Separable Features of the Ligand-Binding Domain Determine the Differential Subcellular Localization and Ligand-Binding Specificity of Glucocorticoid Receptor and Progesterone Receptor

Yihong Wan, Kimberly K. Coxe, Varykina G. Thackray, Paul R. Housley and Steven K. Nordeen

Department of Pathology and Program in Molecular Biology (Y.W., V.G.T., S.K.N.) University of Colorado Health Sciences Center Denver, Colorado 80262
Department of Pharmacology and Physiology (K.K.C., P.R.H.) University of South Carolina School of Medicine Columbia, South Carolina 29208


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoid receptor (GR) and progesterone receptor (PR) are closely related members of the steroid receptor family of transcription factors. The two receptors share a similar domain structure, substantial sequence identity, DNA binding specificity, and the ability to induce many of the same genes. Despite these similarities, the unliganded GR is localized predominantly in the cytoplasm, while unliganded PR is found predominantly in the nucleus. By expressing green fluorescent protein (GFP)-tagged receptors and assessing subcellular localization in living cells by confocal microscopy, we have investigated the structural basis for the differential localization of GR and PR. By constructing a series of GFP-tagged receptor chimeras between GR and PR, we have shown that multiple features in the N-terminal half of the ligand-binding domain (LBD) are the critical determinants that mandate the differential localization of GR and PR. Replacement of residues encompassing helices 1–5 of GR with those of PR yields a receptor that is nuclear. However, this domain is unable to mediate nuclear import by itself when removed from the context of the receptor. The chimeric receptors also indicate that regions encompassing helices 6 and 7 are key determinants of the ligand binding potential and the transactivation potential of receptors. Thus, the determinants specifying localization of hormone-free receptors are separable from those governing ligand binding character.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The steroid hormones progesterone and cortisol have very distinct biological functions. The major physiological role of progesterone in the mammal is to establish and maintain pregnancy in the uterus and ovary, to promote lobular-alveolar development, and to suppress milk protein synthesis in the mammary gland during pregnancy (1). In contrast, classic actions of glucocorticoids include regulation of glucose metabolism, inhibition of bone formation, and antiinflammatory and immunosuppressive actions (2).

The receptors for progesterone (PR) and for glucocorticoids (GR) are two closely related members of the nuclear receptor family of transcription factors. They share very similar domain structures and functional characteristics. Each N-terminal domain of the two receptors, while nonhomologous, has ligand-independent transcription activation function; the DNA-binding domain (DBD) exhibits 86% sequence identity; the short hinge region has little identity between receptors but is the location of a major nuclear localization signal (NLS); and finally, the C-terminal ligand-binding-domain (LBD) exhibits 54% identity between human (h) GR and hPR, overlapping ligand-binding specificity, and a ligand-dependent transcription activation function (3). The DBD and the LBD have been characterized extensively both structurally and functionally. For both receptors an optimal recognition site is an inverted hexameric palindrome separated by 3 bp, PuGNACANNNTGTNCPy (4). In the absence of hormone, receptor monomers form an inactive complex with molecular chaperones, most notably heat shock protein 90 (hsp90) (5). Upon binding of an agonist ligand to the receptor, it dissociates from the chaperones and undergoes dimerization. The dimerized receptor binds to recognition sites in target promoters and recruits coactivators, resulting in increased transcription initiation at those promoters. In a number of cases, both receptors can mediate induction of the same genes (6, 7), although there are poorly understood influences of chromatin that can differentially modulate induction by GR and PR (8). How can two receptors with such remarkable similarities mediate such dissimilar biological effects? The mechanism underlying the distinct biological effects of glucocorticoids and progestins, even in cells where both receptors are present, is a question of significant interest.

