Using inducible vectors to study intracellular trafficking of GFP-tagged steroid/nuclear receptors in living cells

Intracellular trafficking and localization of proteins can now be efficient ly visualized by fusion of a polypeptide to the green fluorescent protein ( GFP). Many spectral variants of this reagent are now available, providing p owerful tools for studies in living cells, This approach is particularly us eful for members of the steroid/nuclear receptor superfamily, since these m olecules frequently undergo rapid subcellular redistribution on ligand acti vation. A major roadblock in the application of this technology concerns pr oblems associated with transient transfections. This technique produces cel l populations that are highly heterogeneous with respect to the newly intro duced protein and usually contain the protein in a highly overexpressed sta te. In addition, long-term studies related to cell cycle and cellular diffe rentiation are essentially impossible with this approach, These problems ca n be overcome by introduction of the GFP fusion into cells under appropriat e induction control, We describe application of the tetracycline regulatory system to inducible control of a glucocorticoid receptor (GR)/GFP chimera, Intracellular concentrations of GFP-GR can be very effectively controlled in this system, providing an ideal environment in which to study subcellula r trafficking of the receptor and interactions with a variety of intracellu lar targets.