, 2 mM MgCl2, 2.03 mM NaH2PO4, 5.6 mM glucose, 10 mM HEPES, and 0.2% bovine serum albumin (BSA), pH 7.4] and cell concentrations were adjusted to 107 cells/ml. Aliquots of 100 µl of cell suspension were mixed with 4 µl of 1:10 diluted FITC-labeled monoclonal antibody against HA (Covance) in the presence of 2 mM Ca2+. After incubation at 4°C for 1 h in the dark, cell suspensions were briefly spun, and the supernatant was discarded. Cells were resuspended in 400 µl of Tyrode solution and analyzed using flow cytometry with FACScan (BD Biosciences). Untransfected CHO-K1 cells were used as negative control.
Measurement of inositol phosphate levels. Inositol phosphate levels were measured as described previously with minor modifications (13, 20, 32). Confluent cultures of cells in 12-well plates were labeled with 1 µCi/ml (0.037 MBq/ml) myo-2-[3H]inositol in inositol-free DMEM for 1624 h. Labeled cells were washed once, the medium was replaced with 970 µl of fresh inositol-free DMEM containing 20 µl of 1 M LiCl, and the cells were incubated at 37°C for 15 min. The cells were then stimulated with 10 µl of 2-MeSADP (varying concentrations) for 10 min, and the reaction was terminated by aspiration of the medium, addition of 0.75 ml of 10 mM formic acid, and incubation at room temperature for 45 min. The solution containing the extracted inositol phosphates was neutralized by dilution with 3 ml of 5 mM NH4OH (yielding a final pH of 89) and then applied directly to a column containing 0.7 ml of the anion exchange resin AG 1-X8 (Bio-Rad, Hercules, CA), equilibrated with 40 mM ammonium formate. The column was washed with 4 ml of 40 mM ammonium formate, pH 5.0, to remove the free inositol and the glyceroinositol. Total inositol phosphates were eluted with 4 ml of 2 M ammonium formate, pH 5.0. The eluate (1 ml) was removed and counted with 9 ml of scintillation fluid.
Measurement of cytoplasmic free Ca2+ concentration. Cultured cells were harvested and incubated for 45 min at room temperature in Hanks' buffered salt solution (HBSS; Mediatech) containing 1 µM fura-2 AM (Molecular Probes, Eugene, OR) and 0.2% BSA. Cells were washed twice and then resuspended in HBSS containing 0.2% BSA, and cytoplasmic free Ca2+ concentration was measured as previously described (46). Briefly, aliquots of 0.7 ml of cell suspension were placed in disposable methacrylate cuvettes under stirring conditions at 37°C. Fluorescence was recorded on an Aminco-Bowman spectrofluorometer (Spectronics Instruments, Rochester, NY) during agonist stimulation by using alternating excitation wavelengths of 340 and 380 nm and monitoring emitted light at 510 nm. Cytoplasmic concentrations of Ca2+ were calculated according to Tsien's ratiometric method (22).
Radioligand binding assay.
Binding of the high-affinity P2Y1 receptor antagonist (5) [3H]MRS2279 to wild-type or truncated P2Y1 receptors was performed as described elsewhere (49). Briefly, cell membranes (125 µg of protein) were incubated at 4°C with 30 nM [3H]MRS2279 (120,000 cpm) in 20 mM Tris, pH 7.5, 145 mM NaCl, and 5 mM MgCl2 in a volume of 50 µl. Specific binding was usually defined as total [3H]MRS2279 binding minus binding occurring in the presence of a 10 µM concentration of the P2Y1 receptor-specific antagonist MRS2179. Incubations proceeded for 30 min at 4°C. Binding reactions were terminated by addition of 4 ml of ice-cold wash buffer (10 mM Tris, pH 7.5 at 4°C, and 145 mM NaCl) and rapid filtration over GF/A glass fiber filters. Each filter was washed with an additional 4 ml of ice-cold wash buffer. Radioactivity was quantitated using liquid scintillation spectrometry.
Statistical analysis. Data are expressed as means ± SE. Statistical significance was determined using Student's t-test and was designated at P values <0.05.
