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

  • Carl P. Nelson
  • R. A. John ChallissEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 746)

Abstract

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.

Key words

Fluorescent biosensor Confocal microscopy Cell signaling G-protein-coupled receptor Green fluorescent protein Phospholipase C 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Cell Physiology and PharmacologyUniversity of LeicesterLeicesterUK

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