Fernando A. González

Src homology 3 binding sites in the P2Y2 nucleotide receptor interact with Src and regulate activities of Src, proline-rich tyrosine kinase 2, and growth factor receptors.

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 P2Y2 nucleotide receptor that directly associate with the tyrosine kinase Src in protein binding assays. Furthermore, Src co-precipitated with the P2Y2 receptor in 1321N1 astrocytoma cells stimulated with the P2Y2 receptor agonist UTP. A mutant P2Y2 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 P2Y2 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 P2Y2 receptor facilitates Src activation, which recruits the EGFR into a protein complex with the P2Y2 receptor and allows Src to efficiently phosphorylate the EGFR.

P2X7 receptors stimulate AKT phosphorylation in astrocytes

Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X7 subtype, are coupled to the phosphoinositide 3‐kinase (PI3K)/Akt pathway in astrocytes. P2Y and P2X receptor agonists ATP, uridine 5′‐triphosphate (UTP) and 2′,3′‐O‐(4‐benzoyl)‐benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration‐ and time‐dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X7 receptor. Activation was maximal at 5 – 10 min and was sustained for 60 min; the EC50 for BzATP was approximately 50 μM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X7 receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso‐pyridoxal‐5′‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid, but was unaffected by trinitrophenyl‐ATP, oxidized ATP, suramin and reactive blue 2. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP‐induced Akt phosphorylation pathway. In conclusion, our data indicate that stimulation of astrocytic P2X7 receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism.

P2Y2 Nucleotide Receptors Enhance α-Secretase-dependent Amyloid Precursor Protein Processing

The amyloid precursor protein (APP) is proteolytically processed by β- and γ-secretases to release amyloid β, the main component in senile plaques found in the brains of patients with Alzheimer disease. Alternatively, APP can be cleaved within the amyloid β domain by α-secretase releasing the non-amyloidogenic product sAPPα, which has been shown to have neuroprotective properties. Several G protein-coupled receptors are known to activate α-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 sAPPα in a time- and dose-dependent manner. P2Y2 R-mediated sAPPα 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 P2Y2 R-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 sAPPα release, indicating a PKC-independent mechanism. U0126, an inhibitor of the mitogen-activated protein kinase pathway, partially inhibited sAPPα release by UTP, whereas inhibitors of Src-dependent epidermal growth factor receptor transactivation by P2Y2 Rs had no effect. The metalloprotease inhibitors phenanthroline and TAPI-2 and the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone also diminished UTP-induced sAPPα release. Furthermore, small interfering RNA silencing of an endogenous adamalysin, ADAM10 or ADAM17/TACE, partially suppressed P2Y2R-activated sAPPα release, whereas treatment of cells with both ADAM10 and ADAM17/TACE small interfering RNAs completely abolished UTP-activated sAPPα release. These results may contribute to an understanding of the non-amyloidogenic processing of APP.

The P2Y2 Nucleotide Receptor Mediates Vascular Cell Adhesion Molecule-1 Expression through Interaction with VEGF Receptor-2 (KDR/Flk-1)

UTP stimulates the expression of pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells through activation of the P2Y2 nucleotide receptor P2Y2R. Here, we demonstrated that activation of the P2Y2R 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 P2Y2R-mediated VCAM-1 expression. Furthermore, VEGFR-2 and the P2Y2R co-localize upon UTP stimulation. Deletion or mutation of two Src homology-3-binding sites in the C-terminal tail of the P2Y2R or inhibition of Src kinase activity abolished the P2Y2R-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 P2Y2R-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.

Functional P2Y2 nucleotide receptors mediate uridine 5′-triphosphate-induced intimal hyperplasia in collared rabbit carotid arteries

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 (Cam) in response to P2Y receptor agonists. In contrast to UDP, activation by UTP or adenosine 5′-triphosphate (ATP) greatly increased Cam, 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 &mgr;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, 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.

P2Y2 receptors activate neuroprotective mechanisms in astrocytic cells

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.

Phospholipase A2 in astrocytes: responses to oxidative stress, inflammation, and G protein-coupled receptor agonists.

