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Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of memory and cognitive ability and is a serious cause of mortality. Many of the pathological characteristics associated with AD are revealed post-mortem, including amyloid-β plaque deposition, neurofibrillary tangles containing hyperphosphorylated tau proteins and neuronal loss in the hippocampus and cortex. Although several genetic mutations and risk factors have been associated with the disease, the causes remain poorly understood. Study of disease-initiating mechanisms and AD progression in humans is inherently difficult as most available tissue specimens are from late-stages of disease. Therefore, AD researchers rely on in vitro studies and the use of AD animal models where neuroinflammation has been shown to be a major characteristic of AD. Purinergic receptors are a diverse family of proteins consisting of P1 adenosine receptors and P2 nucleotide receptors for ATP, UTP and their metabolites. This family of receptors has been shown to regulate a wide range of physiological and pathophysiological processes, including neuroinflammation, and may contribute to the pathogenesis of neurodegenerative diseases like Parkinson's disease, multiple sclerosis and AD. Experimental evidence from human AD tissue has suggested that purinergic receptors may play a role in AD progression and studies using selective purinergic receptor agonists and antagonists in vitro and in AD animal models have demonstrated that purinergic receptors represent novel therapeutic targets for the treatment of AD. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Acute inflammation is important for tissue repair; however, chronic inflammation contributes to neurodegeneration in Alzheimer's disease (AD) and occurs when glial cells undergo prolonged activation. In the brain, stress or damage causes the release of nucleotides and activation of the G(q) protein-coupled P2Y(2) nucleotide receptor subtype (P2Y(2)R) leading to pro-inflammatory responses that can protect neurons from injury, including the stimulation and recruitment of glial cells. P2Y(2)R activation induces the phosphorylation of the epidermal growth factor receptor (EGFR), a response dependent upon the presence of a SH3 binding domain in the intracellular C terminus of the P2Y(2)R that promotes Src binding and transactivation of EGFR, a pathway that regulates the proliferation of cortical astrocytes. Other studies indicate that P2Y(2)R activation increases astrocyte migration. P2Y(2)R activation by UTP increases the expression in astrocytes of alpha(V)beta(3/5) integrins that bind directly to the P2Y(2)R via an Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y(2)R, an interaction required for G(o) and G(12) protein-dependent astrocyte migration. In rat primary cortical neurons (rPCNs) P2Y(2)R expression is increased by stimulation with interleukin-1beta (IL-1beta), a pro-inflammatory cytokine whose levels are elevated in AD, in part due to nucleotide-stimulated release from glial cells. Other results indicate that oligomeric beta-amyloid peptide (Abeta(1-42)), a contributor to AD, increases nucleotide release from astrocytes, which would serve to activate upregulated P2Y(2)Rs in neurons. Data with rPCNs suggest that P2Y(2)R upregulation by IL-1beta and subsequent activation by UTP are neuroprotective, since this increases the non-amyloidogenic cleavage of amyloid precursor protein. Furthermore, activation of IL-1beta-upregulated P2Y(2)Rs in rPCNs increases the phosphorylation of cofilin, a cytoskeletal protein that stabilizes neurite outgrowths. Thus, activation of pro-inflammatory P2Y(2)Rs in glial cells can promote neuroprotective responses, suggesting that P2Y(2)Rs represent a novel pharmacological target in neurodegenerative and other pro-inflammatory diseases.

The G protein-coupled receptor P2Y(2) nucleotide receptor (P2Y(2)R) has been shown to be up-regulated in a variety of tissues in response to stress or injury. Recent studies have suggested that P2Y(2)Rs may play a role in immune responses, wound healing, and tissue regeneration via their ability to activate multiple signaling pathways, including activation of growth factor receptors. Here, we demonstrate that in human salivary gland (HSG) cells, activation of the P2Y(2)R by its agonist induces phosphorylation of ERK1/2 via two distinct mechanisms, a rapid, protein kinase C-dependent pathway and a slower and prolonged, epidermal growth factor receptor (EGFR)-dependent pathway. The EGFR-dependent stimulation of UTP-induced ERK1/2 phosphorylation in HSG cells is inhibited by the adamalysin inhibitor tumor necrosis factor-alpha protease inhibitor or by small interfering RNA that selectively silences ADAM10 and ADAM17 expression, suggesting that ADAM metalloproteases are required for P2Y(2)R-mediated activation of the EGFR. G protein-coupled receptors have been shown to promote proteolytic release of EGFR ligands; however, neutralizing antibodies to known ligands of the EGFR did not inhibit UTP-induced EGFR phosphorylation. Immunoprecipitation experiments indicated that UTP causes association of the EGFR with another member of the EGF receptor family, ErbB3. Furthermore, stimulation of HSG cells with UTP induced phosphorylation of ErbB3, and silencing of ErbB3 expression inhibited UTP-induced phosphorylation of both ErbB3 and EGFR. UTP-induced phosphorylation of ErbB3 and EGFR was also inhibited by silencing the expression of the ErbB3 ligand neuregulin 1 (NRG1). These results suggest that P2Y(2)R activation in salivary gland cells promotes the formation of EGFR/ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3.

