Visualizing protein-protein interactions in the nucleus of the living cell

A question of central importance to the molecular endocrinologist is how hormones function to orchestrate events within cells. The cascades of cellular responses that are triggered by endocrine signals require the formation of specific protein partnerships, and these protein-protein interactions must be coordinated in both space and time. For example, in the absence of ligand, the steroid hormone receptor for estradiol is associated with a multiprotein inhibitory complex (1). The binding of estradiol results in alterations in estrogen receptor conformation that allow it to dissociate from this complex, and the receptor becomes competent to interact with specific DNA elements in the regulatory regions of target genes. The efficient utilization of these regulatory elements by the receptor, however, requires that the receptor associate with other coregulatory proteins (2–4). Biochemical approaches such as coimmunoprecipitation and FarWestern blotting and in vivo approaches such as yeast two-hybrid assay have provided important information regarding the interactions between receptors and coregulatory proteins. These approaches, however, may sometimes implicate nonphysiological associations between proteins that do not normally occur in intact cells. Deciphering where and when specific protein partnerships form within the living cell will be critical to understanding these basic cellular events. The molecular cloning of the jellyfish green fluorescent protein (GFP) and its expression in a variety of cell types have had a major impact on our ability to monitor events within living cells (5–10). GFP retains its fluorescent properties when fused to other proteins, and this allows fluorescence microscopy to be used to monitor the dynamic behavior of the expressed GFP fusion proteins in their natural environment within the living cell. There are now many examples of proteins expressed as GFP chimeras that possess the same subcellular localization and biological function as their endogenous counterparts. For example, the dynamics of nuclear translocation for the glucocorticoid (11, 12) and androgen receptors (13) have been visualized using GFP fusions. GFP fusion proteins have also been used to monitor complex cellular events such as the sorting of proteins between organelles (14) and the dynamics of regulated protein secretion (15, 16). Recently, it was demonstrated that movement of b-arrestin2-GFP fusion proteins serves as a sensitive biosensor for G protein-coupled receptor activation (17). This illustrates the potential for GFP fusion proteins to act as indicators of many different intracellular events. Mutant forms of the GFP protein that emit lights of different colors have been generated that, when coexpressed in the same cell, can be readily distinguished by fluorescence microscopy. This allows the behavior of two independent proteins to be monitored in the intact cell, and the extent to which these proteins colocalize can be assessed. To determine whether these labeled proteins are physically interacting, however, would require resolution beyond the optical limit of the light microscope. Fortunately, this degree of spatial resolution can be achieved with the conventional light microscope using the technique of fluorescence resonance energy transfer (FRET). FRET microscopy involves the detection of increased (sensitized) emission from an acceptor fluorophore that occurs as the result of the direct transfer of excitation energy from an appropriately positioned fluorescent 0888-8809/99/$3.00/0 Molecular Endocrinology Copyright © 1999 by The Endocrine Society