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

Abstract

The pleckstrin homology domain of phospholipase Cδ1 (PH_PLCδ) 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 PH_PLCδ and enhanced green fluorescent protein (EGFP–PH_PLCδ) associates with the plasma membrane due to its association with PtdIns(4,5)P₂. However, PH_PLCδ 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–PH_PLCδ 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.