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Molecular Endocrinology, doi:10.1210/me.2005-0050
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Molecular Endocrinology 19 (6): 1501-1515
Copyright © 2005 by The Endocrine Society

Ligand-Selective Targeting of the Glucocorticoid Receptor to Nuclear Subdomains Is Associated with Decreased Receptor Mobility

Marcel J. M. Schaaf, Laura J. Lewis-Tuffin and John A. Cidlowski

Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: John A. Cidlowski, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, 111 Alexander Drive, P.O. Box 12233, Research Triangle Park, North Carolina 27709. E-mail: cidlowski{at}niehs.nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The association between nuclear distribution and mobility of the human glucocorticoid receptor was examined in living COS-1 cells using yellow fluorescent protein- and cyan fluorescent protein-tagged receptors. Quantitation of the nuclear distribution induced by an array of glucocorticoid ligands revealed a continuum from a random (cortisone) to a nonrandom (triamcinolone acetonide) receptor distribution. Structure-function analysis revealed that the 9-fluoro and 17-hydroxy groups on the steroid significantly impact nuclear receptor distribution. Using time-lapse microsopy, the triamcinolone acetonide-induced receptor distribution did not change significantly over a period of 15 sec. However, using fluorescence recovery after photobleaching, the individual receptors moved at a much faster rate, indicating rapid exchange of receptors on immobile nuclear subdomains. Receptor mobilities for 13 different steroids, measured by fluorescence recovery after photobleaching, appeared to correlate with receptor distribution. Ligands that induced a nonrandom distribution induced slower receptor mobility and vice versa. Finally, application of 2-photon confocal microscopy revealed differences in receptor mobility between nuclear subdomains. Areas of high receptor concentration showed slower mobility than areas of low receptor concentration. Thus, glucocorticoid receptors can be targeted (depending on the ligand) to relatively immobile nuclear subdomains. The transient association of receptor with these domains decreases the mobility of the receptor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ACTIONS OF glucocorticoid hormones are mediated by the glucocorticoid receptor (GR), a steroid receptor that is a member of the nuclear receptor family. In the absence of ligand, GR is located in the cytoplasm, in a complex with heat shock proteins and immunophilins (1). Upon addition of ligand, GR dissociates from this complex and translocates to the nucleus (2, 3). There, the receptor can bind to glucocorticoid-responsive elements (GREs) in the promoters of target genes and induce transcription (4). The transcriptional activity of the GR depends on coactivators that facilitate recruitment of the basal transcription machinery or remodel chromatin (5, 6, 7). Alternatively, GR can repress gene transcription induced by other factors like activator protein-1 and nuclear factor-{kappa}B, probably by physically interacting with these factors (8, 9, 10, 11, 12, 13).

Limited information is available about the nuclear subdomain GR is targeted to or how it is retained there. Van Steensel et al. (14) have shown by immunofluorescence for several types of fixed human and rat cells that upon ligand-induced activation and translocation to the nucleus GR forms approximately 1000–2000 focal domains consisting of 40–50 receptors. This observation was confirmed by Htun et al. (15), who observed a similar pattern while studying the organization of a green fluorescent protein (GFP)-GR chimera in the nucleus upon activation by dexamethasone. Other nuclear receptors appear to distribute within the nucleus in a similar punctate way. For example, GFP-tagged estrogen receptor (ER) (16, 17, 18, 19), androgen receptor (AR) (18, 20, 21, 22), mineralocorticoid receptor (MR) (23, 24), vitamin D receptor (25), thyroid hormone receptor (26), retinoic acid receptor (27) and Aryl hydrocarbon receptor (28) have been observed to distribute in a punctate manner upon agonist-induced activation. However, activation of receptors by an antagonist did not result in a punctate distribution of GR (15), AR (21), and mineralocorticoid receptor (23). ER antagonists induce less pronounced punctate receptor distributions than agonists (16, 17). The determinants of these punctate patterns have not been elucidated.

In addition to the analysis of trafficking and distribution, GFP-tagged proteins have been used to study protein mobility by fluorescence recovery after photobleaching (FRAP). Several nuclear proteins have been investigated by this technique, and these studies have revealed that proteins involved in diverse nuclear processes move rapidly throughout the entire nucleus (29, 30). For example, studies in our laboratory have shown that ligand binding decreases the mobility of a yellow fluorescent protein (YFP)-tagged GR in the nucleus and that this decrease is dependent on the ligand. Similarly, Stenoien et al. (31) have also observed a ligand-dependent decrease in mobility for ER{alpha}. However, the association between the mobility and distribution of any nuclear protein has not yet been investigated. In the present study, we have investigated determinants for targeting of the GR to nuclear subdomains and show that the ligand, as well as the DNA binding domain (DBD) and ligand binding domain (LBD) of the receptor, are important factors in determining the focal distribution of GR in the nucleus. Using fluorescence recovery after photobleaching, we show that there is a strong correlation between a focal distribution pattern and decreased mobility of the receptor as determined by ligand. Finally, we demonstrate that GR mobility at the foci is decreased as compared with its mobility measured in other regions of the nucleus.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ligand-Dependent Nonrandom Nuclear Distribution of YFP-Human GR{alpha} (hGR{alpha})
Previously, we have described the synthesis and characterization of an expression vector for a YFP-tagged hGR{alpha}. This fusion protein translocates to the nucleus upon ligand binding and induces transactivation on a GRE-driven promoter (32). Using this vector, the intracellular localization of YFP-hGR{alpha} could be studied by confocal laser scanning microscopy. After transfection of YFP-hGR{alpha} into COS-1 cells, most of the fluorescence is detected in the cytoplasm in the absence of ligand (Fig. 1AGo). After addition of the GR agonist triamcinolone acetonide [TA (1 µM)], the receptors translocate to the nucleus, where they distribute in an apparently nonrandom, punctate manner (Fig. 1BGo). In contrast, when YFP is transfected into COS-1 cells, it is equally abundant in the nuclear and cytoplasmic compartment, both in presence and absence of TA. YFP is evenly distributed in the nucleus, and its distribution pattern is unaffected by the steroid (Fig. 1Go, C and D).