Despite the remarkable similarities between these two receptors, previous biochemical and immunochemical studies have shown that the subcellular localization of GR vis a vis PR in the absence of ligands is quite distinct. Although both receptors appear to continuously shuttle, at any given moment GR is found largely in the cytoplasm (9–12), while PR localizes to the nucleus (13). Hormone binding results in tight association of the receptors with the nucleus. The multipartite NLSs of GR and PR have been mapped in some detail (14–18). The major NLS is located just C-terminal to the DBD in the hinge domain and is comprised of a stretch of clustered basic residues. Two additional basic clusters in the second zinc finger of the DBD contribute to the overall NLS activity. Interestingly, it has been shown that the NLS in GR, which is cytoplasmic in the absence of hormone, is just as potent as that of PR and ER, which are both nuclear. A mutant PR, whose NLS has been replaced by the NLS of GR, also localized to the nucleus in the absence of ligand (14). Thus, current hypothesis posits that the differential localization of GR and PR is not determined by the NLS but rather that the GR LBD can mask the NLS. This view is based on the following observations: 1) LBD-truncated GR is constitutively nuclear and has constitutive transcriptional activity (19–21); 2) when the LBD is moved to the N terminus of GR, leaving the NLS at the C-terminal end of the receptor, the receptor is constitutively nuclear (22); 3) an antibody (AP64) against the major NLS can react with liganded GR but not unliganded receptor (23). It is unclear whether the LBD itself or the hsp90 bound to the LBD masks the NLS, although some evidence favors hsp90 (24). However, the hypothesis that the LBD or associated proteins masks the NLS has been challenged (25, 26).

In this study, we seek to define the receptor domain that determines the differential localization between GR and PR. We use receptors tagged with green fluorescent protein (GFP) to monitor the subcellular localization of steroid receptors by confocal microscopy. GFP from the jellyfish, Aequorea victoria, has been developed into an extremely useful tool to monitor protein localization and trafficking. Many proteins, when fused to GFP, maintain their normal function and localization (27). The utility of GFP has been extended by the selection of enhanced variants that exhibit increased fluorescence; in addition, codon usage has been humanized to improve translation. Fluorescence can be monitored in living cells, thereby avoiding artifacts caused by biochemical fractionation or fixation. GFP is proving to be an extremely useful means of dissecting the localization of nuclear receptors and the dynamics of receptor trafficking (26, 28–31). Here, we use GFP-tagged receptors to monitor the differential subcellular localization between GR and PR. By assessing a series of GFP-tagged receptor chimeras, we show that it is the LBD that determines the differential localization of the two highly related receptors. Chimeras between the two LBDs implicate multiple features within the N-terminal half of the LBD in the specification of the nuclear or cytoplasmic localization of the receptor in the absence of hormone. However, the context of this region within the receptor is critical since it cannot by itself promote nuclear import or export. Mutagenesis studies of steroid receptors along with ligand-LBD crystal structures from different members of the nuclear receptor family, including PR (32, 33), implicate residues in the N-terminal portion of the LBD in ligand binding specificity. Nonetheless, we show that there is not a strict correlation between the ligand binding specificity of the receptor, its subcellular localization, or capacity for transactivation, implying that distinct structural features determine these properties.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GFP-GR and GFP-PR Can Be Used to Monitor Receptor Localization and Are Biologically Active
To examine whether GFP-GR and GFP-PR exhibited a subcellular localization like that of wild-type GR and PR, expression vectors encoding GFP-GR and GFP-PR fusion proteins were transiently transfected into COS-1 cells, Ltk-, or E82.A3 fibroblasts. Multiple cell lines were employed to assess consistency of results. COS-1 cells have features that optimize visualization, including excellent adherence, a flat morphology, and high levels of protein expression caused by vector replication. E82.A3 is a clonal cell line derived from mouse L cells that has been selected for the absence of endogenous GR expression (34). In addition, these cells do not express PR. The level of receptor expression in transfected E82.A3 cells is lower than in COS-1 cells. Ltk- cells display fibroblast morphology and transfect well, but express low levels of endogenous GR.

