No abstract

No abstract

The Cl- secretory pathway that is defective in cystic fibrosis (CF) can be bypassed by an alternative pathway for Cl- transport that is activated by extracellular nucleotides. Accordingly, the P2 receptor that mediates this effect is a therapeutic target for improving Cl- secretion in CF patients. In this paper, we report the sequence and functional expression of a cDNA cloned from human airway epithelial (CF/T43) cells that encodes a protein with properties of a P2U nucleotide receptor. With a retrovirus system, the human airway clone was stably expressed in 1321N1 astrocytoma cells, a human cell line unresponsive to extracellular nucleotides. Studies of inositol phosphate accumulation and intracellular Ca2+ mobilization induced by extracellular nucleotides in 1321N1 cells expressing the receptor identified this clone as the target receptor in human airway epithelia. In addition, we independently isolated an identical cDNA from human colonic epithelial (HT-29) cells, indicating that this is the same P2U receptor that has been functionally identified in other human tissues. Expression of the human P2U receptor (HP2U) in 1321N1 cells revealed evidence for autocrine ATP release and stimulation of transduced receptors. Thus, HP2U expression in the 1321N1 cell line will be useful for studying autocrine regulatory mechanisms and in screening of potential therapeutic drugs.

P2 receptors for extracellular nucleotides are divided into two categories: the ion channel receptors (P2X) and the G-protein-coupled receptors (P2Y). For the P2X receptors, signal transduction appears to be relatively simple. Upon activation by extracellular ATP, a channel comprised of P2X receptor subunits opens and allows cations to move across the plasma membrane, resulting in changes in the electrical potential of the cell that, in turn, propagates a signal. This regulated flux of ions across the plasma membrane has important signaling functions, especially in impulse propagation in the nervous system and in muscle contractility. In addition, P2X receptor activation causes the accumulation of calcium ions in the cytoplasm, which is responsible for activating numerous signaling molecules. For the P2Y receptors, signal transduction is more complex. Intracellular signaling cascades are the main routes of communication between G-protein-coupled receptors and regulatory targets within the cell. These signaling cascades operate mainly by the sequential activation or deactivation of heterotrimeric and monomeric G proteins, phospholipases, protein kinases, adenylyl and guanylyl cyclases, and phosphodiesterases that regulate many cellular processes, including proliferation, differentiation, apoptosis, metabolism, secretion, and cell migration. In addition, there are numerous ion channels, cell adhesion molecules and receptor tyrosine kinases that are modulated by P2Y receptors and operate to transmit an extracellular signal to an intracellular response. These intracellular signaling pathways and their regulation by P2 receptors are discussed in this review.

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.

Many G protein-coupled receptors activate growth factor receptors, although the mechanisms controlling this transactivation are unclear. We have identified two proline-rich, SH3 binding sites (PXXP) in the carboxyl-terminal tail of the human P2Y 2 nucleotide receptor that directly associate with the tyrosine kinase Src in protein binding assays. Furthermore, Src co-precipitated with the P2Y 2 receptor in 1321N1 astrocytoma cells stimulated with the P2Y 2 receptor agonist UTP. A mutant P2Y 2 receptor lacking the PXXP motifs was found to stimulate calcium mobilization and serine/threonine phosphorylation of the Erk1/2 mitogen-activated protein kinases, like the wild-type receptor, but was defective in its ability to stimulate tyrosine phosphorylation of Src and Srcdependent tyrosine phosphorylation of the proline-rich tyrosine kinase 2, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor. Dual immunofluorescence labeling of the P2Y 2 receptor and the EGFR indicated that UTP caused an increase in the co-localization of these receptors in the plasma membrane that was prevented by the Src inhibitor PP2. Together, these data suggest that agonist-induced binding of Src to the SH3 binding sites in the P2Y 2 receptor facilitates Src activation, which recruits the EGFR into a protein complex with the P2Y 2 receptor and allows Src to efficiently phosphorylate the EGFR.Src and proline-rich tyrosine kinase 2 (Pyk2) 1 are non-receptor tyrosine kinases that have been implicated as intermediates in the signaling pathway by which some G protein-coupled receptors (GPCRs) transactivate growth factor receptors (1-3). Although Src and Pyk2 activities are thought to be necessary for the transactivation of growth factor receptors by GPCRs, there are differing opinions in the literature about the role these kinases play in the activation of downstream mitogenic signaling. For example, inhibition of Src activity by cellular expression of C-terminal Src kinase was found to impair lysophosphatidic acid (LPA) and ␤2-adrenergic receptor-mediated activation of MAP kinases in COS-7 cells (1, 4). In rat-1 fibroblasts, dominant-negative mutants of the epidermal growth factor receptor (EGFR) or Src were used to demonstrate that the EGFR and Src are important for linking GPCR activation with the activation of MAP kinases (5, 6). And in PC12 cells, a dominant-negative mutant of Pyk2 and the EGFR kinase inhibitor AG1478 inhibited GPCR-mediated MAP kinase activation (2, 7). In contrast, experiments performed with embryonic fibroblasts derived from Src Ϫ/Ϫ , Pyk2 Ϫ/Ϫ , or Src Ϫ/Ϫ Pyk2 Ϫ/Ϫ mice indicated that both Src and Pyk2 are essential for GPCRmediated transactivation of the EGFR but are dispensable for GPCR-mediated activation of MAP kinases (3).In the present study, we have expressed wild-type and mutant P2Y 2 nucleotide receptors in human 1321N1 astrocytoma cells to explore how this GPCR transactivates growth factor receptors and affects mitogenic signaling. The P2Y 2 receptor is a G o / q -coupled receptor tha...

