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

The pleckstrin homology domain of phospholipase C delta1 (PHPLC delta) 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 PHPLC delta and enhanced green fluorescent protein (EGFP-PHPLC delta) associates with the plasma membrane due to its association with PtdIns(4,5)P-2. However, PHPLC delta 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 1 alpha 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-mGlu1 alpha cells could be detected with increasing glutamate concentrations, and dual loading with fura 2 and EGFP-PHPLC delta showed that changes in intracellular Ca2+ concentration closely paralleled Ins(1,4,5)P-3 production. Moreover, Ins(1,4,5)P-3 accumulation and intracellular Ca2+ mobilization within single cells is graded in nature and dependent on both agonist concentration and receptor density.