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RESULTS AND DISCUSSION |
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[3H]MRS2279 binds to both wild-type and COOH-terminally truncated P2Y1 receptors.
To confirm that the COOH terminus truncation did not drastically alter the conformation and thereby interfere with the ligand binding ability, we measured the binding of [3H]MRS2279 to cell membranes derived from CHO-K1 cells expressing P2Y1-WT or P2Y1-T330-L373. As shown in Fig. 3, the binding of [3H]MRS2279 to the truncated receptor was not significantly different from the binding to the wild-type receptor. On the basis of these results, we conclude that truncation at the COOH-terminal domain did not result in altered folding of the P2Y1 receptor or lower the affinity of ligands for the receptor. Hence, the inability of the truncated P2Y1 receptor to trigger intracellular signaling is caused by the impaired interaction with the Gq protein. We therefore propose that the human platelet P2Y1 receptor COOH terminus is a Gq-coupling region that is crucial for the interaction between Gq and the P2Y1 receptor.
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Comparison of the COOH-terminal sequence of the human P2Y1 receptor with the P2Y1 receptor COOH-terminal sequence of other species revealed regions of high consensus (Fig. 6A). The human P2Y1 COOH terminus has 97.7% identity with that of bovine and guinea pig, 95.4% identity with that of rat and mouse, and 86.3% with that of chicken. Among the three domains of human P2Y1 receptor COOH terminus investigated in this study (T330-T339, R340-E355, and D356-L373), only the first 10 amino acids in the T330-T339 domain of P2Y1 COOH terminus for human and the other five species (bovine, guinea pig, rat, mouse, and chicken) are 100% identical. The high consensus of amino acids of this domain may be related to the requirement of this domain in Gq activation. It is noteworthy that this region (T330-T339) also contains the BBXXB motif (where B is a basic residue and X is a non-basic residue) that is frequently involved in Gq coupling (34). The two consecutive arginine residues of the BBXXB also exist in the other Gq-coupled human P2Y receptors such as P2Y4 and P2Y6 (Fig. 6B). This implies that these two arginine residues, Arg333 and Arg334, may be critically important for P2Y receptor-mediated Gq activation.
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The P2Y12, P2Y13, and P2Y14 receptors couple to pertussis toxin-sensitive G proteins (8, 26, 52, 53). The functional effects of these three receptors are abolished by pertussis toxin treatment (8, 26, 53). These data and the lack of BBXXB motif in the COOH termini of these receptors (Fig. 6B) are consistent with their inability to activate the Gq class of proteins. Finally, the P2Y11 and P2Y15 receptors couple to both Gq and Gs proteins (10, 27) but lack the BBXXB motif in their COOH termini (Fig. 6B). It is possible that these two receptors use some other motif for Gq coupling.
The BB motif in BBXXB is found not only in P2Y but also in other Gq-coupled receptors. Human TXA2 receptor contains two consecutive arginine residues at the juxtamembrane COOH terminus (47). Moreover, three human 1A-adrenergic receptor isoforms (
1A-1,
1A-2, and
1A-3) (9) as well as the human platelet-activating factor receptor contain two consecutive basic amino acids (KK) at the juxtamembrane position in the COOH terminus (39). However, it is noteworthy that the protease-activated receptor 4 lacks the doublet of basic amino acids in the COOH terminus (51), suggesting that the BB motif is not always required for Gq coupling.
In conclusion, we have shown that the Arg333 and Arg334 residues in the COOH terminus of the P2Y1 receptor are important for the G protein coupling. This is the first report of the identification of an important region and residues in the P2 receptor family for G protein coupling.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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---|
2. Akbar GK, Dasari VR, Webb TE, Ayyanathan K, Pillarisetti K, Sandhu AK, Athwal RS, Daniel JL, Ashby B, Barnard EA, and Kunapuli SP. Molecular cloning of a novel P2 purinoceptor from human erythroleukemia cells. J Biol Chem 271: 1836318367, 1996.