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  		Fig. 1. Intracellular Distribution of YFP-hGR{alpha} and YFP

COS-1 cells were transfected with YFP-hGR{alpha}. In the absence of ligand, most of the fluorescence is detected in the cytoplasm (A). After addition of the GR agonist triamcinolone acetonide (1 µM), the receptors translocate to the nucleus, where they distribute in a nonrandom, punctate manner (B). When YFP is transfected into COS-1 cells, it is equally abundant in the nuclear and cytoplasmic compartment, in the presence (C) and absence of TA (D), and is evenly distributed within the nucleus.

 
To obtain a quantitative measure for the randomness of receptor distribution in the nucleus a method was used that was previously established by Htun et al. (17). Using image analysis software, fluorescence intensity levels were measured along a line through the nucleus [maximal in length without touching a nucleolus (Fig. 2AGo)]. From all individual measurements along this line, a SD and average could be calculated. The quotient of these numbers [the coefficient of variation (CV)] was used as a measure for the degree of randomness of nuclear distribution: the higher this number, the more nonrandom the distribution is. It should be noted that this CV is not an absolute measure for randomness of distribution because it is dependent on the resolution of the image. Cells were selected randomly, but cells with very high or very low YFP-hGR{alpha} expression levels were excluded from the study (for specific criteria, see Materials and Methods).



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  		Fig. 2. Quantitation of Nuclear Distribution of YFP-hGR{alpha}

A, Using image analysis software a line, maximal in length without touching a nucleolus, was drawn through the nucleus. The fluorescence level was measured at all points along this line. From all individual measurements SD and average could be calculated, and the quotient (the CV) was used as a measure for the randomness of nuclear distribution. In each experiment, ten cells were randomly selected and their CV values were averaged. Data shown are average ± SEM of at least two experiments. B, TA time course. CV values were determined at 1, 3, 6, 12, and 24 h after TA administration. No significant change was detected over this time period. C, TA concentration range. CV values were determined between 3 and 6 h after addition of 1,10, 100, and 1000 nM of TA. A maximum level was reached at 100 nM. *, Significantly different from CV at 1 nM (P < 0.05).

 
One hour after addition of TA (1 µM), YFP-hGR{alpha} is located primarily in the nucleus and the CV that was measured for its distribution was 0.191 ± 0.012. At 3, 6, 12, and 24 h after TA administration, the CV was similar, indicating that the randomness of hGR{alpha} distribution in the nucleus is stable over this entire period of time (Fig. 2BGo). Ligand concentration does affect the nuclear distribution of YFP-hGR{alpha}. A dose-response curve (Fig. 2CGo, measured between 3 and 6 h after addition of the steroid) shows that CV values increased from 0.158 ± 0.011 to 0.199 ± 0.004 between 1 and 100 nM TA, where a maximal value was reached. It should be noted that, at all these steroid concentrations, YFP-hGR{alpha} was completely translocated to the nucleus, indicating that complete nuclear translocation is not sufficient for induction of a maximally nonrandom receptor distribution in the nucleus.

Different GR Ligands Induce Different Nuclear Distribution Patterns
We subsequently analyzed the nuclear distribution of the receptor induced by 13 different GR ligands (cortexolone, corticosterone, cortisol, cortisone, 1-dehydrocorticosterone, deltafludrocortisone, desoxy-metasone, dexamethasone, prednisolone, RU486, triamcinolone, triamcinolone acetonide, and ZK98299). These steroids were chosen because of their known differences in affinity for GR and their spectrum of bioactivity, ranging from potent agonist (TA) to pure antagonist (ZK98299). Images were taken and CV was measured between 3 and 6 h after addition of 1 µM of the steroid to YFP-hGR{alpha}-transfected COS-1 cells. Based on the GR binding affinities of these compounds, we can assume that receptor binding is saturated at this concentration.

Representative images of the YFP-hGR{alpha} distribution from at least 20 cells are shown in Fig. 3AGo, and enlarged images of the distributions induced by TA and cortisone are shown in Fig. 3BGo. A large variation in the nuclear distribution of the GR was observed among the evaluated steroids. For example, in the presence of TA the receptor is distributed in many small focal domains, giving the distribution a punctate appearance. In contrast, in the presence of the naturally occurring steroid cortisone the domains of YFP-hGR{alpha} accumulation are absent, but several areas with low fluorescence intensity exist, indicating that the distribution is not entirely random.



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  		Fig. 3. Nuclear Distribution of the Receptor Induced by Different GR Ligands.

A, Images were taken between 3 and 6 h after addition of 1 µM of the steroid to YFP-hGR{alpha}-transfected COS-1 cells. Large variation exists between the distributions induced by these steroids. The more nonrandom distributions are shown in the top row, whereas the more random distributions are shown in the bottom row. B, Nuclear distribution of YFP-hGR{alpha} induced by TA and cortisone. In the presence of TA, the receptor is distributed in many small focal domains, giving the distribution a punctate appearance. In the cortisone-induced nuclear distribution, the focal domains are absent, but several areas with low-fluorescence intensity exist.

 
The distribution patterns induced by the 13 ligands appear to display a continuum, although within this continuum there are roughly three groups. The synthetic ligands deltafludrocortisone, dexamethasone, triamcinolone, and desoxymetasone induce relatively nonrandom receptor distributions, similar to the pattern observed for triamcinolone acetonide. In contrast, the synthetic antagonist ZK98299 and the naturally occurring ligand cortexolone induce a very random distribution like cortisone. Finally, intermediate distributions were detected for the synthetic ligands prednisolone, RU486, and 1-dehydrocorticosterone, as well as the agonists cortisol and corticosterone, the primary endogenous glucocorticoid hormones in man and rodents, respectively. This latter group of compounds distribute the receptor into some domains of high receptor concentration, but the majority of receptors appear diffusely distributed through the nucleus.

