Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells

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 PLCδ1PH-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)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.

Rapid Turnover Rate of Phosphoinositides at the Front of Migrating MDCK Cells

“…On treatment with rapamycin, FKBP-5ptase-dom is recruited to the plasma membrane, where another rapamycin ligand, a fragment of the mammalian target of rapamycin (FRB), is localized by the attached myristylation-moiety ( Figure 2H). The translocation of FKBP-5ptase-dom dephosphorylates PI(4,5)P 2 to yield PI(4)P at the plasma membrane (Varnai et al, 2006). Intriguingly, the increase in PI(4)P was not apparent at the plasma membrane; however, the biosensor was accumulated at perinuclear vesicular structures concomitant with an increase in FRET ( Figure 2H).…”

“…As expected, the change in the FRET/CFP ratio was not observed in cells expressing Raichu-PAK-RhoA (Supplemental Figure S1F). Next, we examined whether Pippi-PI(4)P could monitor the increase in PI(4)P by using a rapamycin-inducible membrane translocation system (Varnai et al, 2006;Aoki et al, 2007). FKBP5ptase-dom is a fusion protein consisting of the catalytic domain of phosphoinositide 5-phosphatase (5ptase) and the FK506-binding protein (FKBP), a ligand for rapamycin.…”

Regulation of EGFR nanocluster formation by ionic protein-lipid interaction

“…To avoid potential artifacts caused by the constitutive PIP2 depletion by SJ2, we established an inducible PIP2 depletion system to modify PIP2 level in the plasma membrane [29]. We transfected COS-7 cells with two constructs: Lyn-FKBP 12 and mCitrine-FRB-SJ2.…”

Hypothesis review: are clathrin-mediated endocytosis and clathrin-dependent membrane and protein trafficking core pathophysiological processes in schizophrenia and bipolar disorder?

“…Depleting PIP2 in living cells results in AP2 mislocalization and inhibition of endocytosis [152][153][154][155][156] . PIP2 is important in psychotic disorders, as it is part of the phosphoinositide-protein kinase C (PI-PKC) pathway, which is inhibited by many psychotropic medications.…”