Laurie Erb

P2 receptors: intracellular signaling

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.

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.

PPADS and suramin as antagonists at cloned P2Y- and P2U-purinoceptors.

1 The effect of suramin and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) on the stimulation of phospholipase C in 1321N1 cells transfected with the human P2U‐purinoceptor (h‐P2U‐1321N1 cells) or with the turkey P2Y‐purinoceptor (t‐P2Y‐1321N1 cells) was investigated. 2‐Methylthioadenosine triphosphate (2MeSATP) was used as the agonist at t‐P2Y‐1321N1 cells and uridine triphosphate (UTP) at h‐P2U‐1321N1 cells. 2 Suramin caused a parallel shift to the right of the concentration‐response curves for 2MeSATP in the t‐P2Y‐1321N1 cells, yielding a Schild plot with a slope of 1.16 ± 0.08 and a pA2 value of 5.77 ± 0.11. 3 Suramin also caused a shift to the right of concentration‐response curves for UTP in the h‐P2U‐1321N1 cells, and on Schild plots gave a slope different from unity (1.57 ± 0.19) and an apparent pA2 value of 4.32 ± 0.13. Suramin was therefore a less potent antagonist at the P2U‐purinoceptor than the P2Y‐purinoceptor. 4 In the presence of the ectonucleotidase inhibitor, ARL 67156 (6‐N,N‐diethyl‐β,γ‐dibromomethylene‐D‐ATP) there was no significant difference in the EC50 or shapes of curves with either cell type, and no difference in pA2 values for suramin. 5 PPADS caused an increase in the EC50 for 2MeSATP in the t‐P2Y‐1321N1 cells. The Schild plot had a slope different from unity (0.55 ± 0.15) and an X‐intercept corresponding to an apparent pA2 of 5.98 ± 0.65. 6 PPADS up to 30 μm had no effect on the concentration‐response curve for UTP with the h‐P2U‐1321N1 cells. 7 In conclusion, suramin and PPADS show clear differences in their action at the 2 receptor types, in each case being substantially more effective as an antagonist at the P2Y‐purinoceptor than at the P2U‐purinoceptor. Ectonucleotidase breakdown had little influence on the nature of the responses at the two receptor types, or in their differential sensitivity to suramin.

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.

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.

Modulation of endothelial cell migration by extracellular nucleotides. Involvement of focal adhesion kinase and phosphatidylinositol 3-kinase - mediated pathways

Extracellular nucleotides bind to type-2 purinergic/pyrimidinergic (P2) receptors that mediate various responses, such as cell activation, proliferation and apoptosis, implicated in inflammatory processes. The role of P2 receptors and their associated signal transduction pathways in endothelial cell responses has not been fully investigated. Here, it is shown that stimulation of human umbilical vein endothelial cells (HUVEC) with extracellular ATP or UTP increased intracellular free calcium ion concentrations ([Ca(2+)](i)), induced phosphorylation of focal adhesion kinase (FAK), p130(cas) and paxillin, and caused cytoskeletal rearrangements with consequent cell migration. Furthermore, UTP increased migration of HUVEC in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. BAPTA or thapsigargin inhibited the extracellular nucleotide-induced increase in [Ca(2+)](i), a response crucial for both FAK phosphorylation and cell migration. Furthermore, long-term exposure of HUVEC to ATP and UTP, agonists of the G protein-coupled P2Y2 and P2Y4 receptor subtypes, caused upregulation of alpha(v) integrin expression, a cell adhesion molecule known to directly interact with P2Y2 receptors. Our results suggest that extracellular nucleotides modulate signaling pathways in HUVEC influencing cell functions, such as cytoskeletal changes, cellular adhesion and motility, typically associated with integrin-activation and the action of growth factors. We propose that P2Y2 and possibly P2Y4 receptors mediate those responses that are important in vascular inflammation, atherosclerosis and angiogenesis.

Cloned and transfected P2Y4 receptors: characterization of a suramin and PPADS-insensitive response to UTP

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.