These different distributions were quantitated and the CVs are shown in Fig. 4Go. Values show a continuum and range between 0.108 (ZK98299) and 0.204 (deltafludrocortisone). Even the lowest measured CV values for YFP-hGR{alpha} were significantly higher than the CV value for YFP (0.065 ± 0.012, measured in a separate experiment), indicating that distributions of YFP-hGR{alpha} are significantly more nonrandom than the distribution of YFP. Remarkably, the five steroids with the highest CVs all have a fluoro-group at the 9{alpha}-position. The effect of this group (which does not occur on natural steroids) could be studied by direct comparison between prednisolone and deltafludrocortisone (the latter having an identical structure except for the presence of the 9{alpha}-fluoro-group). Deltafludrocortisone showed a significantly higher CV (0.204 ± 0.005 compared with 0.1547 ± 0.001, P < 0.05), indicating that the 9{alpha}-fluoro-group enhances a nonrandom distribution of GRs within the nucleus.



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  		Fig. 4. Quantitation of the Experiment Shown in Fig. 3Go

CVs induced by different steroids. Values show a continuum that ranges between 0.108 (ZK98299) and 0.204 (deltafludrocortisone). The effect of the 9{alpha}-fluoro-group can be seen by comparing prednisolone and deltafludrocortisone (the latter having an identical structure except for the presence of the 9{alpha}-fluoro-group). The effect of the 17{alpha}-hydroxy group can be seen by direct comparison corticosterone and cortisol. The latter steroid, containing a 17{alpha}-OH group, induces a more nonrandom receptor distribution. **, Significantly different from deltafludrocortisone. *, Significantly different from steroids deltafludrocortisone to desoxymetasone. #, Significantly different from steroids deltafludrocortisone to cortisol. ##, Significantly different from steroids deltafludrocortisone to RU486.

 
Further analysis of the data revealed that steroids with a hydroxy group at the 17{alpha}-position (which occurs on certain natural glucocorticoids) also induce more nonrandom receptor distributions than similar steroids without this group, although the effect is smaller in magnitude than the effect of the 9{alpha}-fluoro group. Direct comparison between the natural ligands corticosterone and cortisol [same structure, but the latter contains a 17-hydroxy (17-OH) group] demonstrates the magnitude of the 17-OH effect: CVs, respectively, 0.120 ± 0.008 and 0.146 ± 0.005 (significantly different, P < 0.05).

Based on the crystal structure of the dexamethasone-bound LBD of hGR{alpha}, it has been established that the 17-OH group on the steroid forms a hydrogen bond with the glutamine at position 642 of the receptor, and that the 9-fluoro group makes a hydrophobic interaction with the phenylalanine at position 623 (33, 34). To determine the importance of these interactions for receptor distribution, site-directed mutagenesis of these amino acids was performed. For Gln642, we used the previously described mutation into valine, which decreases ligand affinity as well as receptor mobility (32, 35). Phe623 was mutated into alanine. The YFP-tagged mutants were transfected into COS-1 cells, ligands were added, and the randomness of the receptor distribution was measured (Fig. 5Go). The F623A mutant displayed a more random distribution as compared with the wild-type receptor in the presence of steroids that contain a 9-fluoro-group (dexamethasone, triamcinolone acetonide), whereas the distribution in the presence of prednisolone and corticosterone (that do not contain a 9-fluoro-group) was unaltered. Mutant Q642V distributes in a more random way than the wild type when liganded to dexamethasone and prednisolone that contain a 17-OH group. In contrast, in the presence of steroids without this group (triamcinolone acetonide, corticosterone) distribution of Q642V was similar to the wild-type receptor.



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  		Fig. 5. Mutagenesis of Phenylalanine 623 and Glutamine 642

YFP-tagged mutants F623A and Q642V were transfected into COS-1 cells and the distribution was studied in the presence of several ligands. The F623A mutant displayed a more random distribution as compared with the wild-type receptor in the presence of steroids that contain a 9-fluoro-group (dexamethasone, triamcinolone acetonide), whereas the distribution in the presence of prednisolone and corticosterone (that do not contain a 9-fluoro-group) was not significantly affected. Mutant Q642V distributes more randomly than the wild type when liganded to dexamethasone and prednisolone that contain a 17-OH group. In contrast, in the presence of steroids without this group (triamcinolone acetonide, corticosterone) distribution of Q642V was similar to the wild-type receptor. *, Significantly different from wild-type hGR{alpha} for that drug (P < 0.05).

 
The Association between Ligand Affinity and Nuclear Receptor Distribution
The interactions between a steroid and specific amino acids in the LBD are responsible for the conformation of the receptor and also determine the receptor binding affinity of the ligand. Differences in the receptor binding affinity of the ligand resulting in alterations in the stability of the receptor-ligand complex may underlie the differences in the nuclear distribution observed for different steroids. Therefore the relative binding affinity (RBA) of the steroids used in the present study was determined in vitro using a fluorescence polarization-based competition binding assay. The results are shown in Table 1Go. The CV values of all 13 ligands were plotted against the corresponding RBAs (Fig. 6Go). In most cases, a high RBA coincides with a high CV (indicating a nonrandom distribution) and vice versa, and this is reflected in a significant effect of the RBA on CV as analyzed by ANOVA [F(1,11) = 12.86, P = 0.004]. Several outliers exist, however, and the most striking one is triamcinolone, which displays a relatively high CV and low affinity in this assay. Because of the outliers the regression coefficient is relatively low (r2 = 0.54). Nevertheless, these data suggest that in general there is an association between affinity and distribution, but that this is not necessarily true for individual ligands, suggesting that differences in distribution are not completely reflective of differences in receptor binding affinity.


