Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools.
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.
Várnai
P
P
Endocrinology and Reproduction Research Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
Balla
T
T
D016475:3T3 Cells / Q000737:chemistry / Q000201:enzymology; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D019556:COS Cells / Q000737:chemistry / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008164:Luminescent Proteins; D051379:Mice; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D010750:Phosphoproteins; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary; D014162:Transfection; D014316:Tritium; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism
D001798:Blood Proteins; D002614:Chelating Agents; D007202:Indicators and Reagents; D007476:Ionophores; D008164:Luminescent Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins; C039077:platelet protein P47; D014316:Tritium; D049452:Green Fluorescent Proteins; D004533:Egtazic Acid; D015759:Ionomycin; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium