The Use of Translocating Fluorescent Biosensors for Real-Time Monitoring of GPCR-Mediated Signaling

The ability to visualize the subcellular localization of proteins by labeling them with fluorescent proteins is a powerful tool in cell biology. In the G protein-coupled receptor signaling field, this technique has been utilized to examine the various aspects of receptor behavior, including activation, internalization and recycling, as well as alterations in the cellular levels of a variety of second messengers and signaling intermediates. Attaching variants of green fluorescent protein on to protein modules, which possess high affinity and selectivity for specific signaling molecules has allowed the visualization of key signaling pathway intermediates in real time, in living cells. This chapter outlines a protocol for the expression and visualization (by confocal microscopy) of such fluorescent “biosensors” and provides guidance on the analysis and interpretation of data obtained from such experiments.