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  		Table 1. RBAs of Glucocorticoid Ligands

 


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  		Fig. 6. The Effect of Receptor Binding Affinity on Distribution

CV values of 13 different ligands (as shown in Fig. 4Go) were plotted against the RBA of these ligands (as shown in Table 1Go). Regression analysis shows a statistically significant effect of RBA on CV (P = 0.004), but the regression coefficient is relatively low (r2 = 0.54). B, Corticosterone; DEX, dexamethasone; DFC, deltafludrocortisone; 1-DHC, 1-dehydrocorticosterone; DOM, desoxymetasone; E, cortisone; F, cortisol; PDN, prednisolone; RU, RU486; S, cortexolone; TC, triamcinolone; ZK, ZK98299.

 
Several Domains in hGR{alpha} Are Involved in Determining Nonrandom Nuclear Distribution
To study which domains in the GR are involved in its nuclear distribution, we used two YFP-tagged deletion mutants of the receptor ({Delta}9–385, {Delta}428–490) and a YFP-tagged truncated form of the receptor (I550, truncated at amino acid 550). Mutant {Delta}9–385 lacks most of the N-terminal domain, {Delta}428–490 lacks the DBD and I550 the LBD (for a characterization of these YFP-tagged hGR{alpha} mutants, see Ref. 32). The mutants were transfected into COS-1 cells, TA (1 µM) was administered and images were taken (Fig. 7AGo). As shown previously (2, 32), mutants {Delta}9–385 and I550 are exclusively located in the nucleus in the presence of TA, but mutant {Delta}428–490 does not completely translocate to the nucleus under these conditions because it lacks part of a nuclear localization signal.



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  		Fig. 7. Nuclear Distribution of YFP-hGR{alpha} Mutants

Three mutants ({Delta}9–385, {Delta}428–490, and I550) were transfected into COS-1 cells, and TA was administered. A, Mutant {Delta}9–385 displayed a highly nonrandom, punctate distribution. Mutant {Delta}428–490 displayed a different type of nonrandom distribution, and the truncation mutant I550 displayed a very random distribution. B, Quantitation of nuclear distribution of YFP-hGR{alpha} mutants. Mutant {Delta}9–385 showed a CV value similar to that of the wild-type receptor. Mutants {Delta}428–490 and I550 showed a higher and lower CV, respectively. *, Significantly different from wild type (P < 0.05).

 
Interestingly, the three mutants showed remarkable differences in subnuclear distribution of the receptor. Mutant {Delta}9–385 displayed a highly nonrandom, punctate distribution, similar to the wild-type receptor. Mutant {Delta}428–490 displayed a very punctate distribution, but the focal domains were considerably larger in size and fewer in number than we observed with wild-type receptor, suggesting that these foci represent a different nuclear subdomain. In addition, their presence extended into the cytoplasm, also suggesting that these foci represent a different subdomain. A similar distribution pattern was observed when cortisol or RU486 was administered to cells expressing the mutant receptor (data not shown), or when only one zinc-finger was deleted (mutants {Delta}420–451 and {Delta}450–487, data not shown). The accumulation in larger focal domains may be a result of protein misfolding due to the deletion, causing receptor aggregation. This pattern is highly reminiscent of the recently reported distribution observed for GFP-hGR{alpha} after geldanamycin treatment combined with stress (heat, cold, or prolonged imaging) (36). The relatively large focal domains that were observed in this experiment were colocalized with a component of the proteasome (36), suggesting that they are aggregations of misfolded receptor. Truncation mutant I550 displayed a very random distribution that was similar to that observed for the wild-type receptor in the presence of cortisone. No domains of high receptor concentration were present: the receptor was evenly distributed throughout the nucleus, although some small areas displayed a very low receptor concentration. Interestingly, this mutant does have transcriptional activity (Schaaf, M. J. M., and J. A. Cidlowski, unpublished observation). These data were quantitated, and CVs are shown in Fig. 7BGo. Mutant {Delta}9–385 showed a CV similar to that measured for the wild-type receptor in the presence of TA, but {Delta}428–490 displayed a significantly higher CV (0.310 ± 0.018 compared with 0.210 ± 0.007 for the wild type, P < 0.05), and truncation mutant I550 showed a lower CV (0.114 ± 0.013). These data show that both the DBD and the LBD are important contributors for intranuclear targeting of the GR.

YFP-hGR{alpha} Nuclear Distribution Is Relatively Stable, but Individual YFP-hGR{alpha} Molecules Move Rapidly through the Nucleus
Using time-lapse microscopy, the stability of the YFP-hGR{alpha} distribution in the nucleus was studied over a period of 20 sec. COS-1 cells were transfected with YFP-hGR{alpha}, TA (1 µM) was administered and images were taken at a 5-sec interval. Four representative sequential images are shown in Fig. 8AGo. In Fig. 8BGo, a region of the nucleus (indicated by a red box in Fig. 8AGo) is enlarged to facilitate comparison of the distribution pattern at the different time points. Interestingly, the images show that, on this time scale, there is very little change in the distribution of YFP-hGR{alpha} in the nucleus.



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  		Fig. 8. Stability of YFP-hGR{alpha} Nuclear Distribution

A, Time lapse microscopy of a YFP-hGR{alpha} transfected COS-1 cell in the presence of 1 µM TA. Images were taken at a 5-sec interval. Four representative sequential images are shown. Very little change in the distribution of YFP-hGR{alpha} can be observed over this time period. B, Enlarged images of the area indicated by the red square in A.