3. Ayyanathan K, Naylor SL, and Kunapuli SP. Structural characterization and fine chromosomal mapping of the human P2Y1 purinergic receptor gene (P2RY1). Somat Cell Mol Genet 22: 419424, 1996.[ISI][Medline]
4. Ayyanathan K, Webbs TE, Sandhu AK, Athwal RS, Barnard EA, and Kunapuli SP. Cloning and chromosomal localization of the human P2Y1 purinoceptor. Biochem Biophys Res Commun 218: 783788, 1996.[CrossRef][ISI][Medline]
5. Boyer JL, Adams M, Ravi RG, Jacobson KA, and Harden TK. 2-Chloro-N6-methyl-N-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate is a selective high affinity P2Y1 receptor antagonist. Br J Pharmacol 135: 20042010, 2002.[CrossRef][ISI][Medline]
6. Boyer JL, Romero-Avila T, Schachter JB, and Harden TK. Identification of competitive antagonists of the P2Y1 receptor. Mol Pharmacol 50: 13231329, 1996.[Abstract]
7. Burnstock G. Introduction: P2 receptors. Curr Top Med Chem 4: 793803, 2004.[CrossRef][ISI][Medline]
8. Chambers JK, Macdonald LE, Sarau HM, Ames RS, Freeman K, Foley JJ, Zhu Y, McLaughlin MM, Murdock P, McMillan L, Trill J, Swift A, Aiyar N, Taylor P, Vawter L, Naheed S, Szekeres P, Hervieu G, Scott C, Watson JM, Murphy AJ, Duzic E, Klein C, Bergsma DJ, Wilson S, and Livi GP. A G protein-coupled receptor for UDP-glucose. J Biol Chem 275: 1076710771, 2000.
9. Chang DJ, Chang TK, Yamanishi SS, Salazar FH, Kosaka AH, Khare R, Bhakta S, Jasper JR, Shieh IS, Lesnick JD, Ford AP, Daniels DV, Eglen RM, Clarke DE, Bach C, and Chan HW. Molecular cloning, genomic characterization and expression of novel human 1A-adrenoceptor isoforms. FEBS Lett 422: 279283, 1998.[CrossRef][ISI][Medline]
10. Communi D, Govaerts C, Parmentier M, and Boeynaems JM. Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase. J Biol Chem 272: 3196931973, 1997.
11. Communi D, Parmentier M, and Boeynaems JM. Cloning, functional expression and tissue distribution of the human P2Y6 receptor. Biochem Biophys Res Commun 222: 303308, 1996.[CrossRef][ISI][Medline]
12. Communi D, Pirotton S, Parmentier M, and Boeynaems JM. Cloning and functional expression of a human uridine nucleotide receptor. J Biol Chem 270: 3084930852, 1995.
13. Ding Z, Kim S, Dorsam RT, Jin J, and Kunapuli SP. Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues, Cys17 and Cys270. Blood 101: 39083914, 2003.
14. Dubyak GR. Knock-out mice reveal tissue-specific roles of P2Y receptor subtypes in different epithelia. Mol Pharmacol 63: 773776, 2003.
15. Dubyak GR and Cowen DS. Activation of inositol phospholipid-specific phospholipase C by P2-purinergic receptors in human phagocytic leukocytes. Role of pertussis toxin-sensitive G proteins. Ann NY Acad Sci 603: 227245, 1990.[ISI][Medline]
16. Dubyak GR, Cowen DS, and Lazarus HM. Activation of the inositol phospholipid signaling system by receptors for extracellular ATP in human neutrophils, monocytes, and neutrophil/monocyte progenitor cells. Ann NY Acad Sci 551: 218238, 1988.[Abstract]
17. Dubyak GR, Cowen DS, and Meuller LM. Activation of inositol phospholipid breakdown in HL60 cells by P2-purinergic receptors for extracellular ATP. Evidence for mediation by both pertussis toxin-sensitive and pertussis toxin-insensitive mechanisms. J Biol Chem 263: 1810818117, 1988.
18. Dubyak GR and el-Moatassim C. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol Cell Physiol 265: C577C606, 1993.
