A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the rat glucocorticoid receptor (GR). When GFP-GR is expressed in living mouse cells, it is competent for normal transactivation of the GR-responsive mouse mammary tumor virus promoter. The unliganded GFP-GR resides in the cytoplasm and translocates to the nucleus in a hormone-dependent manner with ligand specificity similar to that of the native GR receptor. Due to the resistance of the mutant GFP to photobleaching, the translocation process can be studied by time-lapse video microscopy. Confocal laser scanning microscopy showed nuclear accumulation in a discrete series of foci, excluding nucleoli. Complete receptor translocation is induced with RU486 (a ligand with little agonist activity), although concentration into nuclear foci is not observed. This reproducible pattern of transactivation-competent GR reveals a previously undescribed intranuclear architecture of GR target sites.Steroid receptors are hormone-dependent activators of gene expression. It is generally accepted that the unliganded glucocorticoid receptor (GR) resides in the cytoplasm and that hormone activation leads to both nuclear accumulation and gene activation (see refs. 1-6 and references therein). However, the mechanisms involved in nuclear translocation and targeting of steroid receptors to regulatory sites in chromatin are poorly understood. It has been difficult to discriminate between the ability of a given receptor mutant, or a given receptor-ligand combination, to participate in the separate processes of receptor activation, nuclear translocation, sequence-specific DNA binding, and promoter activation.The paucity of information on these issues stems in part from the lack of appropriate technology to study the various stages in nuclear targeting. Because knowledge of these steps is essential for understanding the mechanism of steroid hormone action, we have taken the approach of tagging GR with a chromophore, allowing us to visualize in vivo, with the least perturbation, the changes in receptor subcellular localization upon exposure to activating ligand.Recent characterization of a chromophore, the green fluorescent protein (GFP), provides a general method to label proteins in living cells. Chimeras formed with a highly efficient variant of GFP (7)

To understand the subcellular localization of the vitamin D receptor (VDR) and to measure VDR content in single cells, we recently developed a fluorescent labeled ligand, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-calcitriol. This tagged hormone has intact biological activity, high affinity and specific binding to the receptor, and enhanced fluorescent emission upon receptor binding. Using BODIPY-calcitriol, here we monitored the subcellular distribution of VDR in living cultured cells by microscopy. Time course studies showed that an equilibrium between the cytoplasmic and nuclear hormone binding developed within 5 min and was maintained thereafter. We found a substantial proportion of VDR residing in the cytoplasm, colocalized with endoplasmic reticulum, the Golgi complex, and microtubules. Confocal microscopy clarified the presence of VDR within discrete regions of the nucleus and along the nuclear envelope. There was no VDR in the plasma membrane. Low affinity BODIPY-calcitriol binding sites were in the mitochondria. Mutations in the VDR gene selectively and specifically altered BODIPYcalcitriol distribution. Defects in the hormone binding region of VDR prevented both nuclear and cytoplasmic hormone binding. Defects in the DNA binding region decreased the nuclear retention of VDR and prevented localization to nuclear foci. These results with BODIPYcalcitriol reveal cytoplasmic VDR localization in living cells and open the possibility of studying the three-dimensional architecture of intranuclear target sites. The vitamin D receptor (VDR)1 belongs to the v-erb-A superfamily of ligand activated transcription factors. The hormonal forms of vitamin D and other steroid hormones act through their receptors to regulate the transcription of target genes and thus modulate a variety of cell functions. These hormones also exert rapid, so-called "nongenomic" actions that take place outside the nucleus. Over the last 30 years we learned many aspects of steroid receptor activation, but the subcellular distribution of the receptors remained controversial. The use of radioligands led to the classical model for steroid receptor activation, which placed the receptors in the cytoplasm (1). Later, antibodies were raised against steroid receptors, and immunocytology suggested that unactivated steroid receptors reside exclusively in the nucleus (2). In the past few years, evidence has accumulated showing that glucocorticoid, mineralocorticoid, and androgen receptors reside both in the cytoplasm and in the nucleus. Nevertheless, the consensus has remained unchanged for the nuclear localization of estrogen receptors, thyroid hormone receptors, and VDR (3). Several studies with immunocytology on aldehyde-fixed cells showed that VDR resides exclusively in the nucleus (4). However, using microwave fixation, we found a significant portion of VDR in the cytoplasm (5). Since then, cytoplasmic VDR has been found by others (6 -10), but the existence of cytoplasmic VDR is still not generally accepted (11,12).While the role of nucle...

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As ligand-regulated transcription factors, glucocorticoid receptors (GR) must traffic through the cytoplasm, traverse the nuclear pores, and subsequently traffic within the the nucleus to reach their target genes. Due to technical difficulties with immunocytology, little is known about the translocation process or the intranuclear localization. The recent characterization of a chromophore, green fluorescent protein (GFP), provided a general tool to fluorescently label proteins in living cells. With the development of a transcriptionally active GFP-GR chimera, it became possible to visualize GR translocation and intranuclear distribution in living cells.This chimeric receptor was transiently transfected into mouse adenocarcinoma cells, allowing the direct visualization of GR using real-time video and confocal laser scanning microscopy. Mobility of GFP-GR was analyzed with fluorescent recovery after photobleaching (FRAP).The hormone-free GFP-GR was localized in the cytoplasm figure 1). Dexamethasone (lOnM) initiated GFP-GR translocation into the nucleus (Figure 2 and 3). The translocation rate was dose- and temperature-dependent, and occurred in a pulsatile manner along cytoplasmic fibrillar structures (Figure 2). FRAP experiments showed that GFP-GR remained in motion within the nucleus after translocation.