Receptor localization was assessed by confocal microscopy in living cells. Similar results were seen in all three lines, and confocal data for COS-1 and E82.A3 cells are shown in Fig. 1Go. GFP itself is distributed throughout the cell; this distribution is unaffected by hormones. GFP-GR is found predominantly in the cytoplasm in the absence of hormone. Treatment of GFP-GR-expressing cells with dexamethasone is accompanied by the rapid movement of receptor to the nucleus (t1/2 ~5 min). Nuclear receptor remains excluded from nucleoli. In contrast, GFP-PR localizes predominantly to the nucleus in the absence of hormone, although cytoplasmic GFP-PR is sometimes seen, especially when the receptor is overexpressed. GFP-PR remains nuclear after hormone addition as expected.



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Figure 1. Subcellular Localization of GFP-Tagged Receptors

COS-1 cells and E82.A3 cells were transfected with the indicated expression vectors. Hormone treatment of transfected cells with vehicle, dexamethasone (100 nM), or R5020 (20 nM) was for 1 h. Cells transfected with GFP-vector and GFP-GR were treated with dexamethasone; cells transfected with GFP-PR were treated with R5020. Confocal microscopy was conducted on live, unfixed cells.

 
Reporter gene transfection experiments indicate that GFP-GR and GFP-PR fusion proteins retain biological activity. The GFP-tagged receptors mediate the induction of a hormone-responsive mouse mammary tumor virus (MMTV)-luciferase reporter as effectively as wild-type GR and PR (Fig. 2CGo). These results demonstrate that the GFP-GR and GFP-PR chimeras recapitulate the cellular distribution expected of GR and PR as well as the wild-type transcriptional activity.



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Figure 2. The LBD Determines Subcellular Localization of Chimeric Receptors

A, Schematics of chimeric receptors in which the LBD of GFP-GR{alpha} and GFP-PR-B have been exchanged. B, Subcellular localization of GFP-tagged chimeric receptors. Cells transfected with P/G were treated with dexamethasone (100 nM); cells transfected with G/P were treated with R5020 (20 nM). Confocal microscopy was conducted on live, unfixed cells. C, The function of GFP-tagged receptors and receptor chimeras was assessed by cotransfection of 1 µg/ml of the indicated receptor expression vector with a reporter plasmid pAHLuc into E82.A3 cells. Reporter gene expression was assessed 20–24 h after the addition of 100 nM dexamethasone (Dex), 20 nM R5020, or vehicle (Veh) to the transfected cells. Luciferase activity has been normalized to the expression of ß-galactosidase directed by the internal transfection control plasmid pCMVß-gal. hGR-I9 represents a fully functional receptor with a small insertion at amino acid 9. I9 was used in the construction of all GFP-hGR (see Materials and Methods). The higher activity of GFP-hGR vs. hGR-I9 is likely due to the higher expression of the receptor from the strong CMV promoter on the GFP expression vector. The figure depicts the results of three independent experiments within which each condition was done in duplicate. Bars represent ± SE.

 
Differential Localization of GR and PR Is Determined by the Ligand-Binding Domain
To determine which functional domain of GR and PR is responsible for the differential localization of the unliganded receptors, expression vectors encoding GFP-tagged receptor chimeras were constructed (Fig. 2AGo). The presence of a unique BclI site adjacent to the hinge-LBD border of hPR (35) facilitates the construction of GFP-tagged receptors in which the LBDs of PR and GR are exchanged. Thus, G/P contains the entire N terminus, DBD, and hinge from hGR and the entire LBD from hPR with GFP at the N terminus of the chimeric receptor. Conversely, P/G is the complement where only the LBD is derived from GR. Unliganded G/P is nuclear in both COS-1 cells and E82.A3 cells, whereas unliganded P/G is cytoplasmic, redistributing to the nucleus on dexamethasone addition (Fig. 2BGo). Thus, while the multipartite NLS is necessary for nuclear import of both GR and PR (14, 15, 16, 17, 18), it is the LBD that determines the differential localization of the unliganded receptor. The chimeric receptors retained transcriptional activity. P/G was fully activated by the synthetic glucocorticoid dexamethasone and G/P by the synthetic progestin R5020 (Fig. 2CGo).

