Cloning and Chromosomal Localization of the Human P2Y1 Purinoceptor

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419

101

ADP is an important platelet agonist causing shape change from smooth discoid shape to spiculated spheres and platelet aggregation. However, the molecular mechanisms involved in ADP-induced platelet activation have not been elucidated. We demonstrated earlier the existence of two distinct ADP receptors on platelets, one coupled to phospholipase C, P2T PLC , and the other to inhibition of adenylyl cyclase, P2T AC (Daniel, J. L., Dangelmaier, C., Jin, J., Ashby, B., Smith, J. B., and Kunapuli, S. P. (1998) J. Biol. Chem. 273, 2024 -2029), in addition to the previously described P2X1 receptor. Here we report the cloning of a cDNA clone encoding the P2Y1 receptor from a human platelet cDNA library by homology screening with radiolabeled P2Y1-P2Y6 receptor cDNAs. ADP or 2-methyl(thio)-ADP-induced intracellular calcium increases were inhibited by the P2Y1 receptor-specific antagonists, adenosine 3-phosphate 5-phosphosulfate (A3P5PS), adenosine 3-phosphate 5-phosphate (A3P5P), and adenosine 2-phosphate 5-phosphate (A2P5P), in a concentration-dependent manner, but not by ARL 66096 or ␣,␤-MeATP. A3P5PS, A3P5P, and A2P5P also inhibited the shape change of aspirinated platelets induced by 10 M ADP or 3 M 2-methyl-(thio)-ADP in a concentration-dependent manner, with complete inhibition occurring at 300 M. On the other hand ARL 66096 (100 nM), a potent P2T AC antagonist and ␣,␤-methylene-ATP (40 M), a P2X1 receptor agonist, had no effect on ADP-induced platelet shape change. On the contrary, ADP-induced inhibition of adenylyl cyclase was blocked by ARL 66096, but not by ␣,␤-MeATP or the P2Y1 receptor-specific antagonists, A3P5PS, A3P5P, or A2P5P. These results demonstrate the role of the P2Y1 receptor in ADP-induced platelet shape change and calcium mobilization and support the idea that several P2 receptors are involved in the regulation of different aspects of platelet stimulus-response coupling. ADP1 has been known to cause platelet activation since 1961(1). When exposed to ADP, platelets undergo shape change from smooth discs to spiculated spheres, activate the fibrinogen receptor, causing platelets to aggregate, release granule contents, and produce thromboxane A 2 (2, 3). These effects have been postulated to be mediated by a platelet ADP receptor, antagonized by ATP (2, 3). Several molecules have been proposed to be the platelet ADP receptor, including aggregin, a 100-kDa protein that covalently binds to 5Ј-p-fluorosulfonylbenzoyladenosine (4), and a platelet glycoprotein IIb, a component of the fibrinogen receptor on platelets (5), but none has been conclusively demonstrated to be a platelet ADP receptor. ADP causes several intracellular responses in platelets: inhibition of adenylyl cyclase, formation of inositol trisphosphate, mobilization of intracellular calcium stores, rapid calcium influx, and activation of phospholipase A 2 (3). Several compounds, including ARL 66096, ARL 67085, ticlopidine, and clopidogrel, have been utilized to block ADP-induced inhibition of adenylyl cyclase and subsequent platelet aggregat...

Section: Discussionmentioning

confidence: 86%

Section: Materials-mentioning

confidence: 99%

Section: Cloning Of the P2y1 Receptor Cdna From A Human Plateletmentioning

confidence: 99%

See 1 more Smart Citation

Molecular Basis for ADP-induced Platelet Activation

Jin¹,

Daniel²,

Kunapuli³

1998

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419

101

“…Oligonucleotides for P2Y 1 were 5Ј-CGGTCCGGGTTCGTCC-3Ј (forward, sense) and 5Ј-CGGACCCCGGTACCT-3Ј (reverse, antisense) as originally designed by Ayyanathan et al (14). The oligonucleotides for P2Y 2 were 5Ј-CGTCATCCTTGTCTGTTACGTGCT-3Ј (forward, sense) and 5Ј-CTACAGCCGAATGTCCTTAGTG-3Ј (reverse, antisense) as originally designed by Bowler et al (15).…”

Section: Methodsmentioning

confidence: 99%

Concomitant Recruitment of ERK1/2 and p38 MAPK Signalling Pathway Is Required for Activation of Cytoplasmic Phospholipase A2via ATP in Articular Chondrocytes

