Single cell analysis and temporal profiling of agonist-mediated inositol 1,4,5-trisphosphate, Ca2+, diacylglycerol, and protein kinase C signaling using fluorescent biosensors

The magnitude and temporal nature of intracellular signaling cascades can now be visualized directly in single cells by the use of protein domains tagged with enhanced green fluorescent protein (eGFP). In this study, signaling downstream of G protein-coupled receptor-mediated phospholipase C (PLC) activation has been investigated in a cell line coexpressing recombinant M-3 muscarinic acetylcholine and alpha(1B)-adrenergic receptors. Confocal measurements of changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3), using the pleckstrin homology domain of PLC delta 1 tagged to eGFP (eGFP-PHPLC delta), and 1,2-diacylglycerol (DAG), using the C1 domain of protein kinase C gamma (PKC gamma) (eGFP-C1(2)-PKC gamma), demonstrated clear translocation responses to methacholine and noradrenaline. Single cell EC50 values calculated for each agonist indicated that responses to downstream signaling targets (Ca2+ mobilization and PKC activation) were approximately 10-fold lower compared with respective Ins(1,4,5)P-3 and DAG EC50 values. Examining the temporal profile of second messenger responses to sub-EC50 concentrations of noradrenaline revealed oscillatory Ins(1,4,5)P-3, DAG, and Ca2+ responses. Oscillatory recruitments of conventional (PKC beta II) and novel (PKC epsilon) PKC isoenzymes were also observed which were synchronous with the Ca2+ response measured simultaneously in the same cell. However, oscillatory PKC activity (as determined by translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate protein) required oscillatory DAG production. We suggest a model that uses regenerative Ca2+ release via Ins(1,4,5)P-3 receptors to initiate oscillatory second messenger production through a positive feedback effect on PLC. By acting on various components of the PLC signaling pathway the frequency-encoded Ca2+ response is able to maintain signal specificity at a level downstream of PKC activation.