The heterologous expression and activation of the human P2Y(2) nucleotide receptor (P2Y(2)R) in human 1321N1 astrocytoma cells stimulates alpha-secretase-dependent cleavage of the amyloid precursor protein (APP), causing extracellular release of the non-amyloidogenic protein secreted amyloid precursor protein (sAPPalpha). To determine whether a similar response occurs in a neuronal cell, we analyzed whether P2Y(2)R-mediated production of sAPPalpha occurs in rat primary cortical neurons (rPCNs). In rPCNs, P2Y(2)R mRNA and receptor activity were virtually absent in quiescent cells, whereas overnight treatment with the pro-inflammatory cytokine interleukin-1beta (IL-1beta) up-regulated both P2Y(2)R mRNA expression and receptor activity by four-fold. The up-regulation of the P2Y(2)R was abrogated by pre-incubation with Bay 11-7085, an IkappaB-alpha phosphorylation inhibitor, which suggests that P2Y(2)R mRNA transcript levels are regulated through nuclear factor-kappa-B (NFkappaB) signaling. Furthermore, the P2Y(2)R agonist Uridine-5'-triphosphate (UTP) enhanced the release of sAPPalpha in rPCNs treated with IL-1beta or transfected with P2Y(2)R cDNA. UTP-induced release of sAPPalpha from rPCNs was completely inhibited by pre-treatment of the cells with the metalloproteinase inhibitor TACE inhibitor (TAPI-2) or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, and was partially inhibited by the MAPK/extracellular signal-regulated kinase inhibitor U0126 and the protein kinase C inhibitor GF109203. These data suggest that P2Y(2)R-mediated release of sAPPalpha from cortical neurons is directly dependent on a disintegrin and metalloproteinase (ADAM) 10/17 and PI3K activity, whereas extracellular signal-regulated kinase 1/2 and PI3K activity may indirectly regulate APP processing. These results demonstrate that elevated levels of pro-inflammatory cytokines associated with neurodegenerative diseases, such as IL-1beta, can enhance non-amyloidogenic APP processing through up-regulation of the P2Y(2)R in neurons.

The functional expression of the G protein-coupled P2Y(2) nucleotide receptor (P2Y(2)R) has been associated with proliferation and migration of vascular smooth muscle cells (SMCs), two processes involved in atherosclerosis and restenosis. Activation of the P2Y(2)R causes dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, suggesting that P2Y(2)Rs may be linked to the actin cytoskeleton. Here, we identified filamin A (FLNa) as a P2Y(2)R-interacting protein using a yeast 2-hybrid system screen with the C-terminal region of the P2Y(2)R as bait. The FLNa binding site in the P2Y(2)R is localized between amino acids 322 and 333. Deletion of this region led to selective loss of FLNa binding to the P2Y(2)R and abolished Tyr phosphorylation of FLNa induced by the P2Y(2)R agonist UTP. Using both time-lapse microscopy and the Transwell cell migration assay, we showed that UTP significantly increased SMC spreading on collagen I (6.8 fold; P < or = 0.01) and migration (3.6 fold; P < or = 0.01) of aortic SMCs isolated from wild-type mice, as compared with unstimulated SMCs. UTP-induced spreading and migration of aortic SMCs did not occur with cells isolated from P2Y(2)R knockout mice. Expression of the full-length P2Y(2)R in SMCs isolated from P2Y(2)R knockout mice restored both UTP-induced spreading and migration. In contrast, UTP-induced spreading and migration did not occur in SMCs isolated from P2Y(2)R knockout mice transfected with a mutant P2Y(2)R that does not bind FLNa. Furthermore, ex vivo studies showed that both ATP and UTP (10 micromol/L) promoted migration of SMCs out of aortic explants isolated from wild-type but not P2Y(2)R knockout mice. Thus, this study demonstrates that P2Y(2)R/FLNa interaction selectively regulates spreading and migration of vascular SMCs.

Nucleotides released from cells due to stress, injury or inflammation, induce mitogenic effects in monocytes via activation of P2Y(2) nucleotide receptors (P2Y(2)Rs). Here we show that P2Y(2) nucleotide receptors in U937 monocytic cells regulate the activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) by inducing the clustering of alpha(v) integrins. The activation of phosphatidylinositol 3-kinase by P2Y(2)R ligands was required for alpha(v) clustering, suggesting a means whereby two different classes of receptors communicate to induce mitogenic responses in monocytic cells. P2Y(2)R-induced alpha(v) clustering was also associated with a flattened phenotype of the U937 cells, consistent with the role of the P2Y(2)R in regulating early events in cell migration., ((c) 2007 Wiley-Liss, Inc.)