Nuclear Localization Specificity Maps to the N-Terminal Half of the LBD but Is Separable from Ligand Binding Specificity
To localize further the region within the LBD that is responsible for the differential localization of GR and PR, additional chimeras within the LBD were created. Portions of the PR LBD were replaced by the homologous portions of GR, taking advantage of three natural restriction sites in PR that divide the LBD into four segments (see Fig. 3AGo and Materials and Methods). Thus, in the expression vectors P/P1-9G1 and P/P1-7G, C-terminal PR sequences have been replaced with 82 or 123 amino acids of GR sequence, respectively. When expressed in cells, both receptors were nuclear without ligand (Fig. 3BGo), like PR itself, indicating that the C-terminal half of the LBD has little role in differential receptor localization. Furthermore, since P/P1-7G differs from the P/G only in the origin of the N-terminal half of the LBD yet exhibits nuclear rather than cytoplasmic localization, it suggests that sequences encompassed by the 123 amino acids of the helices 1–7 segment determine differential localization of GR and PR. The construction and analysis of additional vectors confirmed this suggestion. G/P1-9G and G/P1-7G were nuclear in the absence of ligand just as P/P1-9G and P/P1-7G (Fig. 3Go), confirming that sequences N-terminal to the LBD are not involved in the differential localization of GR and PR.



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Figure 3. Nuclear Localization Specificity Maps to the N-Terminal Half of the LBD but Is Separable from Ligand Binding Specificity

A, Schematics of receptors with chimeric LBDs. The GFP tag at the N terminus of the receptors is not depicted. The distribution of the receptor after transient expression in COS-1 and E82.A3 cells is indicated to the right of the schematic. Treatment with vehicle (Veh), dexamethasone (Dex, 100 nM), or R5020 (20 nM) was for 1 h. The BclI site present at the boundary of the hinge region and the LBD is represented as a solid vertical line. aa, Amino acid. "N" indicates that most or all cells have a distribution that is entirely nuclear or nuclear>>cytoplasmic. "C" indicates that most or all cells have a distribution that is entirely cytoplasmic or cytoplasmic>>nuclear. "N+C" indicates that most or all cells have an approximately equal nuclear and cytoplasmic distribution of fluorescence. B, Representative confocal images of transfected COS-1 cells expressing the chimeric receptors shown in panel A and treated with hormone or vehicle as indicated.

 
Unlike G/P1-7G, the chimera G/P1-3G was cytoplasmic (Fig. 3Go), indicating that sequences within the helices 4–7 segment play a role in specifying localization. However, helices 4–7 are insufficient to specify localization, because the chimera P/GP4-12, the converse of G/P1-3G, is, like G/P1-3G, also cytoplasmic in the absence of hormone (Fig. 3Go). If an LBD chimera and its converse both exhibit a similar localization pattern, one of two possibilities exists: either both chimeras exhibit the default localization phenotype because the localization signal has been disrupted or, conversely, both chimeras exhibit a directed localization because the signal has multiple components that can each act independently. Thus, the finding that the converse pair, P/GP4-12 and G/P1-3G, are both cytoplasmic could signify that this is the default localization, suggesting that in the chimeras an NLS present in PR has been disrupted. This could be either a single, contiguous determinant or a multipartite signal. Alternatively, nuclear localization is the default and determinants from both the GR helices 4–7 and GR helices 1–3 regions can independently specify cytoplasmic localization, e.g. by serving as nuclear export signals. We believe this latter alternative to be less likely, in part because placing an NLS at the N terminus of GR creates a constitutively nuclear receptor (Ref. 22 and L. Taraseviciene and S. K. Nordeen, unpublished results). Additional results below address these possibilities and further localize the determinants.