Bérenbaum¹,

Humbert²,

Béréziat³

et al. 2003

Extracellular ATP is a pro-inflammatory mediator involved in the release of prostaglandin from articular chondrocytes, but little is known about its effects on intracellular signaling. ATP triggered the rapid release of prostaglandin E 2 (PGE 2 ) by acting on P2Y 2 receptors in rabbit articular chondrocytes. We have explored the signaling events involved in this synthesis. ATP significantly increased arachidonic acid production, which involved the activation of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) but not a secreted form of PLA 2 , as demonstrated by various PLA 2 inhibitors and translocation experiments. We also showed that ATP induced the phosphorylation of p38 and ERK1/2 mitogen-activatedprotein kinases (MAPKs). Both PD98059, an inhibitor of the ERK pathway, and SB203580, an inhibitor of p38 MAPK, completely inhibited the ATP-induced release of PGE 2 . Finally, dominant-negative plasmids encoding p38 and ERK transfected alone into the cells impaired the ATP-induced release of PGE 2 to about the same extent as both plasmids transfected together. These results suggest that PGE 2 production induced by ATP requires the activation of both ERK1/2 and p38 MAPKs. Thus, ATP acts via P2Y 2 -purine receptors to recruit cPLA 2 by activating both ERK1/2 and p38 MAPKs and stimulates the release of PGE 2 from articular chondrocytes.

“…This is in sharp contrast to the amino acid sequence conservation between other mammalian and chicken receptors, e.g. the muscarinic receptor M2 with 85% (32), the purinergic receptor P2YR with 84% (33,34), and the melatonin receptor 1a with 79% (35, 36) sequence identity. Notably the intra-species sequence identity between human B1 and B2 receptors is also low, i.e.…”

Section: Discussionmentioning

confidence: 59%

Cloning and Functional Characterization of the Ornithokinin Receptor

Schröeder

Beug

Müller‐Esterl

1997

Kinins are proinflammatory peptides that dilate vessels, increase vascular permeability, contract smooth muscles, and provoke pain. In mammals two principal types of kinin receptors, B1 and B2, with distinct pharmacological properties have been cloned and characterized (1-4). Their ligands are derived from bradykinin, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg. Expression of the B1 receptor is induced under pathophysiological conditions such as injury, infection, or chronic inflammation where it mediates hyperalgesia and sensitization (5, 6). The B2 receptor is constitutively expressed in fibroblasts and in epithelial, endothelial, neuronal, and smooth muscle cells (7,8 (11,12). HOE140, a synthetic peptide derived from the bradykinin sequence, is a selective and tightly binding B2 receptor antagonist (13, 14) that lacks partial agonistic activity in most physiological settings (15).Kinins are well conserved peptides that have been identified in invertebrates, e.g. in the wasp (16). In fact the non-mammalian kinins are identical to their human counterpart including a 4-hydroxylation of a proline residue at position 3 (16). Kinins are present in all the major vertebrate classes including reptiles, amphibia, fish, and birds (8). The avian kinin, first described in 1967 (17), and aptly named ornithokinin, differs from the human bradykinin in two positions: Thr substitutes for Ser at position 6, and Leu for Phe at position 8. Ornithokinin, i.e. [Ser 6 ,Leu 8 ]bradykinin, induces transient hypotension in the chicken and contracts smooth muscle cells of the oviduct (17, 18), two physiological activities reminiscent of the kinin effects in mammals. To date the receptor underlying ornithokinin's effects has not been defined. Unlike most of the insect kinins ornithokinin has no effect on the human kinin system, and vice versa (18). The differential binding profiles are likely to reflect distinct structures of the ligand-binding sites of the corresponding receptors, however, the lack of non-mammalian kinin receptor sequences has precluded such comparisons.Here we describe the cloning of an avian kinin receptor using a PCR 1 -based strategy. Expression of the biologically active chicken ornithokinin receptor revealed the unexpected finding that HOE140, the principal antagonist of the mammalian B2 receptors, is a full agonist of the avian receptor as is ornithokinin, whereas bradykinin and [des-Arg 9 ]bradykinin have no effect. Sequence analyses indicate that avian and mammalian kinin receptors are only distantly related. Our results suggest that the ornithokinin receptor is the first member of a novel type of kinin receptors. More importantly, having available a receptor in which HOE140 acts as a full agonist will make the ornithokinin receptor a unique tool to study ligand binding and receptor function at the molecular level. EXPERIMENTAL PROCEDURESSources of Reagents-The pCDNA3, pVL-1392 vectors, and the Liposome Kit were from Invitrogen; oligonucleotides from MWG-Biotech;