Single-cell imaging of graded Ins(1,4,5)P3 production following G-protein-coupled-receptor activation

The Biochemical Journal
M S NashStefan R Nahorski

Abstract

The pleckstrin homology domain of phospholipase Cdelta1 (PH(PLCdelta)) binds Ins(1,4,5)P(3) and PtdIns(4,5)P(2) specifically, and can be used to detect changes in Ins(1,4,5)P(3) in single cells. A fusion construct of PH(PLCdelta) and enhanced green fluorescent protein (EGFP-PH(PLCdelta)) associates with the plasma membrane due to its association with PtdIns(4,5)P(2). However, PH(PLCdelta) has greater affinity for Ins(1,4,5)P(3) than PtdIns(4,5)P(2), and translocates to the cytosol as Ins(1,4,5)P(3) levels rise. Prolonged activation of group I metabotropic glutamate receptor 1alpha expressed in Chinese-hamster ovary cells or endogenous M(3) muscarinic receptors in SH-SY5Y neuroblastoma cells gave an initial transient peak in translocation, followed by a sustained plateau phase. This closely followed changes in cell population Ins(1,4,5)P(3) mass, but not PtdIns(4,5)P(2) levels, which decreased monophasically, as determined by radioreceptor assay. Translocation thus provides a real-time method to follow increases in Ins(1,4,5)P(3). Graded changes in Ins(1,4,5)P(3) in Chinese-hamster ovary-lac-mGlu1alpha cells could be detected with increasing glutamate concentrations, and dual loading with fura 2 and EGFP-PH(PLCdelta) showed that...Continue Reading

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Citations

Nov 21, 2007·Pflügers Archiv : European journal of physiology·Nicolas FritzJean-Luc Morel
Apr 12, 2011·Neurochemical Research·Songbai ZhangPer Uhlén
Aug 6, 2003·Trends in Neurosciences·Stefan R NahorskiMark S Nash
Jan 5, 2011·PLoS Computational Biology·Andreja JovicShuichi Takayama
Sep 29, 2006·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Daniela BillupsStefan R Nahorski
Sep 2, 2008·Canadian Journal of Physiology and Pharmacology·Nicolas FritzJean-Luc Morel
May 29, 2003·The Journal of Cell Biology·Chang XuLeslie M Loew
Apr 7, 2007·The Journal of Physiology·Nikita Gamper, Mark S Shapiro
Oct 22, 2008·Methods : a Companion to Methods in Enzymology·Péter Várnai, Tamas Balla
Dec 8, 2004·The Journal of Biological Chemistry·Yoko TateishiKatsuhiko Mikoshiba
Apr 3, 2003·The Journal of Biological Chemistry·Kenneth W YoungStefan R Nahorski
Mar 27, 2004·The Journal of Biological Chemistry·Sophia ThoreAnders Tengholm

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