Monitoring changes in membrane phosphatidylinositol 4,5‐bisphosphate in living cells using a domain from the transcription factor tubby

Abstract

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) is a key component in signal transduction, being a precursor to other signalling molecules and itself associated with roles in signal transduction and cell biology. Tubby is a membrane bound transcription factor whose dysfunction results in obesity in mice. It contains a domain that selectively binds PtdIns(4,5)P 2 . We have investigated the use of a fluorescently tagged version of this domain to monitor changes in PtdIns(4,5)P 2 concentration in living cells and compared it to the pleckstrin homology domain of PLCδ1. Our results show that selected mutants of this domain report receptor-mediated changes in cellular PtdIns(4,5)P 2 . In contrast to the pleckstrin homology domain of PLCδ1 it does not have a significant affinity for inositol 1,4,5-trisphosphate (IP 3 ). Using a selected mutant, we examine the regulation of ATP-sensitive K + channels via a G q/11 -coupled receptor. These experiments reveal a correlation between reporter translocation and the onset of current inhibition whilst the recovery of current after agonist removal is delayed when compared to the reporter. Furthermore our studies reveal the importance of Ca 2+ in determining the overall activity of phospholipase C in living cells. This probe may be valuable in examining changes in PtdIns(4,5)P 2 distinct from those of IP 3 in intact cells in a variety of physiological settings.

Phosphoinositides Differentially Regulate Protrudin Localization through the FYVE Domain

“…To study roles of key signaling lipids, PtdIns(4,5)P 2 , and PtdIns(3,4,5)P 3 in the regulation of cellular proteins in a simple, comprehensive, and explicit manner, we generated a library of distinct LBDs obtained from diverse cellular proteins and determined their subcellular localization. Our study showed that most FYVE and PX domains are endosome-localized, whereas ENTH and Tubby domains are PM-associated, as expected from their reported lipid specificity (42)(43)(44). The study also revealed that the FYVE domain of protrudin is localized to PM and the nucleus instead of endosomes, unlike typical FYVE domains (45).…”

“…1, C and D), five of them belonged to the ENTH and Tubby domains (Fig. 1, C and E), which are known to interact with PtdIns(4,5)P 2 (42)(43)(44). Intriguingly, the other PM-localized LBD was the FYVE domain of protrudin (Fig.…”

Phosphatidylinositol 4-Phosphate 5-Kinase α Facilitates Toll-like Receptor 4-mediated Microglial Inflammation through Regulation of the Toll/Interleukin-1 Receptor Domain-containing Adaptor Protein (TIRAP) Location*

“…Mouse type I PIP5Ks cloned into pcDNA3-Myc vector were used as DNA templates for quantitative real time PCR (qRT-PCR) analysis. The YFP-tagged tubby mutant (R332H) was provided by Andrew Tinker (University College London, UK) (38), and TIRAP-GFP and HA-TIRAP were gifts from Ruslan Medzhitov (Yale University). PCR-amplified inserts of Tubby or TIRAP were subcloned into the EcoRI-XhoI sites of the mRFP-pcDNA3 vector to generate mRFP-Tubby-R332H and mRFP-TIRAP.…”

The acute nociceptive signals induced by bradykinin in rat sensory neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+-activated Cl– channels

“…Imaging experiments (Figure 1) also suggest that despite activation of PLC by B 2 R, the overall membrane PIP 2 level in most DRG neurons may not change dramatically, since YFP-tubby (PIP 2 reporter that does not bind IP 3 ; ref. 19) did not translocate in response to BK application (until B 2 Rs were overexpressed).…”

“…However, since PLCδ-PH-GFP has a higher affinity for IP 3 than for PIP 2 , translocation of the probe does not necessarily indicate a significant drop in membrane PIP 2 levels, as even small amounts of hydrolyzed PIP 2 may produce enough IP 3 to cause the probe to translocate (13,18). Thus, we used a new PIP 2 probe (YFP-tubby, R332H mutated), which does not bind IP 3 (19), to probe whether there was a significant depletion of the membrane PIP 2 pool. Like PLCδ-PH-GFP, the YFP-tubby also localized predominantly to the plasma membrane ( Figure 1B); however, unlike PLCδ-PH-GFP, YFP-tubby failed to translocate to the cytosol after BK application in 11 of 12 neurons tested (see also Supplemental Figure 2).…”