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Although the subcellular localization of estrogen receptor a (ERalpha) is generally recognized as in the nucleus, some studies have shown a cytoplasmic distribution. We used a time-lapse imaging technique to examine intracellular and intranuclear localizations of ERalpha using a green fluorescent protein (GFP)-ERalpha chimera protein in living COS-1 cells of different external milieu in a cultured condition. Transfected cells were treated with the ERa agonist, estradiol and the partial antagonist, tamoxifen, to investigate the ligand specific changes of distribution of GFP-ERalpha in a single cell. GFP-ERalpha was accumulated in the nucleus even in the absence of ligand exposure. Cells showed diffuse fine-grained fluorescence of GFP-ERalpha in the nucleus without ligands. These results were confirmed by using anti-ERalpha antibody immunocytochemistry. GFP-ERalpha gradually and clearly formed small clusters or more punctate patterns in the nuclei of cells after ligand incubation for 60 min, while some of the transfected cells showed a finely diffuse pattern. The manner of nuclear redistribution did not depend on the type of ligand. The present study showed that GFP-ERalpha is restricted to the nucleus and accumulates with estradiol and tamoxifen, suggesting that accumulation of ERalpha in the nucleus could be necessary for ERalpha to function with ligands.

Subcellular and Subnuclear Distributions of Estrogen Receptor .ALPHA. in Living Cells Using Green Fluorescent Protein and Immunohistochemistry.