Direct visualization of the human estrogen receptor alpha reveals a role for ligand in the nuclear distribution of the receptor

The human estrogen receptor ct (ER ct) has been tagged at its amino terminu s with the S65T variant of the green fluorescent protein (GFP), allowing su bcellular trafficking and localization to be observed in living cells by fl uorescence microscopy. The tagged receptor, GFP-ER, is functional as a liga nd-dependent transcription factor, responds to both agonist and antagonist ligands, and can associate with the nuclear matrix. Its cellular localizati on was analyzed in four human breast cancer epithelial cell lines, two ER(MCF7 and T47D) and two ER- (MDA-MB-231 and MDA-MB-435A), under a variety o f ligand conditions. In all cell lines, GFP-ER is observed only in the nucl eus in the absence of ligand. Upon the addition of agonist or antagonist li gand, a dramatic redistribution of GFP-ER from a reticular to punctate patt ern occurs within the nucleus. In addition, the full antagonist ICI 182780 alters the nucleocytoplasmic compartmentalization of the receptor and cause s partial accumulation in the cytoplasm in a process requiring continued pr otein synthesis. GFP-ER localization varies between cells, despite being cu ltured and treated in a similar manner. Analysis of the nuclear fluorescenc e intensity for variation in its frequency distribution helped establish lo calization patterns characteristic of cell line and ligand. During the cour se of this study, localization of GFP-ER to the nucleolar region is observe d for ER- but not ER+ human breast cancer epithelial cell lines. Finally, o ur work provides a visual description of the "unoccupied" and ligand-bound receptor and is discussed in the context of the role of ligand in modulatin g receptor activity.