Abstract
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.
Highlights
DAG levels can be monitored in the single cell using the DAG binding domain of protein kinase C (PKC)␥ (4), which translocates to the plasma membrane in response to agonist
Confocal images of Chinese hamster ovary (CHO)-m3/␣1B cells transiently transfected with enhanced green fluorescent protein (eGFP)-PHPLC␦ showing localization of the biosensor under basal and MCh-stimulated conditions
Whether second messenger systems linked to G protein-coupled receptors (GPCRs) activation can encode similar information in signaling networks has yet to be explored thoroughly
Summary
DAG levels can be monitored in the single cell using the DAG binding domain of PKC␥ (eGFP-C12-PKC␥) (4), which translocates to the plasma membrane in response to agonist These fluorescent “biosensors” have revealed oscillatory patterns of Ins(1,4,5)P3 production in response to ATP in Madin-Darby canine kidney cells (10), type 5a metabotropic glutamate (mGlu5a) receptor activation in Chinese hamster ovary (CHO) and human embryonic kidney cells (6, 7, 16), and ␣1B-adrenergic receptor stimulation in CHO cells (8). The oscillatory pattern of signaling observed with threshold stimulation of the ␣1B-adrenergic receptor (8) provides a model to study the synchrony of Ins(1,4,5)P3 and DAG production with Ca2ϩ in single cells These have been compared with temporal patterns of PKC recruitment using conventional (PKCII) and novel (PKC⑀) PKC isoenzymes and to PKC activation using the substrate eGFP-MARCKS. We report here on the presence of oscillatory messenger production and determine the contribution of Ca2ϩ mobilization and entry on the recruitment and activation of PKC isoenzymes
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