Astrocytes comprise the major cell type in the central nervous system (CNS) and they are essential for support of neuronal functions by providing nutrients and regulating cell-to-cell communication. Astrocytes also are immune-like cells that become reactive in response to neuronal injury. Phospholipases A2 (PLA2) are a family of ubiquitous enzymes that degrade membrane phospholipids and produce lipid mediators for regulating cellular functions. Three major classes of PLA2 are expressed in astrocytes: group IV calcium-dependent cytosolic PLA2 (cPLA2), group VI calcium-independent PLA2 (iPLA2), and group II secretory PLA2 (sPLA2). Upregulation of PLA2 in reactive astrocytes has been shown to occur in a number of neurodegenerative diseases, including stroke and Alzheimer’s disease. This review focuses on describing the effects of oxidative stress, inflammation, and activation of G protein-coupled receptors on PLA2 activation, arachidonic acid (AA) release, and production of prostanoids in astrocytes.

The P2Y2 Nucleotide Receptor Interacts with αv Integrins to Activate Go and Induce Cell Migration

Extracellular ATP and UTP induce chemotaxis, or directed cell migration, by stimulating the G protein-coupled P2Y2 nucleotide receptor (P2Y2R). Previously, we found that an arginine-glycine-aspartic acid (RGD) integrin binding domain in the P2Y2R enables this receptor to interact selectively with αvβ3 and αVβ5 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 P2Y2R to Go- but not Gq-mediated intracellular calcium mobilization, leading us to investigate the role of P2Y2R interaction with integrins in nucleotide-induced chemotaxis. Here we show that mutation of the RGD sequence to RGE in the human P2Y2R expressed in 1321N1 astrocytoma cells completely prevented UTP-induced chemotaxis as well as activation of Go, 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 αvβ3/β5 integrins, in cells expressing the wild-type but not the RGE mutant P2Y2R. P2Y2R-mediated chemotaxis, Rac and Vav2 activation, and vitronectin up-regulation were inhibited by pretreatment of the cells with anti-αvβ5 integrin antibodies, αv integrin antisense oligonucleotides, or the Gi/o inhibitor, pertussis toxin. Thus, the RGD-dependent interaction between the P2Y2R and αv integrins is necessary for the P2Y2R to activate Go and to initiate Go-mediated signaling events leading to chemotaxis.

P2X7 nucleotide receptor activation enhances IFNγ‐induced type II nitric oxide synthase activity in BV‐2 microglial cells

Under normal and pathological conditions, brain cells release nucleotides that regulate a wide range of cellular responses due to activation of P2 nucleotide receptors. In this study, the effect of extracellular nucleotides on IFNγ‐induced NO release in murine BV‐2 microglial cells was investigated. BV‐2 cells expressed mRNA for metabotropic P2Y and ionotropic P2X receptors. Among the P2 receptor agonists tested, ATP, ADP, 2′,3′‐O‐(4‐benzoylbenzoyl)‐ATP (BzATP), and 2‐methylthio‐ATP (2‐MeSATP), but not UTP, enhanced IFNγ‐induced iNOS expression and NO production, suggesting that the uridine nucleotide receptors P2Y2 and P2Y6 are not involved in this response. U0126, an antagonist for MEK1/2, a kinase that phosphorylates the extracellular signal‐regulated kinases ERK1/2, decreased IFNγ‐induced NO production. BzATP, a potent P2X7 receptor agonist, was more effective than ATP, ADP, or 2‐MeSATP at enhancing IFNγ‐induced ERK1/2 phosphorylation. Consistent with activation of the P2X7 receptor, periodate‐oxidized ATP, a P2X7 receptor antagonist, and suramin, a non‐specific P2 receptor antagonist, inhibited the effect of ATP or BzATP on IFNγ‐induced NO production, whereas pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), an antagonist of several P2X receptor subtypes, was ineffective. These results suggest that activation of P2X7 receptors may contribute to inflammatory responses in microglial cells seen in neurodegenerative diseases.

P2Y2 nucleotide receptor interaction with αV integrin mediates astrocyte migration

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 P2Y2 receptor for ATP and UTP increases GFAP expression and both chemotactic and chemokinetic cell migration. UTP‐induced astrocyte migration was inhibited by silencing of P2Y2 nucleotide receptor (P2Y2R) expression with siRNA of P2Y2R (P2Y2R siRNA). UTP also increased the expression in astrocytes of αVβ3/5 integrins that are known to interact directly with the P2Y2R to modulate its function. Anti‐αV integrin antibodies prevented UTP‐stimulated astrocyte migration, suggesting that P2Y2R/αV interactions mediate the activation of astrocytes by UTP. P2Y2R‐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‐αV integrin antibody. These results suggest that P2Y2Rs and their associated signaling pathways may be important factors regulating astrogliosis in brain disorders.