Extracellular ATP and UTP induce chemotaxis, or directed cell migration, by stimulating the G protein-coupled P2Y 2 nucleotide receptor (P2Y 2 R). Previously, we found that an arginine-glycineaspartic acid (RGD) integrin binding domain in the P2Y 2 R enables this receptor to interact selectively with ␣ v ␤ 3 and ␣ V ␤ 5 integrins, an interaction that is prevented by mutation of the RGD sequence to arginine-glycine-glutamic acid (RGE) (Erb, L., Liu, J., Ockerhausen, J., Kong, Q., Garrad, R. C., Griffin, K., Neal, C., Krugh, B. Chemotaxis is the movement of a cell in response to a chemical gradient and is required for many physiological events, including embryonic development, immune system function, and wound healing (2-4). The process of chemotaxis is also important for understanding diseases that result from abnormal cell migration, such as chronic inflammation, atherosclerosis, and cancer metastasis. To migrate, cells must establish dynamic and highly regulated adhesive interactions with the extracellular matrix, which are mediated by integrin receptors (3). For example, recent studies have shown that ␣ v integrins play an important role in controlling cell adhesion, spreading, and motility in several cell types, including human vascular smooth muscle cells and pancreatic beta cells (5, 6). Upon activation, many types of integrin receptors cluster together and recruit a host of cytoskeletal and cytoplasmic proteins into specialized adhesive structures called focal adhesions. These focal adhesion complexes not only serve as a physical link between the extracellular and intracellular matrix but also are important sites of signal transduction for integrins and many other types of receptors that mediate cell migration (7). Chemotaxis also requires a cell to assume a polarized morphology that is controlled by cell surface receptors that activate the Rho family of GTPases, including Cdc42, Rac, and Rho (8, 9). Upon activation of a chemoattractant receptor, Cdc42 and Rac localize at the leading edge of a cell and control directional movement and the formation of lamellipodia containing highly branched actin filaments, respectively (8). Rho localizes at the rear and sides of a cell and controls the formation of contractile actin-myosin stress fibers (10). Together, these GTPases promote cell migration toward a chemoattractant by mediating extension of the actin cytoskeleton at the front edge of the cell and retraction of the cytoskeleton at the rear edge of the cell.Recent studies have shown that G protein-coupled receptors (GPCRs) 2 regulate Rac and Rac-dependent lamellipodia formation by activating the G i/o family of heterotrimeric G proteins, whereas activation of Rho and Rho-dependent stress fiber formation are controlled by the G 12/13 family (10). Furthermore, studies have shown that the ␤␥ subunits of G i/o are responsible for activation of Rac guanine nucleotide exchange factors (RacGEFs) that, in turn, activate Rac, whereas the ␣ subunits of G 12/13 are responsible for activation of RhoGEFs (8,11).The P2Y 2 ...