The P2Y2 nucleotide receptor (P2Y2R) interacts with alpha v integrins to activate G(o) and induce chemotaxis in human 1321N1 astrocytoma cells. In this study, it was determined that the P2Y2R also requires interaction with alpha v integrins to activate G12 and associated signaling pathways that control chemotaxis in 1321N1 cells. Mutation of the Arg-Gly-Asp (RGD) integrin-binding sequence in the first extracellular loop of the human P2Y2R to Arg-Gly-Glu (RGE), which prevents integrin interaction, did not inhibit G(q) or ERK1/2 signaling by the P2Y2R agonist UTP but completely inhibited activation of G12 and G12-mediated events, including Rho activation, cofilin and myosin light chain-2 phosphorylation, stress fiber formation and chemotaxis towards UTP. The involvement of G12 in all these events was verified by using a dominant negative G alpha12 construct. G12 activation by the P2Y2R also was inhibited by anti-alpha v beta5 integrin antibodies and alpha v integrin antisense oligonucleotides, suggesting that alpha v integrin activity and expression are required for the P2Y2R to activate G12. Co-immunoprecipitation experiments confirmed that G alpha12 protein associates with the wild-type P2Y2R and with alpha v integrins but not with the RGE mutant P2Y2R or with alpha3 integrins. Collectively, these results suggest that alpha v integrin complexes provide the P2Y2R with access to G12, thereby allowing activation of this heterotrimeric G protein that controls actin cytoskeletal rearrangements required for chemotaxis.

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.

Unlabelled: In rat submandibular gland (SMG), the P2Y1 receptor (P2Y1R) mediates increases in the intracellular calcium concentration, [Ca2+]i that diminish as the animal ages from 1 to 4-6 weeks. However, P2Y1R mRNA levels do not change with age, suggesting that the age-dependent decrease in the [Ca2+]i response to P2Y1R agonists may be due to alterations in the activity of a component of the P2Y1R signalling pathway., Objectives: To assess whether the decrease in P2Y1R-mediated intracellular calcium signalling in SMG cells as rats age is due to a decrease in P2Y1R coupling to G proteins or to a decrease in the expression of a cognate G protein., Design: SMG cells were isolated from Sprague-Dawley rats. P2Y1R function was assessed by measuring 2-MeSADP-induced increases in [Ca2+]i and ERK1/2 activation. P2Y(1)R-mediated activation of G proteins was determined by the [35S]GTPgammaS binding assay. Gq protein expression was determined by RT-PCR, Northern, and Western analysis., Results: In SMG cells from 1-week-old rats, two bands (52 and 42kDa) were detected using anti-Galpha14 antibody, whereas in SMG cells from 4- to 6-week-old rats only the 42 kDa band was detected. Furthermore, 2-MeSADP-induced GTPgamma35S binding to Galpha14 and Galphaq/11 decreases in SMG cells from 4- to 6-week-old rats as compared to 1-week-old rats., Conclusions: These findings suggest that the age-dependent decrease in P2Y1R-mediated intracellular calcium signalling in rat SMG cells is due to a loss of 52 kDa Galpha14 and indicate the differential coupling of the P2Y1R to Galpha14 and Galphaq/11 as the gland develops.

In the past ten years since the discovery and cloning of members of the P2 receptor family, rapid progress has been made in the field regarding the function and pharmacology of different P2 receptor subtypes. This research resulted in identifying these receptors as important players in the pathology of atherosclerosis, cystic fibrosis, neurodegenerative and autoimmune diseases, among other disorders. The signalling mechanisms whereby P2 receptors mediate pathogenesis are not clear in most cases. Future studies in this field will focus on the integration of signalling pathways coupled to P2 receptors and the generation of specific agonists/antagonists for each receptor subtype to provide strategies for the treatment of a variety of diseases.

