Richard C. Garrad

P2Y2 nucleotide receptor signaling in human monocytic cells: Activation, desensitization and coupling to mitogen-activated protein kinases

Activation of P2Y2 receptors by extracellular nucleotides has been shown to induce phenotypic differentiation of human promonocytic U937 cells that is associated with the inflammatory response. The P2Y2 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 P2Y2 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 P2Y2 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 EC50 for receptor activation (from 0.44 to 9.3 μM) and a dramatic (∼75%) decrease in the maximal calcium mobilization induced by a supramaximal dose of UTP. Phorbol ester treatment also caused P2Y2 receptor desensitization (EC50 = 12.3 μM UTP and maximal calcium mobilization reduced by ∼33%). The protein kinase C inhibitor GF 109203X failed to significantly inhibit the UTP‐induced desensitization of the P2Y2 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, P2Y2 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 P2Y2 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 P2Y2 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 P2Y2 receptor activation are independent events in U937 monocytes.

Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor

UTP activates P2Y2 receptors in both 1321N1 cell transfectants expressing the P2Y2 receptor and human HT-29 epithelial cells expressing endogenous P2Y2 receptors with an EC50 of 0.2- 1.0 μM. 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 μM 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 P2Y,2 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 P2Y2 receptor agonists.

Differential agonist-induced desensitization of P2Y2 nucleotide receptors by ATP and UTP

The equal potency and efficacy of the agonists, ATP and UTP, pharmacologically distinguish the P2Y2 receptor from other nucleotide receptors. Investigation of the desensitization of the P2Y2 receptors is complicated by the simultaneous expression of different P2 nucleotide receptor subtypes. The co-expression of multiple P2 receptor subtypes in mammalian cells may have led to contradictory reports on the efficacy of the natural agonists of the P2Y2 receptor to induce desensitization. We decided to investigate the desensitization of human and murine isoforms of the P2Y2 receptor, and to rigorously examine their signaling and desensitization properties. For these purposes, we used 1321N1 astrocytoma cells stably transfected with the human or murine P2Y2 receptor cDNA, as well as human A431 cells that endogenously express the receptor. The mobilization of intracellular calcium by extracellular nucleotides was used as a functional assay for the P2Y2 receptors. While ATP and UTP activated the murine and human P2Y2 receptors with similar potencies (EC50 values were 1.5-5.8 μM), ATP was ~ 10-fold less potent (IC50 = 9.1-21.2 μM) than UTP (IC50 = 0.7-2.9 μM) inducing homologous receptor desensitization in the cell systems examined. Individual cell analyses of the rate and dose dependency of agonist-induced desensitization demonstrated that the murine receptor was slightly more resistant to desensitization than its human counterpart. To our knowledge, this is the first individual cell study that has compared the cellular heterogeneity of the desensitized states of recombinant and endogenously expressed receptors. This comparison demonstrated that the recombinant system conserved the cellular regulatory elements needed to attenuate receptor signaling by desensitization.

The Cloning and Expression of G Protein-Coupled P2Y Nucleotide Receptors

The existence of cell surface receptors for extracellular adenine nucleotides in mammalian cells has been postulated for decades (Burn-stock, 1972), based on scores of reports on diverse responses to these molecules in a wide variety of tissues and cell types (Dubyak and El-Moatassim, 1993). Pharmacological studies have provided evidence for the expression of two major types of nucleotide receptors: those belonging to the G protein-coupled receptor family and those belonging to the ligand-gated ion channel family. Nucleotide receptors (P2 purinergic receptors) are distinct from P1 receptors for adenosine, and, from results of earlier studies, were thought to be selective for purine nucleotides, in particular adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP) and various analogs, including 2MeSATP, α,β-MeATP, and β,γ-MeATP. Recent studies have demonstrated that uridine nucleotides are equally or more effective than adenine nucleotides in activating several G protein-coupled P2 receptor subtypes, raising a question as to the appropriateness of referring to this family of receptors as “purinergic.” Nonetheless, nucleotide receptors have retained the designation “P2,” which now refers to both their purine- and/or pyrimidine-based nucleotide agonists.

