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

Abstract

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.

Agonist-induced phosphorylation and desensitization of the P2Y2 nucleotide receptor

“…EC50 values for Ca 2+ mobilization determined with the HA-tagged P2Y 2 receptor transfected cells (0.32 μM; Table 1) were similar to those obtained by us (20) for endogenous P2Y 2 receptors in human monocytic U-937 cells (0.44 μM). A 15 min treatment with 100 μM UTP produced rapid desensitization showing an IC50 value of 6.3 μM (Table 1).…”

“…Previous reports have suggested that receptor phosphorylation plays a role in the modulation of P2Y 2 receptor desensitization and recovery (9,20). Consistent with this idea, we observed that recovery of P2Y 2 receptor signaling after agonist-induced desensitization was inhibited in 50% upon treatment with 1 μM of the protein phosphatase inhibitor okadaic acid (Figure 3).…”

“…We have previously reported that the P2Y 2 receptor undergoes rapid agonist-induced desensitization (8,9,20). Significantly, treatment of desensitized cells with the protein phosphatase inhibitor, okadaic acid, inhibited resensitization of the receptor, suggesting a role for protein phosphorylation in the regulation of P2Y 2 receptor signaling (9,20).…”

Agonist-Induced Desensitization/Resensitization of Human G Protein-Coupled Receptor 17: A Functional Cross-Talk between Purinergic and Cysteinyl-Leukotriene Ligands

“…Desensitization is a common process that regulates the functional responsiveness of both P2YRs (Otero et al, 2000;Santiago-Pérez et al, 2001) and CysLTs (Winkler et al, 1988). Heterologous regulation between P2YRs and CysLTs has been demonstrated in human monocyte/macrophage-like cells, in which the activation of P2YRs with ATP or UDP induced the desensitization of the CysLT 1 .…”

P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrowhematopoietic stem cells

“…A well-defined effect of nucleotides on platelet activation and vascular smooth muscle migration and growth has suggested participation of P2Y receptors in the response to injury. More recently, these receptors have been noted to affect cellular constituents of the innate immune system altering functional characteristics of monocytes, eosinophils, and dendritic cells and to play a critical role in terminating the inflammatory response in vivo (Mutini et al 1999;Ferrari et al 2000;Idzko et al 2001;Santiago-Perez et al 2001;Warny et al 2001;Wilkin et al 2001). The P2Y receptor specificity originally thought to be restricted to purine (adenine) nucleotides has been extended to pyrimidine nucleotides (uridine) and more recently to a receptor with specificity for UDP, but only when conjugated to glucose or related sugars (Chambers et al 2000).…”