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-glycine-aspartic acid (RGD) integrin binding domain in the P2Y(2)R enables this receptor to interact selectively with alpha(v)beta(3) and alpha(V)beta(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., Santiago-Perez, L. I., Gonzalez, F. A., Gresham, H. D., Turner, J. T., and Weisman, G. A. (2001) J. Cell Biol. 153, 491-501). This RGD domain also was found to be necessary for coupling the P2Y(2)R to G(o)- but not G(q)-mediated intracellular calcium mobilization, leading us to investigate the role of P2Y(2)R interaction with integrins in nucleotide-induced chemotaxis. Here we show that mutation of the RGD sequence to RGE in the human P2Y(2)R expressed in 1321N1 astrocytoma cells completely prevented UTP-induced chemotaxis as well as activation of G(o), Rac, and Vav2, a guanine nucleotide exchange factor for Rac. UTP also increased expression of vitronectin, an extracellular matrix protein that is a ligand for alpha(v)beta(3)/beta(5) integrins, in cells expressing the wild-type but not the RGE mutant P2Y(2)R. P2Y(2)R-mediated chemotaxis, Rac and Vav2 activation, and vitronectin up-regulation were inhibited by pretreatment of the cells with anti-alpha(v)beta(5) integrin antibodies, alpha(v) integrin antisense oligonucleotides, or the G(i/o) inhibitor, pertussis toxin. Thus, the RGD-dependent interaction between the P2Y(2)R and alpha(v) integrins is necessary for the P2Y(2)R to activate G(o) and to initiate G(o)-mediated signaling events leading to chemotaxis.

Astrocytes become activated in response to brain injury, as characterized by increased expression of glial fibrillary acidic protein (GFAP) and increased rates of cell migration and proliferation. Damage to brain cells causes the release of cytoplasmic nucleotides, such as ATP and uridine 5'-triphosphate (UTP), ligands for P2 nucleotide receptors. Results in this study with primary rat astrocytes indicate that activation of a G protein-coupled P2Y(2) receptor for ATP and UTP increases GFAP expression and both chemotactic and chemokinetic cell migration. UTP-induced astrocyte migration was inhibited by silencing of P2Y(2) nucleotide receptor (P2Y(2)R) expression with siRNA of P2Y(2)R (P2Y(2)R siRNA). UTP also increased the expression in astrocytes of alpha(V)beta(3/5) integrins that are known to interact directly with the P2Y(2)R to modulate its function. Anti-alpha(V) integrin antibodies prevented UTP-stimulated astrocyte migration, suggesting that P2Y(2)R/alpha(V) interactions mediate the activation of astrocytes by UTP. P2Y(2)R-mediated astrocyte migration required the activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (Akt) and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways, responses that also were inhibited by anti-alpha(V) integrin antibody. These results suggest that P2Y(2)Rs and their associated signaling pathways may be important factors regulating astrogliosis in brain disorders.

The amyloid precursor protein (APP) is proteolytically processed by beta- and gamma-secretases to release amyloid beta, the main component in senile plaques found in the brains of patients with Alzheimer disease. Alternatively, APP can be cleaved within the amyloid beta domain by alpha-secretase releasing the non-amyloidogenic product sAPP alpha, which has been shown to have neuroprotective properties. Several G protein-coupled receptors are known to activate alpha-secretase-dependent processing of APP; however, the role of G protein-coupled nucleotide receptors in APP processing has not been investigated. Here it is demonstrated that activation of the G protein-coupled P2Y2 receptor (P2Y2R) subtype expressed in human 1321N1 astrocytoma cells enhanced the release of sAPP alpha in a time- and dose-dependent manner. P2Y2 R-mediated sAPP alpha release was dependent on extracellular calcium but was not affected by 1,2-bis(2-aminophenoxy)ethane-N,N,N,-trimethylammonium salt, an intracellular calcium chelator, indicating that P2Y2R-stimulated intracellular calcium mobilization was not involved. Inhibition of protein kinase C (PKC) with GF109203 or by PKC down-regulation with phorbol ester pre-treatment had no effect on UTP-stimulated sAPP alpha release, indicating a PKC-independent mechanism. U0126, an inhibitor of the mitogen-activated protein kinase pathway, partially inhibited sAPPalpha release by UTP, whereas inhibitors of Src-dependent epidermal growth factor receptor transactivation by P2Y2Rs had no effect. The metalloprotease inhibitors phenanthroline and TAPI-2 and the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone also diminished UTP-induced sAPP alpha release. Furthermore, small interfering RNA silencing of an endogenous adamalysin, ADAM10 or ADAM17/TACE, partially suppressed P2Y2R-activated sAPP alpha release, whereas treatment of cells with both ADAM10 and ADAM17/TACE small interfering RNAs completely abolished UTP-activated sAPP alpha release. These results may contribute to an understanding of the non-amyloidogenic processing of APP.