 
To compare the stability of the receptor distribution to the mobility of individual molecules, we applied the FRAP technique as described before (32). A representative FRAP experiment is shown in Fig. 9AGo. COS-1 cells were transfected with YFP-hGR{alpha} and TA was administered. Between 3 and 6 h after addition of the ligand maximal laser power was applied to a selected region in the nucleus, causing bleaching of the fluorescent molecules present in that region. Subsequently, the recovery of the fluorescence intensity in this region was monitored, representing exchange between bleached molecules moving out of the selected region and nonbleached molecules moving in. Fluorescence in the selected region was quantitated and plotted relative to t = 0 and the total fluorescence in the nucleus (see Fig. 9BGo). From these curves, the half-time (t1/2) of maximal recovery can be calculated, which represents the time point after bleaching at which fluorescence recovery is half-maximal. In the experiment shown in Fig. 9Go, t1/2 was 2.52 sec, meaning that half the number of receptors present in the bleached region had moved out of this area and were replaced by receptors from outside this region during this short period of time. These data indicate that individual receptors are moving through the nucleus at a relatively high velocity (32). This relatively high mobility is in sharp contrast to the relatively low rate of change in the overall distribution of YFP-hGR{alpha} as seen in Fig. 8Go. Together, these data suggest that receptors exchange at certain nuclear subdomains in a rapid way, but that these domains are relatively stable.



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  		Fig. 9. Representative FRAP Experiment

A, COS-1 cells were transfected with YFP-hGR{alpha} and TA was administered. Between 3 and 6 h after addition of the ligand, maximal laser power was applied to a selected region in the nucleus, causing bleaching of the fluorescent molecules present in that region at that time point. Subsequently, the recovery was monitored. B, Quantitation of FRAP. Fluorescence in the selected region was quantitated and plotted relative to t = 0 and the total fluorescence in the nucleus. From these curves, the t1/2 of maximal recovery can be calculated.

 
Nonrandom Nuclear Distribution Is Associated with Decreased Receptor Mobility
To examine the association between receptor distribution and mobility, receptor mobility was measured with the FRAP technique for all 13 steroids used in this study. COS-1 cells were transfected with YFP-hGR{alpha}, the steroid (1 µM) was added and FRAP analysis was performed. The t1/2 values, determined from at least 20 cells, which range from 3.73 sec (deltafludrocortisone) to 0.97 sec (cortisone), were then plotted as a function of the previously determined CVs for the respective steroids (as shown in Fig. 4Go). This plot is shown in Fig. 10Go. The data show that there is a strong positive correlation between t1/2 and CV, meaning that when a steroid induces a high CV (indicating highly nonrandom distribution), the t1/2 is relatively high (indicating a low receptor mobility). Regression analysis showed that this correlation was highly significant [r2 = 0.93, F(1,11) = 149.9 (P = 9.5 x 10–8)].



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  		Fig. 10. Correlation between Mobility and Distribution

Mobility was measured with the FRAP technique for all 13 steroids used in this study. The t1/2 values were plotted as a function of the previously determined CVs (as shown in Fig. 4Go) for the respective steroid. The data show that there is a strong positive correlation between t1/2 and CV. B, Corticosterone; DEX, dexamethasone; DFC, deltafludrocortisone; 1-DHC,1-dehydrocorticosterone; DOM, desoxymetasone; E, cortisone; F, cortisol; PDN, prednisolone; RU, RU486; S, cortexolone; TC, triamcinolone; ZK, ZK98299.

 
GRs Display Lower Mobility in Areas with High Receptor Concentration
Because a correlation was observed between the randomness of the distribution of the GR and its mobility, we hypothesized that receptor mobility decreases due to receptor association with certain nuclear subdomains. We therefore designed a series of experiments to investigate the difference in receptor mobility between high-fluorescence intensity areas and areas of low intensity.

We initially performed two experiments to examine the relationship between relative fluorescence intensity (high or low) and either t1/2 or bleach efficiency. The latter is defined as the percent decrease that is observed in the fluorescence intensity immediately after bleaching. We first evaluated the linearity of our fluorescence detection system by performing FRAP several times on the same cell using different gain adjustments and thus different fluorescence intensities. In this experiment, receptor number does not change, although the measured fluorescence intensity does. We performed FRAP three times per cell, with gain adjustments between each FRAP to give a high (200)-, medium (150)-, or low (100)-standardized fluorescence intensity. ANOVA indicated that neither the measured bleach efficiencies nor the t1/2s differed between low-, medium-, or high-fluorescence intensity nuclei (Fig. 11Go, A and B; n = 72; F values of 0.239 and 1.418, respectively).



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  		Fig. 11. Control Experiments Demonstrating that Fluorescence Intensity Does Not Affect Bleaching Efficiency or t1/2

In the first experiment (A and B), FRAP was performed on a series of cells, three times per cell, with gain adjustments between each FRAP to give a high (200), medium (150), or low (100) standardized fluorescence intensity. Bleach efficiencies and t1/2s did not differ between low-, medium-, or high-fluorescence intensity. In the second experiment (C and D), FRAP was performed on a series of cells with different intrinsic fluorescence intensities, while keeping the gain constant. No effect of the fluorescence intensity on bleach efficiency or t1/2 was observed.

 
We next determined whether t1/2 or bleach efficiency is affected by the intrinsic fluorescence of a nucleus, which is proportional to the number of receptors. FRAP was performed on a series of cells with different intrinsic fluorescence intensities, while keeping the detector gain constant. ANOVA indicated no effect of fluorescence intensity on bleach efficiency or on t1/2 in this experiment (Fig. 11Go, C and D; n = 55; F values of 0.273 and 0.048, respectively). Taken together, these experiments indicate that the relative fluorescence intensity, whether affected by the detector gain or the intrinsic fluorescence, does not alter bleach efficiency or t1/2, both of which reflect receptor mobility.

