RECEPTOR-INDUCED TRANSIENT REDUCTION IN PLASMA-MEMBRANE PTDINS(4,5)P-2 CONCENTRATION MONITORED IN LIVING CELLS

Although phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2) is a w ell-characterized precursor for the second messengers inositol 1,4,5-t risphosphate, diacylglycerol [1] and phosphatidylinositol 3,4,5-trisph osphate [2], it also interacts with the actin-binding proteins profili n and gelsolin [3], as well as with many signaling molecules that cont ain pleckstrin homology (PH) domains [4]. It is conceivable that stimu li received by receptors in the plasma membrane could be sufficiently strong to decrease the PtdIns(4,5)P-2 concentration; this decrease cou ld alter the structure of the cortical cytoskeleton and modulate the a ctivity of signaling molecules that have PH domains. Here, we tested t his hypothesis by using an in vivo fluorescent indicator for PtdIns(4, 5)P-2, by tagging the PH domain of phospholipase C delta 1 (PLC-delta 1) with the green fluorescent protein (GFP-PH). When expressed in cell s, GFP-PH was found to be enriched at the plasma membrane. Binding stu dies in vitro and mutant analysis suggested that GFP-PH bound PtdIns(4 ,5)P-2 selectively over other phosphatidylinositol lipids. Strikingly, receptor stimulation induced a transient dissociation of GFP-PH from the plasma membrane, suggesting that the concentration of PtdIns(4,5)P -2 in the plasma membrane was effectively lowered. This transient diss ociation was blocked by the PLC inhibitor U73122 but was not affected by the phosphoinositide (PI) 3-kinase inhibitor wortmannin, suggesting that it is mostly mediated by PLC and not by PI 3-kinase activation. Overall, our studies show that PtdIns(4,5)P-2 can have second messenge r functions of its own, by mediating a transient dissociation of prote ins anchored in the plasma membrane. (C) Current Biology Ltd ISSN 0960 -9822.