Trophic factors are required to ensure neuronal viability and regeneration after neural injury. Although abundant information is available on the factors that cause the activation of astrocytes, little is known about the molecular mechanisms underlying the regulation of this process. Nucleotides released into the extracellular space from injured or dying neural cells can activate astrocytes via P2 nucleotide receptors. After a brief historical review and update of novel P2 receptor antagonists, this article focuses on recent advancements toward understanding molecular mechanisms that regulate G protein-coupled P2Y receptor signaling. Among P2Y receptor subtypes, the heptahelical P2Y2 nucleotide receptor interacts with vitronectin receptors via an RGD sequence in the first extracellular loop, and this interaction is required for effective signal transduction to activate mitogen-activated protein kinases ERK1/2, to mobilize intracellular calcium stores via activation of phospholipase C, protein kinase C isoforms, and to activate focal adhesion kinase and other signaling events. Ligation of vitronectin receptors with specific antibodies caused an inhibition of P2Y2 receptor-induced ERK1/2 and p38 phosphorylation and P2Y2 receptor-induced cytoskeleton rearrangement and DNA synthesis. Structure-function studies have identified agonist-induced phosphorylation of the C-terminus of the P2Y2 receptor, an important mechanism for receptor desensitization. Understanding selective mechanisms for regulating P2Y2 receptor signaling could provide novel targets for therapeutic strategies in the management of brain injury, synaptogenesis, and neurological disorders.

In the mammalian nervous system, P2 nucleotide receptors mediate neurotransmission, release of proinflammatory cytokines, and reactive astrogliosis. Extracellular nucleotides activate multiple P2 receptors in neurons and glial cells, including G protein-coupled P2Y receptors and P2X receptors, which are ligand-gated ion channels. In glial cells, the P2Y2 receptor subtype, distinguished by its ability to be equipotently activated by ATP and UTP, is coupled to pro-inflammatory signaling pathways. In situ hybridization studies with rodent brain slices indicate that P2Y2 receptors are expressed primarily in the hippocampus and cerebellum. Astrocytes express several P2 receptor subtypes, including P2Y2 receptors whose activation stimulates cell proliferation and migration. P2Y2 receptors, via an RGD (Arg-Gly-Asp) motif in their first extracellular loop, bind to alphavbeta3/beta5 integrins, whereupon P2Y2 receptor activation stimulates integrin signaling pathways that regulate cytoskeletal reorganization and cell motility. The C-terminus of the P2Y2 receptor contains two Src-homology-3 (SH3)-binding domains that upon receptor activation, promote association with Src and transactivation of growth factor receptors. Together, our results indicate that P2Y2 receptors complex with both integrins and growth factor receptors to activate multiple signaling pathways. Thus, P2Y2 receptors present novel targets to control reactive astrogliosis in neurodegenerative diseases.

Mechanical or ischemic trauma to the CNS causes the release of nucleotides and other neurotransmitters into the extracellular space. Nucleotides can activate nucleotide receptors that modulate the expression of genes implicated in cellular adaptive responses. In this investigation, we used human 1321N1 astrocytoma cells expressing a recombinant P2Y2 receptor to assess the role of this receptor in the regulation of anti-apoptotic (bcl-2 and bcl-xl) and pro-apoptotic (bax) gene expression. Acute treatment with the P2Y2 receptor agonist UTP up-regulated bcl-2 and bcl-xl, and down-regulated bax, gene expression. Activation of P2Y2 receptors was also coupled to the phosphorylation of cyclic AMP responsive element binding protein that positively regulates bcl-2 and bcl-xl gene expression. Cyclic AMP responsive element decoy oligonucleotides markedly attenuated the UTP-induced increase in bcl-2 and bcl-xl mRNA levels. Activation of P2Y2 receptors induced the phosphorylation of the pro-apoptotic factor Bad and caused a reduction in bax/bcl-2 mRNA expression ratio. All these signaling pathways are known to be involved in cell survival mechanisms. Using cDNA microarray analysis and RT-PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor 2; neurotrophin 3; glia-derived neurite-promoting factor, as well as extracellular matrix proteins CD44 and fibronectin precursor--genes known to regulate neuroprotection. Consistent with this observation, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Taken together, our results suggest an important novel role for the P2Y2 receptor in survival and neuroprotective mechanisms under pathological conditions.

UTP stimulates the expression of pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells through activation of the P2Y(2) nucleotide receptor P2Y(2)R. Here, we demonstrated that activation of the P2Y(2)R induced rapid tyrosine phosphorylation of vascular endothelial growth factor receptor (VEGFR)-2 in human coronary artery endothelial cells (HCAEC). RNA interference targeting VEGFR-2 or inhibition of VEGFR-2 tyrosine kinase activity abolishes P2Y(2)R-mediated VCAM-1 expression. Furthermore, VEGFR-2 and the P2Y(2)R co-localize upon UTP stimulation. Deletion or mutation of two Src homology-3-binding sites in the C-terminal tail of the P2Y(2)R or inhibition of Src kinase activity abolished the P2Y(2)R-mediated transactivation of VEGFR-2 and subsequently inhibited UTP-induced VCAM-1 expression. Moreover, activation of VEGFR-2 by UTP leads to the phosphorylation of Vav2, a guanine nucleotide exchange factor for Rho family GTPases. Using a binding assay to measure the activity of the small GTPases Rho, we found that stimulation of HCAEC by UTP increased the activity of RhoA and Rac1 (but not Cdc42). Significantly, a dominant negative form of RhoA inhibited P2Y(2)R-mediated VCAM-1 expression, whereas expression of dominant negative forms of Cdc42 and Rac1 had no effect. These data indicate a novel mechanism whereby a nucleotide receptor transactivates a receptor tyrosine kinase to generate an inflammatory response associated with atherosclerosis.