Chimera G/P1-3G redistributes to the nucleus upon addition of the glucocorticoid dexamethasone. In contrast, P/GP4-12 does not. However, P/GP4-12 can bind the progestin R5020, albeit poorly, since it only partially redistributes to the nucleus after exposure to ligand. Since P/GP4-12 binds progestins in preference to glucocorticoids yet is cytoplasmic in the absence of ligand, it suggests that localization and hormone binding specificity are separable properties of receptors. Additional chimeras confirm this suggestion as detailed below.

Multiple Subdomains of the LBD Contribute to Localization
The next series of chimeras were constructed to determine which domains within the N-terminal half of the LBD are involved in receptor localization (Fig. 4AGo). P/P1-speG is a chimera in which the fusion was made at an introduced SpeI site within the third helix. The converse chimera is G/GPspe-12 in which the only LBD sequences from GR are the first helix, spacer, and the first 4 amino acids of the third helix. When expressed in cells, both of these chimeric receptors are cytoplasmic like GR (Fig. 4BGo). The fusion of PR and GR sequences at the introduced SpeI site within helix 3 may have disrupted an NLS. To test this, the four GR residues of the third helix of G/GPspe-12 LBD (WRIM) were mutated to PR sequence (SSLL) to create G/GP3-12 where the entire helix 3 to C-terminal sequence is from PR. In the absence of ligand, this chimera is still largely cytoplasmic. This suggests that PR LBD sequence N-terminal to helix 3, along with the helices 4–7 domain implicated above, contribute to the specification of a PR-like nuclear localization.



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Figure 4. Multiple Subdomains of the N-Terminal Portion of the LBD Contribute to Localization

A, Schematics of receptors with chimeric LBDs. The GFP-tag at the N terminus of the receptors is not depicted. The distribution of the receptor after transient expression in COS-1 and E82.A3 cells is indicated to the right of the schematic. Treatment with vehicle (Veh), dexamethasone (Dex, 100 nM), or R5020 (20 nM) was for 1 h. "H3" stands for helix 3; aa, amino acid. "N" indicates that most or all cells have a distribution that is entirely nuclear or nuclear>>cytoplasmic. "C" indicates that most or all cells have a distribution that is entirely cytoplasmic or cytoplasmic>>nuclear. B, Representative confocal images of transfected COS-1 cells expressing the chimeric receptors shown in panel A and treated with hormone or vehicle as indicated.

 
While both P/P1-speG and the converse chimera, G/GPspe-12, are cytoplasmic in the absence of ligand, they exhibit distinct ligand specificities. Dexamethasone drives nuclear redistribution of the former while the latter redistributes in response to progestins. Like G/GPspe-12, G/GP3-12 also redistributes completely to the nucleus upon addition of R5020. Together these results indicate that although LBD sequences N-terminal to helix 3 are involved in subcellular localization of the ligand-free receptor, they do not play a significant role in the specification of ligand recognition.

Results presented above (Fig. 3Go) demonstrated that G/P1-3G is cytoplasmic and that G/P1-7G is nuclear. The next series of constructs attempted to refine the requirement for PR sequences from helices 4 through 7 in nuclear localization. Some of the greatest sequence disparity between the LBD of GR and PR occurs in helix 7. Furthermore, sequences in helix 7, helix 6, and the ß-turn structure preceding helix 6 have been implicated in differential ligand recognition (36). We therefore constructed chimeras based on G/P1-3G and G/P1-speG replacing GR sequence with PR helix 7 or a larger PR block encompassing the ß-turn, helix 6, and helix 7 (Fig. 5Go, A and B). All of the resulting double chimeras, G/P1-3GP7G, G/P1-3GP6-7G, G/P1-speGP7G, and G/P1-speGP6-7G, retained a cytoplasmic phenotype in the absence of ligand (Fig. 5CGo), indicating a role for PR sequences in helix 4 and/or helix 5 in the acquisition of a nuclear phenotype. The double LBD chimeras all had altered ligand recognition specificity compared with the progenitors, G/P1-3G and G/P1-speG. All double chimeras could translocate to the nucleus in response to R5020, and all exhibited a reduced ability of dexamethasone to induce translocation (Fig. 5CGo).




