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 μM) and a dramatic (∼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 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 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. © 2001 Wiley‐Liss, Inc.