Abstract
Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)delta 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 PLCdelta PH domain known to form critical contacts with PtdIns(4, 5)P2. 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 Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution closely correlated with changes in absolute mass of PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after Ca2+ chelation in ionomycin-treated cells or during agonist stimulation. In contrast, the synthesis of the PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been found inhibitory of the synthesis of myo-[3H]inositol- labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains will further our understanding of the regulation of such proteins by inositol phospholipids.
Highlights
Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)␦ PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells
To investigate whether the PtdIns(4,5)P2 pools that are visualized by PHPLC␦–green fluorescent protein (GFP) show any change after agonist stimulation, we examined the effect of the calcium mobilizing hormone, Angiotensin II (Ang II), on the redistribution of the fluorescent probe in various cells
10 M WT had no effect on the Ang II–induced transient translocation of the fluorescent signal. This result contrasted our previous finding that the metabolically labeled hormone-sensitive PtdIns(4,5)P2 pools are maintained by WT-sensitive PtdIns 4-kinase(s) in several agonist-stimulated cells and that stimulation of these cells in the presence of 10 M WT leads to the depletion of these phosphoinositide pools in myo-[3H]inositol–labeled cells (27). These findings suggest that the myo-[3H]inositol–labeled, agonist-sensitive PtdIns(4,5)P2 pools are formed by WT-sensitive PtdIns 4-kinase(s), additional PtdIns(4,5)P2 pools that bind PH domains are synthesized by WT-insensitive mechanisms that are only inhibited by less-specific PI kinase inhibitors, such as quercetin or phenylarsine oxide (PAO) (34)
Summary
Phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) pools that bind pleckstrin homology (PH) domains were visualized by cellular expression of a phospholipase C (PLC)␦ PH domain–green fluorescent protein fusion construct and analysis of confocal images in living cells. Whereas in vitro binding studies have been valuable in determining the ability of PH domains to associate with inositol phospholipids, the dependency of the specificity of their binding on lipid micelle composition and detergent environment (21) emphasizes the need to assess these interactions in intact cells. Another question that is raised by studies on PH domain–phosphoinositide interactions is how the binding of PH domains to the inositide headgroup affects the primary signaling functions of PtdIns(4,5)P2, its availability to PLC enzymes or to PtdIns 3 kinases. It is known that other proteins that bind PtdIns(4,5)P2, such as profilin, greatly impair the ability of PLC enzymes to hydrolyze this lipid (14)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