P2Y nucleotide receptors in the immune system : Signaling by a P2Y2 receptor in U937 monocytes

G protein‐coupled P2Y nucleotide receptors have been described in cells of the immune system, including neutrophils, monocytes, macrophages, B‐ and T‐lymphocytes, granulocytes, and myeloblasts. In the monocyte/macrophage lineage, a P2Y2 receptor subtype activated equipotently by adenosine 5′‐triphosphate (ATP) and uridine 5′‐triphosphate (UTP) is coupled to phospholipase C and regulates low density lipoprotein uptake, superoxide production, gating of calcium channels, and phagocytosis. In U937 monocytes, P2Y2 receptor activation leads to phosphorylation of MKK3 and p38, mitogen‐activated protein kinases. P2Y2 receptors in U937 monocytes undergo agonist‐induced desensitization that decreases the potency and efficacy of subsequent doses of agonist. Cells recover rapidly from desensitization after short‐term (<30 minutes) agonist treatments, whereas long‐term (>1‐hour) treatments produced sustained desensitization correlating with a decrease in P2Y2 receptor mRNA levels. To investigate the molecular determinants of desensitization, a recombinant P2Y2 receptor was expressed in human astrocytoma cells in which it exhibited agonist‐induced desensitization and sequestration. P2Y2 receptors containing C‐terminal deletions of potential phosphorylation sites for protein kinases were resistant to desensitization and sequestration. Other results indicate that an integrin‐binding domain, arginine‐glycine‐aspartate (RGD), in the first extracellular loop of the P2Y2 receptor binds specifically to αvβ3 and αvβ5 integrins (vitronectin receptors), an intriguing finding considering the wide distribution of these receptors among immune cells. The RGD domain was necessary for localizing the receptor to focal adhesion complexes to promote efficient receptor signaling. Finally, positively charged amino acids were identified in the ligand binding site of the P2Y2 receptor, information that could promote the design of compounds for selective modulation of immune function. Drug Dev. Res. 45:222–228, 1998.

Chapter 4 P2Y receptors in the nervous system: Molecular studies of a P2Y2 receptor subtype from NG108–15 neuroblastoma x glioma hybrid cells

Publisher Summary Among the variety of potential P2Y receptor subtypes that may be expressed in the nervous system, P2Y 1 and P2Y 2 receptors are the first to be cloned. A P2Y 2 receptor is cloned from a NG108-15 neuroblastoma x glioma cell cDNA library by expression cloning in Xenopus luevis oocytes and functionally expressed in mammalian cells. The cloning and expression of P2Y receptors have provided the means to address the relevance of these receptors in neurological function. There is also a need to determine P2Y receptor subtype expression patterns in vivo under a variety of physiological and pathological conditions, including cell growth and differentiation, tissue damage and neurological disorders. Ultimately, P2Y receptors may prove to be ideal targets for drug therapies in the nervous system once a fuller understanding of the structural basis for receptor function has evolved. The delineation of P2Y receptor interactions leading to receptor desensitization, sequestration, and downregulation may help control these processes to increase the effectiveness of receptor agonists. The potential that P2Y 2 receptors, in particular, may be localized to focal adhesions to participate in cell-to-cell communication suggests that these receptors may have functions yet to be recognized.

P2 Receptor Modeling and Identification of Ligand Binding Sites

As of now, seven subtypes of P2X receptors and four subtypes of P2Y receptors have been cloned and functionally identified. Analysis of the predicted amino acid sequences of these structurally diverse subtypes of nucleotide receptors has allowed construction of models that have aided in two- and three-dimensional visualization of P2X and P2Y receptors, respectively. Based on these models and on site-directed mutagenesis studies primarily with P2Y receptors, the ligand binding site of P2 receptors is being delineated. In this chapter, we present the current information on P2X and P2Y receptor structures and discuss the potential for amino acids in these receptors to interact with the nucleotide ligands.

P2 Nucleotide Receptor Structure and Function

Mammalian cells respond to extracellular ATP via a variety of proteins including ectoATPases, ectokinases and nucleotide receptors. Nucleotide receptors, also termed P2 purinoceptors, bind extracellular ATP to transduce signals in cells of most tissues. Nucleotide receptors can be classified on a structural and functional basis into two families, the P2X and P2Y receptors. The P2 receptor classification system has recently been amended due to the availability of receptor clones from both families, adding a new dimension to a nomenclature that previously relied primarily on the rank order of agonist potencies for a variety of nucleotides and synthetic analogs at each P2 receptor subtype.