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Figure 5. Helices 1–5 of the LBD Determine Steroid Receptor Localization

A, Schematics of receptors with chimeric LBDs. The GFP tag at the N terminus of the receptors is not depicted. The distribution of the receptor after transient expression in COS-1 and E82.A3 cells is indicated to the right of the schematic. Treatment with vehicle (Veh), dexamethasone (Dex, 100 nM), or R5020 (20 nM) was for 1 h. aa, Amino acid. "N" indicates that most or all cells have a distribution that is entirely nuclear or nuclear>>cytoplasmic. "C" indicates that most or all cells have a distribution that is entirely cytoplasmic or cytoplasmic>>nuclear. "N>C" indicates that most or all cells have a distribution that is nuclear>cytoplasmic but there is a significant level of cytoplasmic distribution B, Amino acid sequences of the helices 1–7 region of hGR, hPR, and the double chimeras shown in panel A. hPR sequences are in bold and underlined. Amino acids included in each helix or the ß-turn region are indicated by an overline. The location of each restriction site used in receptor chimera construction is indicated by an arrow. C, Representative confocal images of transfected COS-1 cells expressing the chimeric receptors shown in panel A and treated with hormone or vehicle as indicated. Fig. 5CGo can be found on the facing page.

 
The final chimera that was examined was G/P1-5G in which the first 87 amino acids (through helix 5) of the LBD are derived from PR (Fig. 5Go, A and B). It exhibits largely nuclear localization in the absence of ligand (Fig. 5CGo). This chimera represented the receptor with the smallest region of PR that retained a predominantly nuclear phenotype in the absence of ligand. This chimera binds glucocorticoids, as evidenced by the fact that the remaining cytoplasmic receptor could be driven to the nucleus upon addition of dexamethasone and is transcriptionally active in response to dexamethasone (see below). Thus, several different chimeras demonstrated that a receptor binding a glucocorticoid ligand could be nuclear in the absence of ligand and, conversely, that a progestin binding receptor could be cytoplasmic in the ligand-free state. Therefore, the structural features that determine localization in the ligand-free state and those that determine ligand specificity are distinct and separable.

The Sequences That Specify Nuclear Localization Do Not Act as an Independent NLS
To test whether a region of PR that can specify nuclear localization contains an independent NLS, the 123-amino acid domain encompassing helices 1–7 was fused to a dimer of GFP (Fig. 6AGo). A GFP dimer has a molecular mass of about 54 kDa and is distributed mostly in cytoplasm (C>N). When expressed in COS cells, the fusion protein (GFP)2-PR1-7 is predominantly cytoplasmic (Fig. 6BGo). This result indicates that the helices 1–7 domain does not itself serve as an independent NLS. However, this conclusion must be tempered by the uncertainty that helices 1–7 can fold properly in the absence of the rest of the LBD.



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Figure 6. The Sequences That Specify Nuclear Localization Do Not Act as an Independent NLS

A, Schematics of GFP fusion proteins. B, Representative confocal images of transfected COS-1 cells expressing the chimeric proteins shown in panel A.

 
Ligand Binding and Transactivation Properties of Receptors with Chimeric LBDs
To ensure that LBD chimeras were, in fact, active receptors and, therefore, that localization results were not in some way artifactual, many of the chimeras were assessed for their capacity to be activated by a specific glucocorticoid, dexamethasone, and a specific progestin, R5020. Receptor expression vectors were transiently transfected into E82.A3 cells together with an MMTV-luciferase reporter plasmid and a cytomegalovirus (CMV)-ß galactosidase internal control plasmid. Many of the chimeras can transactivate gene expression in response to the appropriate steroid (Fig. 7AGo). In parallel, direct ligand binding assays were performed to examine the suppositions made with regard to ligand specificity based on ligand-mediated receptor redistribution. Ligand binding was assessed by whole-cell binding assays after transient expression of receptors in COS-1 cells (Fig. 7BGo). These studies confirm that the structural features governing ligand binding and activation capacity of a receptor are not coincident with those that determine its distribution in the absence of hormone. Also, as detailed below, a number of inferences can be made concerning the role of different domains in ligand specificity and transactivation.



