Inhibitors of PI(4,5)P2 Synthesis Reveal Dynamic Regulation of IgE Receptor Signaling by Phosphoinositides in RBL Mast Cells

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) is a versatile phospholipid that participates in many membrane-associated signaling processes. PI(4,5)P-2 production at the plasma membrane (PM) depends on levels of its precursor, phosphatidylinositol 4-phosphate (PI4P), synthesized principally by two intracellular enzymes, PI4-kinases III alpha 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)P-2 synthesis by PAO and quercetin. To characterize the direct effects of these agents on PI(4,5)P-2 synthesis, we monitored the reappearance of the PI(4,5)P-2-specific PH domain PH-phospholipase C delta-EGFP at the PM after Ca2+ ionophore (A23187)-induced PI(4,5)P-2 hydrolysis, followed by Ca2+ chelation with excess EGTA. Resynthesized PI(4,5)P-2 initially appears as micron-sized patches near the PM. Addition of quercetin subsequent to A23187-induced PI(4,5)P-2 hydrolysis reduces PI(4,5)P-2 resynthesis in PM-associated patches, and PAO reduces PI(4,5)P-2 at the PM while enhancing PI(4,5)P-2 accumulation at the Golgi complex. Taken together, these results provide evidence that PI4P generated by PI4-kinase III alpha is dynamically coupled to PI(4,5)P-2 pools at the PM that are important for downstream signaling processes activated by IgE receptors.