Visualization of Phosphoinositides That Bind Pleckstrin Homology Domains: Calcium- and Agonist-induced Dynamic Changes and Relationship to Myo-[

Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P 2 ) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC) d PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of the fluorescent probe required the presence of three basic residues within the PLC d PH domain known to form critical contacts with PtdIns(4,5)P 2 . Activation of endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent signal from the membrane to cytosol, which was reversed after Ca 2 1 chelation. In both ionomycinand agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P 2 . Inhibition of PtdIns(4,5)P 2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca 2 1 chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P 2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[ 3 H]inositol– labeled PtdIns(4,5)P 2 . Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.