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.
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.