Direct Visualization of the Human Estrogen Receptor α Reveals a Role for Ligand in the Nuclear Distribution of the Receptor

Abstract

The human estrogen receptor ␣ (ER ␣) has been tagged at its amino terminus with the S65T variant of the green fluorescent protein (GFP), allowing subcellular trafficking and localization to be observed in living cells by fluorescence microscopy. The tagged receptor, GFP-ER, is functional as a ligand-dependent transcription factor, responds to both agonist and antagonist ligands, and can associate with the nuclear matrix. Its cellular localization was analyzed in four human breast cancer epithelial cell lines, two ERϩ (MCF7 and T47D) and two ERϪ (MDA-MB-231 and MDA-MB-435A), under a variety of ligand conditions. In all cell lines, GFP-ER is observed only in the nucleus in the absence of ligand. Upon the addition of agonist or antagonist ligand, a dramatic redistribution of GFP-ER from a reticular to punctate pattern occurs within the nucleus. In addition, the full antagonist ICI 182780 alters the nucleocytoplasmic compartmentalization of the receptor and causes partial accumulation in the cytoplasm in a process requiring continued protein synthesis. GFP-ER localization varies between cells, despite being cultured and treated in a similar manner. Analysis of the nuclear fluorescence intensity for variation in its frequency distribution helped establish localization patterns characteristic of cell line and ligand. During the course of this study, localization of GFP-ER to the nucleolar region is observed for ERϪ but not ERϩ human breast cancer epithelial cell lines. Finally, our work provides a visual description of the "unoccupied" and ligand-bound receptor and is discussed in the context of the role of ligand in modulating receptor activity. INTRODUCTIONSteroid hormones elicit diverse biological responses, important during growth, differentiation, inflammation, pregnancy, and homeostasis among many other processes. The genomic actions of steroid hormones are mediated by steroid receptors, members of the nuclear receptor superfamily of ligand-dependent transcription factors. In the absence of hormone, steroid receptors exist in a complex with chaperone proteins capable of high-affinity binding to steroid hormones. Hormone binding leads to a conformational change in the receptor that results in its dissociation from chaperone proteins and ultimately in the binding of the receptor as a homodimer to cognate sites in steroid-responsive genes (reviewed in Tsai and O'Malley, 1994;Mangelsdorf et al., 1995;Beato et al., 1996).Immunohistochemistry and biochemical fractionation show the unoccupied steroid receptors to reside ‡ These authors contributed equally to this work. Corresponding author. E-mail address: hagerg@exchange.nih.gov. predominantly in the cytoplasm, the nucleus, or both compartments, depending on the receptor, in a complex with chaperone proteins (Jensen, 1991;DeFranco et al., 1995;Beato et al., 1996;Pratt and Toft, 1997). For the predominantly nuclear receptors, such as the estrogen receptor (ER), the unoccupied receptor exists in the nucleus either bound or not bound to its cognate site in target genes. ...

The Human Estrogen Receptor-α Is a Ubiquitinated Protein Whose Stability Is Affected Differentially by Agonists, Antagonists, and Selective Estrogen Receptor Modulators

“…Recent studies have demonstrated that ER␣ is distributed in a reticular pattern within the nuclei in the absence of ligand (54). The addition of either estradiol or 4OH-tamoxifen results in a rapid and dramatic redistribution of ER␣ into a punctate pattern, whereas the addition of ICI 182,780 results in trapping of ER␣ in the cytoplasm (37,54).…”

“…Recent studies have demonstrated that ER␣ is distributed in a reticular pattern within the nuclei in the absence of ligand (54). The addition of either estradiol or 4OH-tamoxifen results in a rapid and dramatic redistribution of ER␣ into a punctate pattern, whereas the addition of ICI 182,780 results in trapping of ER␣ in the cytoplasm (37,54). Since the distribution of proteasome factors within the cytoplasm and the nucleus is distinct (55,56), it is possible that these distinct ER␣-ligand complexes interact with distinct proteasome complexes that degrade them at different rates.…”

Fluorescence Imaging Reveals the Nuclear Behavior of Peroxisome Proliferator-activated Receptor/Retinoid X Receptor Heterodimers in the Absence and Presence of Ligand*♦

“…2C and data not shown). As a control, a similar experiment was conducted with ER␣, which was shown to form speckles in the nuclei of cells treated with estradiol (16,17). Indeed, in MCF-7 cells (Fig.…”

Nanoscale Intracellular Organization and Functional Architecture Mediating Cellular Behavior

“…This transport is linked to the cytoskeleton, but the exact mechanism is not known. 38 One postulate is that chaperones, proteins that are associated with both the glucocorticoid receptors and the cytoskeleton, alter the transport properties of the cell. The glucocorticoid receptor is a gene regulatory protein that is bound in the cytoplasm to the chaperone heat shock protein 90 (Hsp90) as well as interacting with cochaperones such as hsp40, and other peptides including cyp40; Hsp90 also binds to both actin and tubulin.…”