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 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.
Santiago-Pérez
L I
LI
Department of Chemistry, University of Puerto Rico, San Juan, Puerto Rico.
Flores
R V
RV
Santos-Berríos
C
C
Chorna
N E
NE
Krugh
B
B
Garrad
R C
RC
Erb
L
L
Weisman
G A
GA
González
F A
FA
D002118:Calcium / Q000378:metabolism; D006801:Humans; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D020935:MAP Kinase Signaling System / Q000276:immunology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009000:Monocytes / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D009711:Nucleotides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D020298:U937 Cells
D002118:Calcium; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D009711:Nucleotides; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; C546385:P2RY2 protein, human; C482128:MAP2K2 protein, human; C482125:MAP2K1 protein, human