P2Y(2) nucleotide receptor signaling in human monocytic cells: Activation,desensitization, and coupling to mitogen-activated protein kinases

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 cou ple to MAP kinases via phosphatidylinositol 3-kinase (P13K) and c-src. Acco rdingly, ERK1/2 phosphorylation induced by UTP was inhibited by the P13K in hibitors, wortmannin and LY294002, and the c-src inhibitors, radicicol and PP2, but not by inhibitors of protein kinase C (PKC). The phosphorylation o f ERK1/2 was independent of the ability of P2Y(2) receptors to increase the concentration of intracellular free calcium, since chelation of intracellu lar 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 b y an increase in the EC50 for receptor activation (from 0.44 to 9.3 muM) an d a dramatic (similar to 75%) decrease in the maximal calcium mobilization induced by a supramaximal dose of UTP. Phorbol ester treatment also caused P2Y(2) receptor desensitization (EC50 = 12.3 muM UTP and maximal calcium mo bilization reduced by similar to 33%). The protein kinase C inhibitor GF 10 9203X failed to significantly inhibit the UTP-induced desensitization of th e P2Y(2) receptor, whereas the protein phosphatase inhibitor okadaic acid b locked receptor resensitization. Recovery of receptor activity after UTP-in duced desensitization was evident in cells treated with agonist for 5 or 30 min. However, P2Y2 receptor activity remained partially desensitized 30 mi n after pretreatment of cells with UTP for 1 h or longer. This sustained de sensitized state correlated with a decrease in P2Y(2) receptor mRNA levels. Desensitization of ERK1/2 phosphorylation was induced by a 5-minute pretre atment with UTP, and cell responsiveness did not return even after a 30-min ute 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 fro m the calcium signaling pathway, and that transcriptional regulation may pl ay a role in long-term desensitization. Our results indicate that calcium m obilization and ERK1/2 phosphorylation induced by P2Y(2) receptor activatio n are independent events in U937 monocytes. (C) 2001 Wiley-Liss, Inr.