TY - JOUR T1 - Inhibitors of PI(4,5)P<sub>2</sub> Synthesis Reveal Dynamic Regulation of IgE Receptor Signaling by Phosphoinositides in RBL Mast Cells JF - Molecular Pharmacology JO - Mol Pharmacol SP - 793 LP - 804 DO - 10.1124/mol.112.082834 VL - 83 IS - 4 AU - Marcela de Souza Santos AU - Rose Mary Zumstein Georgetto Naal AU - Barbara Baird AU - David Holowka Y1 - 2013/04/01 UR - http://molpharm.aspetjournals.org/content/83/4/793.abstract N2 - Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a versatile phospholipid that participates in many membrane-associated signaling processes. PI(4,5)P2 production at the plasma membrane (PM) depends on levels of its precursor, phosphatidylinositol 4-phosphate (PI4P), synthesized principally by two intracellular enzymes, PI4-kinases IIIα and IIIb; the former is preferentially inhibited by phenylarsine oxide (PAO). We found that PAO and quercetin, another lipid kinase inhibitor, rapidly inhibit Ca2+ responses to antigen in IgE-sensitized rat basophilic leukemia mast cells. Quercetin also rapidly inhibits store-operated Ca2+ influx stimulated by thapsigargin. In addition, quercetin and PAO effectively inhibit antigen-stimulated ruffling and spreading in these cells, and they inhibit endocytosis of crosslinked IgE receptor complexes, evidently by inhibiting pinching off of endocytic vesicles containing the clustered IgE receptors. A minimal model to account for these diverse effects is inhibition of PI(4,5)P2 synthesis by PAO and quercetin. To characterize the direct effects of these agents on PI(4,5)P2 synthesis, we monitored the reappearance of the PI(4,5)P2-specific PH domain PH-phospholipase C δ-EGFP at the PM after Ca2+ ionophore (A23187)-induced PI(4,5)P2 hydrolysis, followed by Ca2+ chelation with excess EGTA. Resynthesized PI(4,5)P2 initially appears as micron-sized patches near the PM. Addition of quercetin subsequent to A23187-induced PI(4,5)P2 hydrolysis reduces PI(4,5)P2 resynthesis in PM-associated patches, and PAO reduces PI(4,5)P2 at the PM while enhancing PI(4,5)P2 accumulation at the Golgi complex. Taken together, these results provide evidence that PI4P generated by PI4-kinase IIIα is dynamically coupled to PI(4,5)P2 pools at the PM that are important for downstream signaling processes activated by IgE receptors. ER -