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 PLCdelta1 tagged to eGFP (eGFP-PH(PLCdelta)), and 1,2-diacylglycerol (DAG), using the C1 domain of protein kinase Cgamma (PKCgamma) (eGFP-C1(2)-PKCgamma), demonstrated clear translocation responses to methacholine and noradrenaline. Single cell EC(50) values calculated for each agonist indicated that responses to downstream signaling targets (Ca(2+) mobilization and PKC activation) were approximately 10-fold lower compared with respective Ins(1,4,5)P(3) and DAG EC(50) values. Examining the temporal profile of second messenger responses to sub-EC(50) concentrations of noradrenaline revealed oscillatory Ins(1,4,5)P(3), DAG, and Ca(2+) responses. Oscillatory recruitments of conventional (PKCbetaII) and novel (PKCepsilon) PKC isoenzymes were also observed which were synchronous with the Ca(2+) 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 Ca(2+) 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 Ca(2+) response is able to maintain signal specificity at a level downstream of PKC activation.