RT Journal Article SR Electronic T1 Inhibitors of PI(4,5)P2 Synthesis Reveal Dynamic Regulation of IgE Receptor Signaling by Phosphoinositides in RBL Mast Cells JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 793 OP 804 DO 10.1124/mol.112.082834 VO 83 IS 4 A1 Marcela de Souza Santos A1 Rose Mary Zumstein Georgetto Naal A1 Barbara Baird A1 David Holowka YR 2013 UL http://molpharm.aspetjournals.org/content/83/4/793.abstract AB 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.