To study whether there are indeed regional differences in receptor mobility due to association with certain nuclear domains, we used 2-photon FRAP. This technique is identical to the conventional FRAP as described above, but uses 2-photon excitation for bleaching and imaging. For FRAP, this means that the laser bleaches fluorophores that are present in an approximately 1 µm range in the z-direction, in contrast to conventional lasers that cause bleaching of large areas outside the confocal plane. For 2-photon FRAP, cyan fluorescent protein (CFP) was used as a fluorescent tag because the effective wavelength of the laser is 420 nm. COS-1 cells were transfected with CFP-hGR{alpha}, treated with ligand for 3–6 h, and FRAP analysis was subsequently performed. Pictures from a representative experiment using a TA-treated cell are shown in Fig. 12Go. In Fig. 12AGo, a small area (indicated by a red box) that contains a high receptor concentration is bleached. Two seconds later, most of the fluorescence has already returned to this area. Figure 12BGo shows a similar experiment, but an area with low receptor concentration is bleached.



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  		Fig. 12. Two-Photon FRAP

COS-1 cells were transfected with CFP-hGR{alpha}, treated with ligand, and FRAP analysis was performed. A, Representative experiment using a TA-treated cell in which a small area (indicated by a red box) that contains a high receptor concentration is bleached. Two seconds later, most of the fluorescence has already returned to this area, which still represents an area of high receptor concentration. B, A similar experiment, but an area with low receptor concentration is bleached.

 
Thirty cells treated with TA and thirty cells treated with cortisol were analyzed by 2-photon FRAP. For each cell, a low- and a high-intensity fluorescence area was bleached and the recovery monitored over 10 sec after bleaching. Data were quantified and fluorescence recovery curves are shown in Fig. 13Go. In Fig. 13AGo, curves are shown for TA-treated cells. The curves for the low- and high-intensity area exhibit two significant differences. First, the efficiency of bleaching is greater in the high-intensity area. Immediately after bleaching fluorescence is at 56% of the prebleach level in the high intensity area vs. 73% in the low-intensity area. Bleaching efficiency is considered a measure of mobility because during the bleach pulse molecules exchange between the bleached and nonbleached area; faster moving molecules result in a lower bleach efficiency (32). Therefore, this result indicates a lower receptor mobility in the high-intensity area. In addition to the difference in bleach efficiency, the t1/2 of the high-intensity area is longer (1.75 sec vs. 1.33 sec for the low-intensity area), also indicating a lower receptor mobility. The results for the cortisol-treated cells are shown in Fig. 13BGo. The results are similar, although the data indicate an overall higher mobility in both the low- and high-intensity areas compared with TA. After bleaching, fluorescence was 64% of the prebleach level in the high-intensity area and at 76% in the low-intensity area. For the high-intensity area, t1/2 was 1.46 sec vs. 1.11 sec for the low-intensity area. These data indicate that there are regional differences in receptor mobility in the cell nucleus that correlate with the receptor concentration. Areas with a low receptor concentration contain rapidly moving receptors, whereas areas with high receptor concentration contain receptors with a lower mobility.



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  		Fig. 13. Differences in Mobility between Areas with Low and High Receptor Concentration Revealed by 2-Photon FRAP

Thirty cells treated with TA and thirty cells treated with cortisol were analyzed. For each cell, a low- and a high-intensity fluorescence area was bleached and the recovery monitored over 10 sec after bleaching. A, FRAP curves for TA-treated cells. The efficiency of bleaching is larger in the high-intensity area, and t1/2 of the high-intensity area is higher, which both indicate a lower receptor mobility for the high-intensity area. B, FRAP curves for cortisol-treated cells. The results were similar, although the data show higher mobility in both the low- and high-intensity area compared with TA.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study, we have shown that the intranuclear distribution of the GR is highly dependent on the ligand with which it is associated. Some ligands, especially high-affinity synthetic ligands like dexamethasone and triamcinolone acetonide, induce a highly punctate distribution in which the GR is organized in discrete domains of high receptor concentration and virtually excluded from others. In contrast, other ligands, mainly naturally occurring low-affinity ligands like cortisone and cortexolone, induce a more even, although still not entirely random, distribution. In a similar experiment, Htun et al. (15) previously described that dexamethasone induces GR to organize into nuclear foci, whereas RU486 does not. However, we demonstrate here that receptor distribution is not bimodal as they suggested, but rather a continuum, with RU486 inducing a more random distribution than dexamethasone.

Because it was observed that different ligands induce such different distributions and mobilities, and that deletion of the LBD results in a highly random distribution and a high receptor mobility (32), we suggest that the ligand-induced conformational changes of the LBD are major determinants of the intranuclear targeting of GR. In particular, we have demonstrated that interaction of the 17{alpha}-OH and the 9{alpha}-fluoro group on the ligand with amino acids Phe623 and Gln642 appears to contribute to a more nonrandom receptor distribution. The crystal structure of the hGR{alpha} LBD in the presence of dexamethasone (33, 34) has revealed that Phe623 and Gln642 are localized in a region of the LBD between helix 5 and 6 that contains two ß strands. This region could be directly or indirectly involved in receptor interactions with nuclear structures, and affinity for these structures could be determined by the ligand-induced conformation of this region. However, because a putative dimerization domain has been defined in this region (33), with important roles for Ile628 and Pro625, this region could also alter receptor distribution by altering receptor homodimerization.

The randomness of receptor distribution induced by a certain ligand correlates with the mobility of the receptor in the presence of this ligand: the more random the distribution induced by a ligand, the higher the receptor mobility. Furthermore, by studying regional differences in receptor mobility, we found that in areas with high receptor concentration, the mobility was low, whereas in regions with low receptor concentration the mobility was high. These results indicate that the areas of high receptor concentration contain either more GR binding sites or sites with higher affinity for GR as compared with the areas with low receptor concentration. Because we also showed that receptor distribution hardly changes over a time period of 15 sec, whereas individual receptors move on the seconds scale, we suggest that GRs associate transiently with a relatively immobile nuclear structure.

