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Research article

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

Mark S. NASH, Kenneth W. YOUNG, Gary B. WILLARS, R. A. John CHALLISS, Stefan R. NAHORSKI
Biochemical Journal May 08, 2001, 356 (1) 137-142; DOI: 10.1042/bj3560137
Mark S. NASH
Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, P.O. Box 138, University Road, Leicester LE1 9HN, U.K.
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Kenneth W. YOUNG
Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, P.O. Box 138, University Road, Leicester LE1 9HN, U.K.
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Gary B. WILLARS
Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, P.O. Box 138, University Road, Leicester LE1 9HN, U.K.
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R. A. John CHALLISS
Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, P.O. Box 138, University Road, Leicester LE1 9HN, U.K.
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Stefan R. NAHORSKI
Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, P.O. Box 138, University Road, Leicester LE1 9HN, U.K.
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Abstract

The pleckstrin homology domain of phospholipase Cδ1 (PHPLCδ) binds Ins(1,4,5)P3 and PtdIns(4,5)P2 specifically, and can be used to detect changes in Ins(1,4,5)P3 in single cells. A fusion construct of PHPLCδ and enhanced green fluorescent protein (EGFP–PHPLCδ) associates with the plasma membrane due to its association with PtdIns(4,5)P2. However, PHPLCδ has greater affinity for Ins(1,4,5)P3 than PtdIns(4,5)P2, and translocates to the cytosol as Ins(1,4,5)P3 levels rise. Prolonged activation of group I metabotropic glutamate receptor 1α expressed in Chinese-hamster ovary cells or endogenous M3 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)P3 mass, but not PtdIns(4,5)P2 levels, which decreased monophasically, as determined by radioreceptor assay. Translocation thus provides a real-time method to follow increases in Ins(1,4,5)P3. Graded changes in Ins(1,4,5)P3 in Chinese-hamster ovary-lac-mGlu1α cells could be detected with increasing glutamate concentrations, and dual loading with fura 2 and EGFP–PHPLCδ showed that changes in intracellular Ca2+ concentration closely paralleled Ins(1,4,5)P3 production. Moreover, Ins(1,4,5)P3 accumulation and intracellular Ca2+ mobilization within single cells is graded in nature and dependent on both agonist concentration and receptor density.

  • group I metabotropic glutamate receptor 1α
  • M3 muscarinic receptor
  • pleckstrin homology domain

Footnotes

  • Abbreviations used: PLC, phospholipase C; PHPLCδ, pleckstrin homology domain of PLCδ1; EGFP, enhanced green fluorescent protein; mGlu1α, group I metabotropic glutamate receptor 1α; CHO, Chinese-hamster ovary; GPCR, G-protein-coupled receptor; [Ca2+]i, intracellular calcium concentration; IPTG, isopropyl β-d-thiogalactoside; KHB, Krebs–Henseleit buffer.

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May 2001

Volume: 356 Issue: 1

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Single-cell imaging of graded Ins(1,4,5)P3 production following G-protein-coupled-receptor activation
Mark S. NASH, Kenneth W. YOUNG, Gary B. WILLARS, R. A. John CHALLISS, Stefan R. NAHORSKI
Biochemical Journal May 2001, 356 (1) 137-142; DOI: 10.1042/bj3560137
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Single-cell imaging of graded Ins(1,4,5)P3 production following G-protein-coupled-receptor activation
Mark S. NASH, Kenneth W. YOUNG, Gary B. WILLARS, R. A. John CHALLISS, Stefan R. NAHORSKI
Biochemical Journal May 2001, 356 (1) 137-142; DOI: 10.1042/bj3560137

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Keywords

group I metabotropic glutamate receptor 1α
M3 muscarinic receptor
pleckstrin homology domain

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