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 Src-dependent 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.

P2Y2 receptor up-regulation and activation induces intimal hyperplasia and monocyte/macrophage infiltration in the collared rabbit carotid artery model of vascular injury, suggesting a potential role for P2Y2 receptors in monocyte recruitment by vascular endothelium. In this study, we addressed the hypothesis that activation of P2Y2 receptors by extracellular nucleotides modulates the expression of adhesion molecules on vascular endothelial cells that are important for monocyte recruitment. Results indicated that the equipotent P2Y2 receptor agonists UTP or ATP (1-100 microm) stimulated the expression of vascular cell adhesion molecule-1 (VCAM-1) in human coronary artery endothelial cells (HCAEC) in a time- and dose-dependent manner. P2Y2 antisense oligonucleotides inhibited VCAM-1 expression induced by UTP but not by tumor necrosis factor-alpha. Furthermore, UTP induced VCAM-1 expression in human 1321N1 astrocytoma cell transfectants expressing the recombinant P2Y2 receptor, whereas vector-transfected control cells did not respond to UTP. The effect of UTP on VCAM-1 expression in HCAEC was prevented by depletion of intracellular calcium stores with thapsigargin or by inhibition of p38 mitogen-activated protein kinase or Rho kinase, but was not affected by inhibitors of the mitogen-activated protein/extracellular signal-regulated kinase pathway (i.e. MEK1/2). Consistent with a role for VCAM-1 in the recruitment of monocytes, UTP or ATP increased the adherence of monocytic U937 cells to HCAEC, an effect that was inhibited by anti-VCAM-1 antibodies. These findings suggest a novel role for the P2Y2 receptor in the p38- and Rho kinase-dependent expression of VCAM-1 that mediates the recruitment of monocytes by vascular endothelium associated with the development of atherosclerosis.

Background: Extracellular uridine 5'-triphosphate (UTP) induces mitogenic activation of smooth muscle cells (SMCs) through binding to P2Y2 nucleotide receptors. P2Y2 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 P2Y2 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 5'-triphosphate (ATP) greatly increased Ca(m), which indicates upregulation of functional P2Y2 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 micromol/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, P2Y2 antisense oligonucleotide inhibited osteopontin expression induced by UTP., Conclusions: These findings indicate for the first time a role for the UTP/ATP receptor, P2Y2, in development of intimal hyperplasia associated with atherosclerosis and restenosis.

Activation of P2Y(2) receptors by extracellular nucleotides has been shown to induce phenotypic differentiation of human promonocytic U937 cells that is associated with the inflammatory response. The P2Y(2) receptor agonist, UTP, induced the phosphorylation of the MAP kinases MEK1/2 and ERK1/2 in a sequential manner, since ERK1/2 phosphorylation was abolished by the MEK1/2 inhibitor PD 098059. Other results indicated that P2Y(2) receptors can couple to MAP kinases via phosphatidylinositol 3-kinase (PI3K) and c-src. Accordingly, ERK1/2 phosphorylation induced by UTP was inhibited by the PI3K inhibitors, wortmannin and LY294002, and the c-src inhibitors, radicicol and PP2, but not by inhibitors of protein kinase C (PKC). The phosphorylation of ERK1/2 was independent of the ability of P2Y(2) receptors to increase the concentration of intracellular free calcium, since chelation of intracellular calcium by BAPTA did not diminish the phosphorylation of ERK1/2 induced by UTP. A 5-minute treatment with UTP reduced U937 cell responsiveness to a subsequent UTP challenge. UTP-induced desensitization was characterized by an increase in the EC(50) for receptor activation (from 0.44 to 9.3 microM) and a dramatic ( approximately 75%) decrease in the maximal calcium mobilization induced by a supramaximal dose of UTP. Phorbol ester treatment also caused P2Y(2) receptor desensitization (EC(50) = 12.3 microM UTP and maximal calcium mobilization reduced by approximately 33%). The protein kinase C inhibitor GF 109203X failed to significantly inhibit the UTP-induced desensitization of the P2Y(2) receptor, whereas the protein phosphatase inhibitor okadaic acid blocked receptor resensitization. Recovery of receptor activity after UTP-induced desensitization was evident in cells treated with agonist for 5 or 30 min. However, P2Y(2) receptor activity remained partially desensitized 30 min after pretreatment of cells with UTP for 1 h or longer. This sustained desensitized state correlated with a decrease in P2Y(2) receptor mRNA levels. Desensitization of ERK1/2 phosphorylation was induced by a 5-minute pretreatment with UTP, and cell responsiveness did not return even after a 30-minute incubation of cells in the absence of an agonist. Results suggest that desensitization of the P2Y(2) receptor may involve covalent modifications (i.e., receptor phosphorylation) that functionally uncouple the receptor from the calcium signaling pathway, and that transcriptional regulation may play a role in long-term desensitization. Our results indicate that calcium mobilization and ERK1/2 phosphorylation induced by P2Y(2) receptor activation are independent events in U937 monocytes., (Copyright 2001 Wiley-Liss, Inc.)