We suggest that only two large structures in the nucleus are as immobile as the domains observed in the present study: the chromatin or the nuclear matrix. Chromatin binding could be an important determinant for receptor mobility because deletion of the DBD of GR (32) and AR (37), as well as mutation of DNA-interacting domains in chromatin-interacting proteins like high-mobility group N-1 (38), dramatically increases protein mobility. GRs bind with high affinity to GREs in the genome through the GR DBD. However, Van Steensel et al. (14) have shown that the areas of high GR concentration do not colocalize with RNA polymerase II or with newly synthesized RNA, suggesting that these domains do not reflect sites of gene transcription. In addition, it is unlikely that there are enough active GREs in the genome at a given time point (estimates vary around 100) to affect the mobility or distribution of the bulk of hGR{alpha} protein. However, GR may be otherwise associated with active promoters, for example by being tethered to response elements through physical interaction with transcription factors like nuclear factor-{kappa}B and activator protein-1. In addition, an attractive hypothesis has recently been formulated stating that receptor mobility may be determined by low affinity binding to random sites in the chromatin. This would reflect the receptor scanning the entire genome for the relatively few available GREs (39).

Another relatively immobile nuclear structure is formed by the nuclear matrix, which was originally defined as the nonchromatin structural components of the nucleus. In most experimental reports, it is referred to as the structure that is left after extraction of most of the chromatin and soluble and loosely bound proteins. Although a nuclear matrix remains a somewhat controversial concept (40), the idea is accepted by many researchers (41). Ligand-bound GR has been shown to be present in nuclear matrix preparations in several studies and the DBD and LBD appear to be required (42, 43, 44). It is still unclear how GR would accumulate at certain domains of the nuclear matrix [although a GR-binding constituent of the nuclear matrix has been found (45, 46)], and what the function of this accumulation is. It has been demonstrated that transcription takes place at certain domains of the nuclear matrix (47), which could coincide with domains of receptor accumulation. Alternatively, transcription complexes may be assembled at these domains, or this complex could be degraded at these sites. An important role for proteasome activity in the mobility of glucocorticoid and estrogen receptors has been demonstrated in several reports (31, 32, 36, 48), suggesting receptor degradation and subnuclear targeting may be linked. Transcriptional coactivators like steroid receptor coactivator-1 (21, 49), GR-interacting protein 1 (26), cAMP response element binding protein-binding protein (21, 49), and Brahma related gene-1 (50) have been shown to be organized in nuclear foci as well, and cAMP response element binding protein-binding protein, transcription intermediary factor-2 and steroid receptor coactivator-1 have been shown to be colocalized with AR (21).

In summary, our data show that GR ligands differ in their ability to target the receptor to focal subdomains in the nucleus. The receptor exchanges rapidly on these relatively immobile domains, and the transient association with these domains decreases its mobility.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
The synthesis of expression plasmids for yellow fluorescent protein (YFP)- and cyan fluorescent protein (CFP)-tagged hGR{alpha} and hGR{alpha} mutants was described previously (32). Briefly, plasmids pEYFP-C1 and pECFP-C1 were purchased from BD Biosciences Clontech (Palo Alto, CA). Using PCR amplification of hGR{alpha} cDNA and subsequent cloning of the PCR product into pEYFP-C1 and pECFP-C1, plasmids pEYFP-hGR{alpha} and pECFP-hGR{alpha} were constructed, encoding an in-frame N-terminal fusion protein of hGR{alpha} with YFP or CFP. Mutated versions of this vector were made in our laboratory. Mutants F623A and Q642V were created by site-directed mutagenesis of pEYFP-hGR{alpha} using the QuikChange kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Expression vectors for YFP-tagged deletion mutants {Delta}9–385 and {Delta}428–490 and truncation mutant I550 were generated like pEYFP-hGR{alpha}, using vectors for the respective nontagged mutants of pRShGR{alpha} (51, 52) obtained from Dr. R. Evans (The Salk Institute, San Diego, CA).

Compounds
The following steroids were used in the present study: cortexolone (4-pregnen-17, 21-diol-3, 20-dione), corticosterone (4-pregnen-11ß, 21-diol-3, 20-dione), cortisol (4-pregnen-11ß, 17, 21-triol-3, 20-dione), cortisone (4-pregnen-17, 21-diol-3, 11, 20-trione), 1-dehydrocorticosterone (1, 4-pregnadien-11ß, 21-diol-3, 20-dione), deltafludrocortisone (1, 4-pregnadien-9{alpha}-fluoro-11ß, 17, 21-triol-3, 20-dione), desoxymetasone (1, 4-pregnadien-9{alpha}-fluoro-16{alpha}-methyl-11ß, 21-diol-3, 20-dione), dexamethasone (1, 4-pregnadien-9{alpha}-fluoro-16{alpha}-methyl-11ß, 17, 21-triol-3, 20-dione), prednisolone (1, 4-pregnadien-11ß, 17, 21-triol-3, 20-dione), RU486 (4, 9-estradien-17{alpha}-propynyl, 11ß-[4-dimethylaminophenyl]-17ß-ol-3-one), triamcinolone (1, 4-pregnadien-9{alpha}-fluoro-11ß, 16{alpha}, 17, 21-tetrol-3, 20-dione), triamcinolone acetonide (1, 4-pregnadien-9{alpha}-fluoro-11ß, 16{alpha}, 17, 21-tetrol-3, 20-dione-16, 17-acetonide), and ZK98299 (4,9-gonadien-11ß-[4-dimethylaminophenyl]-17{alpha}-ol-17ß-[3-hydroxypropyl]-13{alpha}-methyl-3-one). All compounds were purchased from Steraloids Inc. (Newport, RI), except ZK98299, which was a kind gift from Schering (Berlin, Germany).

