The P2Y2 nucleotide receptor (P2Y2R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein–coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y2R to K562 erythroleukemia cells was inhibited by antibodies against αVβ3/β5 integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y2Rs indicated that αV integrins colocalized 10-fold better with the wild-type P2Y2R than with a mutant P2Y2R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y2R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal–regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca2+. Furthermore, an anti-αV integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y2R. Pertussis toxin, an inhibitor of Gi/o proteins, partially inhibited Ca2+ mobilization mediated by the wild-type P2Y2R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y2R-mediated activation of Go, but not Gq. Since CD47 has been shown to associate directly with Gi/o family proteins, these results suggest that interactions between P2Y2Rs, integrins, and CD47 may be important for coupling the P2Y2R to Go.
Background-Extracellular uridine 5Ј-triphosphate (UTP) induces mitogenic activation of smooth muscle cells (SMCs) through binding to P2Y 2 nucleotide receptors. P2Y 2 receptor mRNA is upregulated in intimal lesions of rat aorta, but it is unclear how this G-protein-coupled receptor contributes to development of intimal hyperplasia. Methods and Results-This study used a silicone collar placed around rabbit carotid arteries to induce vascular injury and intimal thickening. Collar placement caused rapid upregulation of P2Y 2 receptor mRNA in medial SMCs before appearance of neointima. Fura-2 digital imaging of single SMCs was used to measure changes in myoplasmic calcium concentration (Ca m ) in response to P2Y receptor agonists. In contrast to UDP, activation by UTP or adenosine 5Ј-triphosphate (ATP) greatly increased Ca m , which indicates upregulation of functional P2Y 2 receptors at which UTP and ATP are equipotent agonists. The number of responsive cells was significantly greater for freshly dispersed SMCs from collared arteries than for controls. Perivascular infusion of UTP (100 mol/L) within the collar significantly enhanced neointimal development. Intimas that resulted from UTP exposure were infiltrated by macrophages. Moreover, increased expression of osteopontin occurred in response to in situ application of UTP. ATP or UTP also stimulated osteopontin expression in cultured SMCs in a dose-dependent manner. Furthermore, P2Y 2 antisense oligonucleotide inhibited osteopontin expression induced by UTP. Conclusions-These findings indicate for the first time a role for the UTP/ATP receptor, P2Y 2 , in development of intimal hyperplasia associated with atherosclerosis and restenosis.
Interest for extracellular nucleotides has increased since the pioneer work of Burnstock in the early seventies. Research on cellular functions modulated by purines and pyrimidines has led to the identification and characterization of the different components of purine signaling, namely purinoceptors and ecto-nucleotidases. Receptors for tri- and diphosphonucleosides, known as P2 nucleotide receptors, are designated either P2Y receptors, for those coupled to G-proteins, or P2X for those which are ligand gated-ion channels. Ecto-nucleoside triphosphate diphosphohydrolase (NTPDase; EC 3.6.1.5), previously identified as ecto-ATPase, ecto-ATPDase or CD39, is now considered as the main ecto-nucleotidase responsible for the sequential hydrolysis of beta and gamma phosphates of tri- and diphosphonucleosides. More recently, research has been focused on the development of specific agonists and antagonists to P2 purinoceptors. The need to develop specific inhibitors for NTPDase to understand the role of this enzyme has clearly emerged. This paper covers the development of specific molecules targeting purinergic signaling, more specifically the inhibition of NTPDase and their impact on the different physiological systems.
Although Group IV cytosolic phospholipase A 2 (cPLA 2 ) in astrocytes has been implicated in a number of neurodegenerative diseases, mechanisms leading to its activation and release of arachidonic acid (AA) have not been clearly elucidated. In primary murine astrocytes, phorbol myristate acetate (PMA) and ATP stimulated phosphorylation of ERK1/2 and cPLA 2 as well as evoked AA release. However, complete inhibition of phospho-ERK by U0126, an inhibitor of mitogenactivated protein kinase kinase (MEK), did not completely inhibit PMA-stimulated cPLA 2 and AA release. Epidermal growth factor (EGF) also stimulated phosphorylation of ERK1/2 and cPLA 2 [largely through a protein kinase C (PKC)-independent pathway], but EGF did not evoke AA release. These results suggest that phosphorylation of cPLA 2 due to phospho-ERK is not sufficient to evoke AA release. However, complete inhibition of ATP-induced cPLA 2 phosphorylation and AA release was observed when astrocytes were treated with GF109203x, a general PKC inhibitor, together with U0126, indicating the important role for both PKC and ERK in mediating the ATP-induced AA response. There is evidence that PMA and ATP stimulated AA release through different PKC isoforms in astrocytes. In agreement with the sensitivity of PMA-induced responses to PKC down-regulation, prolonged treatment with PMA resulted in down-regulation of PKCa and e in these cells. Furthermore, PMA but not ATP stimulated rapid translocation of PKCa from cytosol to membranes. Together, our results provided evidence for an important role of PKC in mediating cPLA 2 phosphorylation and AA release in astrocytes through both ERK1/2-dependent and ERK1/2-independent pathways.
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