Abstract
Rapamycin (rapa)-induced heterodimerization of the FRB domain of the mammalian target of rapa and FKBP12 was used to translocate a phosphoinositide 5-phosphatase (5-ptase) enzyme to the plasma membrane (PM) to evoke rapid changes in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) levels. Rapa-induced PM recruitment of a truncated type IV 5-ptase containing only the 5-ptase domain fused to FKBP12 rapidly decreased PM PtdIns(4,5)P(2) as monitored by the PLCdelta1PH-GFP fusion construct. This decrease was paralleled by rapid termination of the ATP-induced Ca(2+) signal and the prompt inactivation of menthol-activated transient receptor potential melastatin 8 (TRPM8) channels. Depletion of PM PtdIns(4,5)P(2) was associated with a complete blockade of transferrin uptake and inhibition of epidermal growth factor internalization. None of these changes were observed upon rapa-induced translocation of an mRFP-FKBP12 fusion protein that was used as a control. These data demonstrate that rapid inducible depletion of PM PtdIns(4,5)P(2) is a powerful tool to study the multiple regulatory roles of this phospholipid and to study differential sensitivities of various processes to PtdIns(4,5)P(2) depletion.
Publication types
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Research Support, N.I.H., Intramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Adenosine Triphosphate / pharmacology
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Animals
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COS Cells
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Calcium Signaling / drug effects
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Cell Membrane / drug effects
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Cell Membrane / metabolism*
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Chlorocebus aethiops
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ErbB Receptors / metabolism
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Green Fluorescent Proteins / metabolism
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Humans
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Lipid Metabolism* / drug effects
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Luminescent Proteins / metabolism
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Phosphatidylinositol 4,5-Diphosphate / metabolism*
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Phospholipase C gamma / metabolism
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Phosphoric Monoester Hydrolases / genetics
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Phosphoric Monoester Hydrolases / metabolism*
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Protein Kinases / metabolism
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Protein Structure, Tertiary
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Protein Transport / drug effects
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Receptors, Transferrin / metabolism
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Recombinant Fusion Proteins / metabolism
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Red Fluorescent Protein
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Sirolimus / pharmacology
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TOR Serine-Threonine Kinases
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TRPM Cation Channels / metabolism
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Tacrolimus Binding Protein 1A / genetics
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Transfection
Substances
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Cyan Fluorescent Protein
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Luminescent Proteins
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Phosphatidylinositol 4,5-Diphosphate
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Receptors, Transferrin
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Recombinant Fusion Proteins
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TRPM Cation Channels
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TRPM8 protein, human
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Green Fluorescent Proteins
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Adenosine Triphosphate
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Protein Kinases
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MTOR protein, human
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ErbB Receptors
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TOR Serine-Threonine Kinases
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Phosphoric Monoester Hydrolases
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phosphoinositide 5-phosphatase
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Phospholipase C gamma
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Tacrolimus Binding Protein 1A
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Sirolimus