Abstract
Current studies involve an investigation of the role of the pleckstrin homology (PH) domain in membrane targeting and activation of phospholipase Cbeta(1) (PLCbeta(1)). Here we report studies on the membrane localization of the isolated PH domain from the amino terminus of PLCbeta(1) (PLCbeta(1)-PH) using fluorescence microscopy of a green fluorescent protein fusion protein. Whereas PLCbeta(1)-PH does not localize to the plasma membrane in serum-starved cells, it undergoes a rapid but transient migration to the plasma membrane upon stimulation of cells with serum or lysophosphatidic acid (LPA). Regulation of the plasma membrane localization of PLCbeta(1)-PH by phosphoinositides was also investigated. PLCbeta(1)-PH was found to bind phosphatidylinositol 3-phosphate most strongly, whereas other phosphoinositides were bound with lower affinity. The plasma membrane localization of PLCbeta(1)-PH induced by serum and LPA was blocked by wortmannin pretreatment and by LY294002. In parallel, activation of PLCbeta by LPA was inhibited by wortmannin, by LY294002, or by the overexpression of PLCbeta(1)-PH. Microinjection of betagamma subunits of G proteins in serum-starved cells induced the translocation of PLCbeta(1)-PH to the plasma membrane. These results demonstrate that a cooperative mechanism involving phosphatidylinositol 3-phosphate and the Gbetagamma subunit regulates the plasma membrane localization and activation of PLCbeta(1)-PH.
Highlights
Many hormones and neurotransmitters elicit intracellular responses through their receptors by inducing the rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)1
The pleckstrin homology (PH) Domain of phospholipase C1 (PLC1) Is Localized to the Plasma Membrane—Previous studies have demonstrated that the binding of phosphoinositides to the PH domains of the PLC␥ and PLC␦
The green fluorescent protein (GFP)-PLC1-PH fusion protein was transiently overexpressed in COS-7 and HeLa cells, and the distribution of the fluorescent label was examined by confocal fluorescence microscopy
Summary
Many hormones and neurotransmitters elicit intracellular responses through their receptors by inducing the rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)1. Microinjection of ␥ subunits of G proteins in serum-starved cells induced the translocation of PLC1-PH to the plasma membrane.
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