| Abstract |
Green fluorescent protein (GFP)-tagged phospholipase C (PLC)-delta1 and its mutants were expressed in Madin-Darby canine kidney (MDCK) cells. GFP-PLC-delta1 or the GFP-tagged pleckstrin homology (PH) domain of PLC-delta1 itself was found to be predominantly localized at the plasma membrane. The DeltaPH mutant or a site-directed mutant containing a PH domain which does not bind inositol 1,4, 5-trisphosphate and cannot hydrolyze phosphatidylinositol 4, 5-bisphosphate in vitro was seen only in the cytosol. In living MDCK cells hypo-osmotic stress caused a rapid dissociation of GFP-PLC-delta1 from the plasma membrane, which coincided with phosphoinositide breakdown. A PLC inhibitor, U73122, blocked this translocation, but depletion of extracellular Ca2+ had no effect. The translocation was reversed by replacement with an iso-osmotic buffer. Our results demonstrate that the PH domain plays a critical role in the membrane targeting of PLC-delta1 and that the intracellular distribution of the enzyme is regulated by osmotic stress-driven phosphoinositide turnover.
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