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Figure 7. Transactivation and Ligand Binding Properties of Receptors with Chimeric LBDs

A, Transactivation by chimeric receptors. Each indicated receptor expression vector was transfected into E82.A3 cells and cotransfected with a reporter plasmid pAHLuc and an internal control plasmid pCMVß-gal as described in Materials and Methods. Induction of luciferase was assessed after a 24-h exposure to dexamethasone (Dex, 1 µM), R5020 (1 µM), or vehicle (Veh). Luciferase activity has been normalized to the expression of ß-galactosidase. The figure depicts the results of two independent experiments within which each condition was done in duplicate. Bars represent ± range. B, Whole-cell ligand binding assays. COS-1 cells were transiently transfected to express the indicated receptor chimeras. Specific hormone binding for dexamethasone (Dex, 20 nM) and R5020 (1 nM) are shown as a percentage of that seen with GFP-GR or GFP-PR, respectively. The figure depicts the results of two independent experiments where each condition was done in triplicate. Bars represent ± range.

 
The nuclear chimeric receptor, G/P1-7G, in which the C-terminal 123 amino acids of PR sequence have been replaced by the homologous GR sequence, still binds and is activated by R5020. Although this receptor exhibits some glucocorticoid binding (~20% of wild type), it is only minimally activated even at very high levels of dexamethasone. Replacement with additional GR sequence, G/P1-5G, still results in a receptor with a largely nuclear distribution. However, this chimeric receptor now exhibits a strong preference for binding to and activation by glucocorticoids. These data concur with the implications of the ligand-mediated redistribution data that the ß-turn helices 6–7 region possesses key determinants for steroid binding specificity.

Additional chimeras confirm the key role of this region. Chimera G/P1-3G binds glucocorticoids similarly to GR and is transcriptionally activated by dexamethasone. When the ß-turn-helix 6-helix 7 region is changed to PR sequence (G/P1-3GP6-7G), the resulting double chimera does not bind to glucocorticoids nor respond to them. Instead, this chimera displays significant binding to R5020 (albeit reduced from wild-type PR) and a proportionate transactivation response. When only helix 7 instead of the ß-turn-helix 6-helix 7 region is changed to PR sequence in another double chimera, G/P1-3GP7G, the receptors can still relocalize in response to high levels of R5020 although neither R5020 nor dexamethasone binding or transactivation is observed. The observation that this double chimeric receptor can redistribute to the nucleus in response to ligand but fail to transactivate will be addressed in the Discussion.

Along with the ß-turn-helices 6–7 region, helix 3, which forms part of the ligand binding pocket (32, 33), contributes to ligand binding and specificity. G/GP3-12 binds well to progestins and undetectably to glucocorticoids. Replacing helix 3 with the homologous GR sequence (P/GP4-12) abrogates R5020 binding. Even changing only four amino acids at the N terminus of helix 3 to make G/GPspe-12 reduces R5020 binding 4-fold compared with G/GP3-12. Thus, together these chimeras indicate that the best R5020 binding is seen when the entire helix 3 through helix 7 region is derived from PR. Interestingly, the origin of helix 3 is of lesser consequence to glucocorticoid binding. For example, G/P1-3G binds dexamethasone well. However, now changing the helices 6–7 domain to PR sequence (G/P1-3GP6-7G) abrogates glucocorticoid binding. This binding can be partially recovered by changing part of helix 3 to GR sequence (G/P1-speGP6-7G) as can a proportionate degree of transactivation. Thus, while the helix 3 sequences may be less important in glucocorticoid specificity than progestin specificity, they do contribute in the context of additional domains of the LBD. Together, these data indicate that sequences governing ligand specificity overlap, but are not coincident with, the functional domains that govern differential localization of the two receptors.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
As a matter of convenience, throughout this manuscript, we have referred to the subcellular distribution of GR and PR at a given moment as localization. By no means is this intended to imply that the distribution of these receptors is static. Indeed, the current understanding is one of exquisite dynamism as recent results from several laboratories indicate. Both in the presence and absence of ligand, GR and PR shuttle continuously between nucleus and cytoplasm (25, 37, 38). Thus, the equilibrium distribution at any moment represents the net of the nuclear import and export steps. The mechanisms and mediators of these steps are not well understood for steroid receptors. One recent report suggests both the nuclear import and export of rabbit PR can be competed by the SV-40 NLS coupled to BSA. Several lines of evidence also indicate that export of PR does not occur through an NES-CRM-1-mediated pathway (37). Results with rat GR have implicated NES-CRM-1 in nuclear export (26) although more recent work does not support the involvement of this pathway in GR trafficking (39).