The P2Y(2) nucleotide receptor (P2Y(2)R) 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 P2Y(2)R to K562 erythroleukemia cells was inhibited by antibodies against alpha(V)beta(3)/beta(5) integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y(2)Rs indicated that alpha(V) integrins colocalized 10-fold better with the wild-type P2Y(2)R than with a mutant P2Y(2)R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y(2)R, 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 Ca(2+). Furthermore, an anti-alpha(V) integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y(2)R. Pertussis toxin, an inhibitor of G(i/o) proteins, partially inhibited Ca(2+) mobilization mediated by the wild-type P2Y(2)R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y(2)R-mediated activation of G(o), but not G(q). Since CD47 has been shown to associate directly with G(i/o) family proteins, these results suggest that interactions between P2Y(2)Rs, integrins, and CD47 may be important for coupling the P2Y(2)R to G(o).

UTP activates P2Y, receptors in both 1321N1 cell transfectants expressing the P2Y2 receptor and human HT-29 epithelial cells expressing endogenous P2Y, receptors with an EC50 of 0.2-1.0 microM. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC50 for UTP-induced receptor desensitization was 0.3-1.0 microM for both systems). Desensitization and down-regulation of the P2Y2 nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y2 receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y2 receptor. In these cells, potent P2Y2 receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca2+-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca2+]i, after addition of desensitizing concentrations of UTP, indicating that P2Y2 receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca2+]i induced by UTP in 1321N1 cell transfectants expressing the P2Y2 receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y2 receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50%, whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases-1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y2 receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y2 receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y2 nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y, receptor agonists.

P2 nucleotide receptor expression in cultured human retinal pigment epithelial (RPE) cells was investigated using the photoaffinity ATP analog BzATP, polymerase chain reaction of reverse-transcribed RNA (RT-PCR) and fura-2 fluorescence measurement of changes in intracellular free calcium concentration ([Ca2+]i). In experiments carried out in RPE cells at passage 10-15, addition of micromolar concentrations of ATP, UTP, and ATPgammaS to RPE cells resulted in a rapid, transient 3.5-fold increase in [Ca2+]i followed by a prolonged elevation that was twofold above the original baseline. Similar results were obtained from cells at passage 2. Characteristics of nucleotide-stimulated calcium mobilization in RPE cells, including partial inhibition by pertussis toxin, suggest that a G protein-coupled receptor mediates this response. Consistent with the expression of a P2Y2 nucleotide receptor subtype in RPE cells, [alpha-32P]BzATP labeled a 53-kDa protein in plasma membranes, and RT-PCR revealed the presence of P2Y2 receptor RNA. Adenosine had no effect on [Ca2+]i in RPE cells, indicating that the A2 subtype of P1 receptor described previously in human RPE is not involved in the response to nucleotides. Together the results indicate that human RPE cells express functional P2Y2 nucleotide receptors.

In studies designed to understand the roles of P2 nucleotide receptors in differentiation of T lymphocytes, we observed a transient and protein synthesis-independent enhancement of mRNA expression for the G protein-coupled P2Y2 receptor in mouse thymocytes after the addition of steroid hormone or T cell receptor (TCR) crosslinking by anti-TCR mAb. Conversely, dexamethasone-induced increases in mRNA expression for the ligand-gated ion channel P2X1 receptor was detected in rat, but not mouse, thymocytes, raising questions about the previously suggested role of P2X1 receptors in thymocyte apoptosis. Flow cytometry analysis of thymocyte subsets excluded the possibility that the observed increases in P2Y2 receptor mRNA expression were due to the enrichment of steroid-treated cells with an P2Y2 mRNA-rich thymocyte subset. Triggering of TCR-mediated intracellular signaling pathways through crosslinking of TCR or by addition of phorbol ester and Ca2+ ionophore also resulted in the up-regulation of P2Y2, but not P2X1, receptor mRNA. It is proposed that the rapid increase of P2Y2 receptor mRNA expression could be a common early event in responses of T cells to different activating stimuli. Taken together with the recently discovered ability of nucleotide receptor-initiated signaling to antagonize or enhance the effects of TCR crosslinking or steroids on thymocytes, the observed rapid up-regulation of P2Y2 receptor mRNA expression may reflect an immediate early gene response where newly expressed cell surface nucleotide receptors provide regulatory feedback signaling from extracellular ATP in the T cell differentiation process.