19. Fabre JE, Nguyen M, Latour A, Keifer JA, Audoly LP, Coffman TM, and Koller BH. Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice. Nat Med 5: 11991202, 1999.[CrossRef][ISI][Medline]
20. Filtz TM, Li Q, Boyer JL, Nicholas RA, and Harden TK. Expression of a cloned P2Y purinergic receptor that couples to phospholipase C. Mol Pharmacol 46: 814, 1994.[Abstract]
21. Garrad RC, Otero MA, Erb L, Theiss PM, Clarke LL, Gonzalez FA, Turner JT, and Weisman GA. Structural basis of agonist-induced desensitization and sequestration of the P2Y2 nucleotide receptor. Consequences of truncation of the C terminus. J Biol Chem 273: 2943729444, 1998.
22. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 34403450, 1985.[Abstract]
23. He W, Miao FJ, Lin DC, Schwandner RT, Wang Z, Gao J, Chen JL, Tian H, and Ling L. Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors. Nature 429: 188193, 2004.[CrossRef][ISI][Medline]
24. Henderson DJ, Elliot DG, Smith GM, Webb TE, and Dainty IA. Cloning and characterisation of a bovine P2Y receptor. Biochem Biophys Res Commun 212: 648656, 1995.[CrossRef][ISI][Medline]
25. Hoffmann C, Moro S, Nicholas RA, Harden TK, and Jacobson KA. The role of amino acids in extracellular loops of the human P2Y1 receptor in surface expression and activation processes. J Biol Chem 274: 1463914647, 1999.
26. Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden P, Nurden A, Julius D, and Conley PB. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 409: 202207, 2001.[CrossRef][ISI][Medline]
27. Inbe H, Watanabe S, Miyawaki M, Tanabe E, and Encinas JA. Identification and characterization of a cell-surface receptor, P2Y15, for AMP and adenosine. J Biol Chem 279: 1979019799, 2004.
28. Jiang Q, Guo D, Lee BX, Van Rhee AM, Kim YC, Nicholas RA, Schachter JB, Harden TK, and Jacobson KA. A mutational analysis of residues essential for ligand recognition at the human P2Y1 receptor. Mol Pharmacol 52: 499507, 1997.
29. Jin J, Daniel JL, and Kunapuli SP. Molecular basis for ADP-induced platelet activation. II. The P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. J Biol Chem 273: 20302034, 1998.
30. Jin J and Kunapuli SP. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci USA 95: 80708074, 1998.
31. Jin J, Quinton TM, Zhang J, Rittenhouse SE, and Kunapuli SP. Adenosine diphosphate (ADP)-induced thromboxane A2 generation in human platelets requires coordinated signaling through integrin IIb
3 and ADP receptors. Blood 99: 193198, 2002.
32. Kunapuli P, Lawson JA, Rokach J, and FitzGerald GA. Functional characterization of the ocular prostaglandin F2 (PGF2
) receptor. Activation by the isoprostane, 12-iso-PGF2
. J Biol Chem 272: 2714727154, 1997.
33. Kunapuli SP, Dorsam RT, Kim S, and Quinton TM. Platelet purinergic receptors. Curr Opin Pharmacol 3: 175180, 2003.[CrossRef][ISI][Medline]
34. Lee NH, Geoghagen NS, Cheng E, Cline RT, and Fraser CM. Alanine scanning mutagenesis of conserved arginine/lysine-arginine/lysine-X-X-arginine/lysine G protein-activating motifs on m1 muscarinic acetylcholine receptors. Mol Pharmacol 50: 140148, 1996.[Abstract]
35. Lee SY, Wolff SC, Nicholas RA, and O'Grady SM. P2Y receptors modulate ion channel function through interactions involving the C-terminal domain. Mol Pharmacol 63: 878885, 2003.
36. Leon C, Hechler B, Freund M, Eckly A, Vial C, Ohlmann P, Dierich A, LeMeur M, Cazenave JP, and Gachet C. Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y1 receptor-null mice. J Clin Invest 104: 17311737, 1999.
