Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells.
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.
Varnai
Peter
P
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Thyagarajan
Baskaran
B
Rohacs
Tibor
T
Balla
Tamas
T
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D020013:Calcium Signaling / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002522:Chlorocebus aethiops; D066246:ErbB Receptors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism / Q000187:drug effects; D008164:Luminescent Proteins / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D051966:Phospholipase C gamma / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D011990:Receptors, Transferrin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D050053:TRPM Cation Channels / Q000378:metabolism; D022061:Tacrolimus Binding Protein 1A / Q000235:genetics; D014162:Transfection; D000097573:Red Fluorescent Protein; D000099182:Phosphoinositide 5-Phosphatases
D000255:Adenosine Triphosphate; D066246:ErbB Receptors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D051966:Phospholipase C gamma; D010744:Phosphoric Monoester Hydrolases; D011494:Protein Kinases; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D050053:TRPM Cation Channels; D022061:Tacrolimus Binding Protein 1A; D000097573:Red Fluorescent Protein; D000099182:Phosphoinositide 5-Phosphatases; C485182:Cyan Fluorescent Protein; C425966:TRPM8 protein, human; C546842:MTOR protein, human