Application of Green Fluorescent Protein to the Study of Dynamic Protein-Protein Interactions and Subcellular Trafficking of Steroid Receptors

The green fluorescent protein (GFP) from the jellyfish, Aequoria victoria , converts blue light to green fluorescence when expressed in intact cells and transgenic animals, and has proven to be a powerful tool for biological and medical research. This chapter describes the application of spectrally distinguishable variants of GFP to the investigation of steroid hormone receptor action. Topics that are covered include the design of GFP-receptor chimeras, the expression of GFP-fusion proteins in cells in culture, the detection of the GFP-tagged receptors in living and fixed cells, and the use of GFP-variants to study the colocalization and interaction of steroid receptors and other proteins. Specifically, the authors describe the application of GFP-tagged steroid receptors to assess issues in receptor trafficking and receptor interaction with coactivator proteins. The latter approach employs fluorescence resonance energy transfer (FRET), a technique that effectively permits a 100-fold enhancement beyond the inherent resolving power of the light microscope.