37. Leon C, Vial C, Cazenave JP, and Gachet C. Cloning and sequencing of a human cDNA encoding endothelial P2Y1 purinoceptor. Gene 171: 295297, 1996.[CrossRef][ISI][Medline]
38. Meshki J, Tuluc F, Bredetean O, Ding Z, and Kunapuli SP. Molecular mechanism of nucleotide-induced primary granule release in human neutrophils: role for the P2Y2 receptor. Am J Physiol Cell Physiol 286: C264C271, 2004.
39. Nakamura M, Honda Z, Izumi T, Sakanaka C, Mutoh H, Minami M, Bito H, Seyama Y, Matsumoto T, and Noma M. Molecular cloning and expression of platelet-activating factor receptor from human leukocytes. J Biol Chem 266: 2040020405, 1991.
40. Offermanns S, Toombs CF, Hu YH, and Simon MI. Defective platelet activation in Gq-deficient mice. Nature 389: 183186, 1997.[CrossRef][ISI][Medline]
41. Otero M, Garrad RC, Velazquez B, Hernandez-Perez MG, Camden JM, Erb L, Clarke LL, Turner JT, Weisman GA, and Gonzalez FA. Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor. Mol Cell Biochem 205: 115123, 2000.[CrossRef][ISI][Medline]
42. Parr CE, Sullivan DM, Paradiso AM, Lazarowski ER, Burch LH, Olsen JC, Erb L, Weisman GA, Boucher RC, Turner JT. Cloning and expression of a human P2U nucleotide receptor, a target for cystic fibrosis pharmacotherapy. Proc Natl Acad Sci USA 91: 32753279, 1994 [Corrigendum. Proc Natl Acad Sci USA 91: December 20, 1994, p. 13067.]
43. Ralevic V and Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50: 413492, 1998.
44. Schachter JB, Li Q, Boyer JL, Nicholas RA, and Harden TK. Second messenger cascade specificity and pharmacological selectivity of the human P2Y1-purinoceptor. Br J Pharmacol 118: 167173, 1996.[ISI][Medline]
45. Tokuyama Y, Hara M, Jones EM, Fan Z, and Bell GI. Cloning of rat and mouse P2Y purinoceptors. Biochem Biophys Res Commun 211: 211218, 1995.[CrossRef][ISI][Medline]
46. Tuluc F, Meshki J, and Kunapuli SP. Membrane lipid microdomains differentially regulate intracellular signaling events in human neutrophils. Int Immunopharmacol 3: 17751790, 2003.[CrossRef][ISI][Medline]
47. Turek JW, Halmos T, Sullivan NL, Antonakis K, and Le Breton GC. Mapping of a ligand-binding site for the human thromboxane A2 receptor protein. J Biol Chem 277: 1679116797, 2002.
48. Von Kugelgen I and Wetter A. Molecular pharmacology of P2Y-receptors. Naunyn Schmiedebergs Arch Pharmacol 362: 310323, 2000.[CrossRef][ISI][Medline]
49. Waldo GL, Corbitt J, Boyer JL, Ravi G, Kim HS, Ji XD, Lacy J, Jacobson KA, and Harden TK. Quantitation of the P2Y1 receptor with a high affinity radiolabeled antagonist. Mol Pharmacol 62: 12491257, 2002.
50. Webb TE, Simon J, Krishek BJ, Bateson AN, Smart TG, King BF, Burnstock G, and Barnard EA. Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Lett 324: 219225, 1993.[CrossRef][ISI][Medline]
51. Xu WF, Andersen H, Whitmore TE, Presnell SR, Yee DP, Ching A, Gilbert T, Davie EW, and Foster DC. Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci USA 95: 66426646, 1998.
52. Zhang FL, Luo L, Gustafson E, Lachowicz J, Smith M, Qiao X, Liu YH, Chen G, Pramanik B, Laz TM, Palmer K, Bayne M, and Monsma FJ Jr. ADP is the cognate ligand for the orphan G protein-coupled receptor SP1999. J Biol Chem 276: 86088615, 2001.
53. Zhang FL, Luo L, Gustafson E, Palmer K, Qiao X, Fan X, Yang S, Laz TM, Bayne M, and Monsma F Jr. P2Y13: identification and characterization of a novel Gi-coupled ADP receptor from human and mouse. J Pharmacol Exp Ther 301: 705713, 2002.
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