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Published 6 November 2006. doi:10.1083/jcb.200607116
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 175, Number 3, 377-382
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Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells



Peter Varnai1,2, Baskaran Thyagarajan3, Tibor Rohacs3, and Tamas Balla1

1 Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
2 Department of Physiology, Semmelweis University, School of Medicine, H-1085 Budapest, Hungary
3 Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103

Correspondence to Tamas Balla: ballat{at}mail.nih.gov

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)P2) 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)P2 as monitored by the PLC{delta}1PH-GFP fusion construct. This decrease was paralleled by rapid termination of the ATP-induced Ca2+ signal and the prompt inactivation of menthol-activated transient receptor potential melastatin 8 (TRPM8) channels. Depletion of PM PtdIns(4,5)P2 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)P2 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)P2 depletion.

Abbreviations used in this paper: [Ca2+]i; cytoplasmic Ca2+; HEK, human embryonic kidney; mRFP, monomeric red fluorescent protein; mTOR, mammalian target of rapamycin; PM, plasma membrane; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; 5-ptase, phosphoinositide 5-phosphatase; rapa, rapamycin; Tf, transferrin; Tg, thapsigargin; TRPM8, transient receptor potential melastatin 8.


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