The P2Y family of receptors are G protein-coupled receptors for ATP, ADP, UTP and UDP. Recently several members of this family have been cloned, including the P2Y4, which is activated by UTP but not by ATP. In the present report, using receptors stably transfected into 1321N1 cells, we show that suramin acts as an antagonist at cloned P2Y1 and (less potently) P2Y2 receptors, but not at the cloned P2Y4 receptor. Furthermore, PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid), a potent antagonist at the P2Y1 receptor, is a relatively inneffective antagonist at the cloned P2Y4 receptor. This work moves us closer to the goal of classifying the native P2Y receptors on the basis of agonist and antagonist profiles.

The cloning of a P2U purinoceptor cDNA has made it possible to use molecular biological approaches to investigate P2U purinoceptor function. Expression of recombinant P2U purinoceptors in mammalian cells lacking endogenous P2U purinoceptors has enabled us to characterize the receptor protein and its downstream effectors, and has allowed a partial analysis of the role of certain amino acid residues in ligand binding. These approaches have placed the pharmacological classification of the P2U purinoceptor on a firm molecular footing and have generated model systems that can be used to investigate receptor-ligand binding, regulation and signal transduction.

Two subtypes of G protein-coupled receptors for nucleotides (P2U and P2Y purinoreceptors) contain several conserved positively charged amino acids in the third, sixth, and seventh putative transmembrane helices (TMHs). Since the fully ionized form of nucleotides has been shown to be an activating ligand for both P2U and P2Y purinoceptors (P2UR and P2YR), we postulated that some of these positively charged amino acids are involved in binding of the negatively charged phosphate groups of nucleotides. To investigate the role of the conserved positively charged amino acids in purinoceptor function, a series of mutant P2UR cDNAs were constructed so that lysine 107 and arginine 110 in TMH 3, histidine 262 and arginine 265 in TMH 6, and arginine 292 in TMH 7 were changed to the neutral amino acid leucine or isoleucine. The mutated P2UR cDNAs were stably expressed in 1321N1 astrocytoma cells and receptor activity was monitored by quantitating changes in the concentration of intracellular Ca2+ upon stimulation with full (ATP, UTP) or partial (ADP, UDP) P2UR agonists. Neutralization of His262, Arg265, or Arg292 caused a 100-850-fold decrease in the potency of ATP and UTP relative to the unmutated P2UR and rendered ADP and UDP ineffective. In contrast, neutralization of Lys107 or Arg110 did not alter the agonist potency or specificity of the P2UR. Neutralization of Lys289 in the P2UR, which is expressed as a glutamine residue in the P2Y subtype, did not alter receptor activity; however, a conservative change from lysine to arginine at this position altered the rank order of agonist potency so that ADP and UDP were approximately 100-fold more potent than ATP and UTP. A three-dimensional model of the P2UR indicates the feasibility of His262, Arg265, and Arg292 interactions with the phosphate groups of nucleotides.

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.

ATP and UTP can function as extracellular signaling molecules by activating plasma membrane receptors termed P2 purinergic receptors. In the present study a P2U receptor cDNA has been expressed in K-562 human leukemia cells, one of the few available mammalian cell lines that lacks an endogenous P2U receptor. In stably transfected cells, low micromolar concentrations of ATP or UTP activated the receptor, resulting in the mobilization of intracellular calcium but not the influx of extracellular calcium. A photoaffinity agonist of the P2U receptor, 3'-O-(4-benzoylbenzoyl)adenosine 5'-[alpha-32P]triphosphate ([alpha-32P]BzATP), photolabeled several proteins in plasma membranes from the stable transfectant or from untransfected K-562 cells. The photolabeling of a 53-kDa protein was significantly greater in plasma membranes from the stable transfectant than from untransfected cells. A mutant receptor containing six consecutive histidine residues at its carboxyl terminus was constructed and used to verify that this 53-kDa protein was the P2U receptor. In plasma membranes from cells expressing the histidine-tagged P2U receptor, but not from cells expressing the wild-type receptor, a single [alpha-32P]BzATP-labeled protein with a molecular mass of 53 kDa was retained on a Ni(2+)-charged Sepharose column, which binds many proteins containing a polyhistidine tag. Photolabeling of the 53-kDa protein by [alpha-32P]BzATP was inhibited by ATP but not by UTP, raising the possibility that the P2U receptor may have distinct binding sites for each nucleotide.