ABSTRACT
Nucleotides released from cells due to stress, injury or inflammation, induce mitogenic effects in monocytes via activation of P2Y(2) nucleotide receptors (P2Y(2)Rs). Here we show that P2Y(2) nucleotide receptors in U937 monocytic cells regulate the activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) by inducing the clustering of alpha(v) integrins. The activation of phosphatidylinositol 3-kinase by P2Y(2)R ligands was required for alpha(v) clustering, suggesting a means whereby two different classes of receptors communicate to induce mitogenic responses in monocytic cells. P2Y(2)R-induced alpha(v) clustering was also associated with a flattened phenotype of the U937 cells, consistent with the role of the P2Y(2)R in regulating early events in cell migration.
Subject(s)
Cell Membrane/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Integrin alphaV/metabolism , Monocytes/metabolism , Receptors, Purinergic P2/metabolism , Uridine Triphosphate/metabolism , Calcium/metabolism , Cell Membrane/drug effects , Cell Movement , Cell Proliferation , Cell Shape , Enzyme Activation , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Monocytes/cytology , Monocytes/drug effects , Phenotype , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Transport , Purinergic P2 Receptor Agonists , RNA, Messenger/metabolism , Receptor Aggregation , Receptor Cross-Talk , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2Y2 , Time Factors , U937 Cells , Uridine Triphosphate/pharmacologyABSTRACT
IEX-1 (immediate early response gene X-1) is a stress-inducible gene. Its overexpression can suppress or enhance apoptosis dependent on the nature of stress, yet the polypeptide does not possess any of the functional domains that are homologous to those present in well characterized effectors or inhibitors of apoptosis. This study using sequence-targeting mutagenesis reveals a transmembrane-like integrated region of the protein to be critical for both pro-apoptotic and anti-apoptotic functions. Substitution of the key hydrophobic residues with hydrophilic ones within this region impairs the capacity IEX-1 to positively and negatively regulate apoptosis. Mutations at N-linked glycosylation and phosphorylation sites or truncation of the C terminus of IEX-1 also abrogated its potential to promote cell survival. However, distinguished from the transmembrane-like domain, these mutants preserved pro-apoptotic activity of IEX-1 fully. On the contrary, mutation of nuclear localization sequence, despite its importance in apoptosis, did not impede IEX-1-mediated cell survival. Strikingly, all the mutants that lose their anti-apoptotic ability are unable to prevent acute increases in production of intracellular reactive oxygen species (ROS) at the initial onset of apoptosis, whereas those mutants that can sustain anti-death function also control acute ROS production as sufficiently as wild-type IEX-1. These findings suggest a critical role of IEX-1 in regulation of intracellular ROS homeostasis, providing new insight into the mechanism underlying IEX-1-mediated cell survival.