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)

Our recent animal and human studies revealed that chronic hyponatremia is a previously unrecognized cause of osteoporosis that is associated with increased osteoclast numbers in a rat model of the human disease of the syndrome of inappropriate antidiuretic hormone secretion (SIADH). We used cellular and molecular approaches to demonstrate that sustained low extracellular sodium ion concentrations ( Hyponatremia, defined as serum sodium ion concentration ([Na ϩ ]) less than 135 mmol/liter, is a frequently encountered electrolyte abnormality in patients. Chronic hyponatremia is an especially common disorder in elderly people, often due to the dysregulation of hypothalamic osmoregulatory mechanisms, leading to the syndrome of inappropriate antidiuretic hormone secretion (SIADH).2 Hyponatremia may also arise from chronic heart failure or cirrhosis and from treatment with a large number of drugs, including diuretics and selective serotonin reuptake inhibitors. The estimated prevalence of chronic hyponatremia in the United States is in the range of 3.2-6.1 million persons annually, 75-80% of whom are without obvious neurological symptoms. Although chronic hyponatremia is often considered to be "asymptomatic," recent reports have shown its adverse effects, namely impaired gait stability and neurocognitive functions and, therefore, a greater risk of falls and fractures. In one recent case-controlled study of asymptomatic chronic hyponatremic patients, even mild hyponatremia was associated with a 67-fold increased odds ratio for falling compared with normonatremic controls. Even more alarming, a recent study from Belgium found that mild asymptomatic hyponatremia was associated with bone fractures in ambulatory elderly subjects (adjusted odds ratio of 4.16, 95% confidence interval: 2.24 -7.71) (1), and the incidence of hyponatremia in patients aged 65 or older with skeletal fractures was more than double that of patients with no fracture (9.1 and 4.1%, respectively; p ϭ 0.007) (2). Recent studies from our laboratory have indicated that hyponatremia is also associated with osteoporosis due to increased osteoclastic bone resorption in a rat model of SIADH (3). Histomorphometric analysis indicated that osteoclast number was increased 5-10-fold in excised femurs, tibia, and spine from hyponatremic rats, and analysis of blood samples revealed no significant metabolic or hormonal change that could account for the increased osteoclastic bone resorption (3). Early studies of radionucleotide distribution indicated that approximately one-third of the body's sodium is stored in the bone matrix along with calcium and phosphorus and is released during osteoclastic resorption (4). A more recent study has also shown that large amounts of sodium are stored in an osmotically inactive compartment in the bones of dogs and are released from this store during prolonged dietary sodium deprivation (5). The primary components of bone matrix are removed by osteoclasts first by demineralization of the inorganic mineral through acidification of the...

Liganded and unliganded vitamin D receptors (VDRs) carry out distinct functions; both types of functions require heterodimerization with retinoid X receptors (RXRs). Our recent studies with fluorescent protein chimeras of VDR and RXR, termed GFP-VDR, YFP-RXR, and RXR-BFP, indicated that RXR regulates VDR functions in part by regulating subcellular localization. Here we explored the mechanisms of this regulation. Photobleaching experiments demonstrated that YFP-RXR and both unliganded and liganded GFP-VDR shuttle constantly between nucleus and cytoplasm. To characterize RXR import, we identified a nuclear localization sequence (NLS) in the DNA-binding domain. Mutations in this NLS caused predominant cytoplasmic localization of nlsYFP-RXR and prevented transcriptional activity. The nlsRXR-BFP retained unliganded GFP-VDR in the cytoplasm and reduced baseline transcriptional activity. After calcitriol exposure, however, both GFP-VDR and nlsRXR-BFP entered the nucleus. We characterized receptor export rates and mechanisms using permeabilization experiments. Mutations in the calreticulin binding region slowed both GFP-VDR and YFP-RXR export. Coexpression of RXR-BFP slowed the export of unliganded GFP-VDR, whereas calcitriol treatment tripled the rate of GFP-VDR export. Treatment with leptomycin B, an inhibitor of CRM-1 receptor-mediated export, inhibited export of unliganded GFP-VDR but did not influence export of liganded GFP-VDR or YFP-RXR. Leptomycin B added before calcitriol similarly decreased hormone-induced luciferase activity but was ineffective when added subsequent to calcitriol. These results indicate that the unliganded and liganded VDR interact differently with the import and export receptors and with RXR. Most likely, the regulation of VDR nuclear import by RXR is essential for ligand-independent functions.

The vitamin D receptor (VDR) acts as heterodimer with the retinoid X receptor ␣ (RXR) to control transcriptional activity of target genes. To explore the influence of heterodimerization on the subcellular distribution of these receptors in living cells, we developed a series of fluorescent-protein chimeras. The steady-state distribution of the yellow fluorescent protein-RXR was more nuclear than the unliganded green fluorescent protein (GFP)-VDR. Coexpression of RXR-blue fluorescent protein (BFP) promoted nuclear accumulation of GFP-VDR by influencing both nuclear import and retention. Fluorescence resonance energy transfer microscopy (FRET) demonstrated that the unliganded GFP-VDR and RXR-BFP form heterodimers. The increase in nuclear heterodimer content correlated with an increase in basal transcriptional activity. FRET also revealed that calcitriol induces formation of multiple nuclear foci of heterodimers. Mutational analysis showed a correlation between hormone-dependent nuclear VDR foci formation and DNA binding. RXR-BFP also promoted hormone-dependent nuclear accumulation and intranuclear foci formation of a nuclear localization signal mutant receptor (nlsGFP-VDR) and rescued its transcriptional activity. Heterodimerization mutant RXR failed to alter GFP-VDR and nlsGFP-VDR distribution or activity. These experiments suggest that RXR has a profound effect on VDR distribution. This effect of RXR to promote nuclear accumulation and intranuclear targeting contributes to the regulation of VDR activity and probably the activity of other heterodimerization partners.Proteins of the nuclear receptor superfamily mediate response to hormones or intracellular signals into transcriptional responses and regulate an array of important cellular functions. A member of the nuclear receptor superfamily, the vitamin D receptor (VDR) 1 , mediates effects of calcitriol on bone development and maintenance, calcium homeostasis, immune functions, endocrine functions, vitamin D metabolism, and cellular proliferation and differentiation. Like other class II nuclear receptors, such as the thyroid hormone receptor, the retinoic acid receptor, and many orphan receptors, VDR requires heterodimerization with the retinoid X receptor (RXR) for high affinity binding to target genes (1, 2). VDR and RXR can heterodimerize in the absence of calcitriol, and these heterodimers regulate basal transcriptional activity of target genes and exert transcriptional silencing functions (3). The addition of calcitriol stabilizes the heterodimers and promotes their binding to the vitamin D response elements (4). The importance of heterodimerization in VDR functions led us to investigate the spatial and temporal relationships between these receptors in living cells.Recently we and others have used green fluorescent protein chimeras of VDR to study the receptor distribution in living cells (5-7). Unlike the glucocorticoid receptor (GR), which stays in the cytoplasm without the ligand, the unliganded VDR distributes evenly between the cytoplasm and the nucle...