REGULATION AND INTRACELLULAR LOCALISATION OF PHOSPHOIN OSITIDE-BINDING PROTEINS.

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Ligand recognition, intracellular localization and physiological importance of phosphoinositide-binding proteins have been examined. It was found that certain basic amino acids in the pleckstrin homology (PH) domain are important for specific ligand recognition, and therefore, responsible for interaction of PH-containing proteins with the plasma membrane. This was especially the case for phospholipase C-δ1 (PLCδ1). We demonstrated that osmotic stress caused a rapid dissociation of the fluorescence from the plasma membrane of renal epithelial cells expressing GFP-tagged PLCδ1 or its PH domain. The dissociation rate increased when cells were treated with cytoskeleton-disrupting reagents, suggesting the PtdIns(4,5)P2 hydrolysing actuvity is regulated by juxtamembrane structures. Released GFP-PLCδ1 translocated to perinuclear regions, presumably ER, but the mechanism of targeting may be different from those of PLA2 (and PKC) whose C2 domain may play a role. Although little is present in the nucleus under normal conditions, PLCδ1 does shuttle between the cytosol (plus the plasma membrane) and the nucleus. We have shown that PLCδ1 has a functional nuclear export signal sequence, and disruption of it or inhibition of the crm1/exportin-dependent nuclear exp ort could let the enzyme stay in the nucleus. This finding would provide further insights into the nuclear phosphoinositide signalling, since new roles of inositol polyphosphates and their kinases have been postulated. We then explored roles of PtdIns(4,5)P2/PLC in cell movements using a free living amoeba as a model. When membrane sheets prepared from amoebae were treated with PLCδ1-PH or PLCδ1, a mesh work structure of actin filaments gradually disappeared. Furthermore, a specific inhibitor of PLC reversibly blocked the pseudopod formation and the cell movement in vivo, suggesting that remodelling of cytoskeleton is regulated by Ca^<2+>-sensitive PLC that hy droly ses PtdIns(4,5)P2.