Cell Culture and Transfection
COS-1 cells were grown as described previously (32, 53). One day before transfection, cells were transferred to 78.5 cm2 dishes (7.5 x 105 cells per dish). Cells were transfected using TransIt reagent (Mirus, Madison, WI) with either the YFP-hGR{alpha} expression vector pEYFP-hGR{alpha}, or a mutated version of this vector. Per dish, 0.4 µg of plasmid and 20 µl of TransIt Reagent were used. After a 5-h incubation with the TransIt reagent/DNA mixture, cells were re-fed with supplemented DMEM. One day after transfection, cells were transferred to 9.6 cm2 dishes containing glass bottoms (MatTek Corp., Ashland, MA; 1.5 x 105 cells per dish). The next day, the cells were studied by confocal microscopy.

Confocal Microscopy
Cells were observed using a Zeiss LSM 510 confocal laser-scanning microscope (Carl Zeiss, Jena, Germany), using a Plan-Apochromat 100x oil immersion objective (1.4 numeric aperture). Cells expressing YFP-tagged proteins were excited with an Argon laser at 514 nm, and emission was collected using a 530-nm-long pass filter. Cells with very high and very low YFP-hGR{alpha} expression levels were excluded from the study by using the following criteria. The average fluorescence intensity in the nucleus (after ligand addition) as indicated by the detection software had to be between 100 and 200 (arbitrary units), with the detector gain between 750 and 900 V. Images were taken at a resolution of 512 x 512 pixels (pixel size 0.07 µm x 0.07 µm, pixel time 6.4 µsec), except for the pictures shown in Figs. 3BGo (2048 x 2048 pixels).

Image Analysis
To quantitate randomness of distribution image analysis was performed using the program NIH-Image 1.63 (developed at the National Institutes of Health and available at http://rsb.info.nih.gov/nih-image). In each experiment, ten cells were randomly selected per treatment using the criteria described above. The cells were taken from two separate dishes (five cells per dish). Data shown are average ± SEM of at least two experiments.

FRAP
FRAP was performed as described previously (32). For determining the mobility of YFP-tagged proteins, images were taken every 196.6 msec at a resolution of 128 x 128 pixels (pixel size 0.29 µm x 0.29 µm, pixel time 3.52 µsec). After the first image, a selected rectangular region of fixed size (50 x 10 pixels) in the nucleus was bleached at a set laser power of 15 mW for 40 iterations. Fluorescence in the bleached region and in the total nucleus was quantified at every time point using LSM software (Zeiss). In each experiment, two dishes (five cells per dish) were analyzed per treatment. To correct for differences in expression level between individual cells, fluorescence data for the bleached region and the total nucleus were normalized to the prebleaching level. In addition, at all time points data were normalized to the fluorescence in the total nucleus to correct for the loss in fluorescence due to the bleach pulse and the imaging. Using these data, the t1/2 of maximal recovery was determined, which is defined as the time point after bleaching at which the normalized fluorescence has increased to half the amount of the maximal recovery. Every t1/2 shown is an average ± SEM of at least two experiments.

Analyzing the mobility of CFP-tagged proteins was done similarly using a 2-photon laser at 840 nm. Images were taken every 496 msec at a resolution of 128 x 128 pixels (pixel size 0.14 µm x 0.14 µm, pixel time 12.8 µsec). After the first image, a selected rectangular region of fixed size (10 x 10 pixels) in the nucleus was bleached at a set laser power for 20 iterations. In each experiment, three dishes (five cells per dish) were analyzed per treatment.

GR Competitor Binding Assay
The relative GR binding affinity was determined for all ligands used in the present study using the GR Competitor Assay kit (PanVera, Madison, WI). The assay was performed according to the manufacturer’s instructions. Briefly, in microwell plate wells, a fixed concentration of a fluorescent GR ligand (Fluormone GS1) was mixed with ten different concentrations of the ligand of interest. Purified human recombinant GR was added, and the plate was incubated for 4 h in the dark at room temperature. Increased receptor binding by the ligand of interest causes a decrease in fluorescence polarization levels. For each well, fluorescence polarization values were measured using the Polarion fluorescence polarization system (Tecan, Durham, NC) using 485-nm excitation and 535-nm emission interference filters. In each individual experiment, dose-response curves were generated and relative binding affinities calculated. Data shown are averages (±SEM) of at least three individual experiments.

Statistical Analysis
Statistical analysis was performed using JMP 5.0.1 software (SAS Institute, Cary, NC) and consisted of one- or two-way ANOVA performed on log-transformed data. Where ANOVA indicated statistical significance, the Tukey-Kramer HSD post hoc test was used to compare individual groups, except for the experiment described in Fig. 7Go, where Dunnett’s test was used to compare mutant vs. wild-type receptors. Regression analysis was performed by ANOVA. Statistical significance was accepted at P < 0.05.


   ACKNOWLEDGMENTS
 
The authors would like to thank Jeff Reece for his assistance with the confocal microscopy, and Drs. Gary Bird and Tatsuya Sueyoshi for critical reading of the manuscript.


   FOOTNOTES
 
Present address for M.J.M.S.: Institute of Biology, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. E-mail: schaaf{at}rulbim.leidenuniv.nl.

First Published Online February 10, 2005

Abbreviations: 17-OH, 17-hydroxy; AR, androgen receptor; CFP, cyan fluorescent protein; CV, coefficient of variation; DBD, DNA binding domain; ER, estrogen receptor; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; GR, glucocorticoid receptor; GRE, glucocorticoid-responsive elements; hGR{alpha}, human GR{alpha}; LBD, ligand binding domain; MR, mineralocorticoid receptor; RBA, relative binding affinity; TA, triamcinolone acetonide; t1/2, half-time of maximal recovery; YFP, yellow fluorescent protein.

Received for publication October 15, 2004. Accepted for publication January 12, 2005.


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Nuclear Receptors:   GR
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