The molecular mechanisms underlying G protein signaling have so far been elucidated using purified proteins and lysed cells. Little information is available about the behavior of G protein pathways in a living mammalian cell. The broad aim is to visualize the functioning of a G protein signaling pathway and its cross-talk with other signaling pathways both spatially and temporally in live cells. G protein based biosensors have been designed containing signaling proteins fused to cyan and yellow fluorescent proteins (CFP & YFP). The sensors respond to receptor stimulation or inactivation with a corresponding fluorescence resonance energy transfer (FRET) signal change. Images of cells expressing the sensors acquired during signal processing provide direct information in real time on the properties of the pathway in different parts of the cell.
The specific aims will be (i) to develop methods for imaging G protein signaling activity in mammalian cells. (ii) To test models about (a) the spatial distribution of signaling machinery and (b) the spatio-temporal progression of localized G protein activation. (iii) To test the hypotheses that a G protein pathway affects the spatio-temporal dynamics of (a) another G protein signaling activity and (b) ras and rap activity. Experiments will be performed with transfected and endogenous receptors. A vascular smooth muscle cell line will be used to examine other endogenous pathways. The majority of commercially available drugs are targeted at G protein coupled receptors. The biosensors developed here can be of considerable value pharmacologically. They can be used to screen for novel drugs and identify ligands for orphan receptors in non-invasive assays. In the longterm, this approach can help observe networks of signaling pathways functioning in a cell as it senses, processes and responds to a signal.
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