Among the steroid receptors, there is general agreement that hormone-free PR and the estrogen receptor are nuclear (13, 40). Recent studies employed GFP-tagged receptors to compare the localization of the two isoforms of PR (31). In this work, the distribution of the two isoforms differed somewhat, with GFP-PR-B on average showing slightly more nuclear than cytoplasmic fluorescence and GFP-PR-A predominantly nuclear staining. We also find some GFP-PR-B transfected cells with detectable cytoplasmic fluorescence, although most of the cells exhibit predominantly or entirely nuclear fluorescence. In contrast to PR, GR and the mineralocorticoid receptor are cytoplasmic (9, 10, 11, 12, 41, 42). Both nuclear and cytoplasmic localization of the androgen receptor have been reported (43, 44, 45, 46, 47). In this work we have mapped the sequence domains of GR and PR that control the differential distribution of the two receptors in a ligand-free state. Although the sequences of the GR and PR that render the receptor capable of nuclear localization upon hormone addition have been mapped to two clusters of basic amino acids in the DBD and another in the hinge (14, 15, 16, 17, 18), previous work in which the LBD of the PR was replaced with that of GR implicated the LBD in determining the differential distribution of the two receptors in the absence of hormone (14). We have extended this work using receptor chimeras to delineate the sequences that control the differential distribution. Our approach has employed an enhanced GFP tag to follow the receptor in contrast to most previous studies on receptor localization and trafficking that have used indirect immunofluorescence. A green fluorescent protein tag permits the visualization of receptor proteins in living, unfixed cells and therefore represents a valuable alternative approach to studies on receptor trafficking. In addition, due to the similarity of the LBDs of GR and PR, receptor chimeras used in our study maintain the receptor integrity and overall conformation compared with receptor deletion mutants, as evidenced by the ability to bind hormone and, in most cases, to transactivate in response. This is not unexpected. The crystal structure of the LBD of hPR bound to progesterone has been solved to a 1.8 Å resolution (32, 33). Despite amino acid identity as low as 15%, PR exhibits an overall structure very similar to other members of the nuclear receptor family for whom the crystal structure is available.

Notwithstanding the overall conformational similarity and extensive sequence identity of the LBD, GR and PR exhibit distinct ligand binding properties and distinct subcellular distribution patterns in the ligand-free state. The examination of many chimeric LBDs has shown that determinants of ligand binding specificity and differential localization are separable. The region of helices 1–5 determines subcellular localization specificity, while helix 7 and the larger ß-turn-helix 6-helix 7 domain along with helix 3 make important contributions to ligand recognition. Therefore, a glucocorticoid binding chimeric receptor could be nuclear, and conversely a progestin binding chimeric receptor could be cytoplasmic in the ligand-free state.

Interestingly, a subset of chimeras (the double chimeras, Figs. 5Go and 7Go) could redistribute from cytoplasm to nucleus upon addition of ligand, yet they exhibited greatly reduced or ablated transactivation capability. GR is driven to the nucleus by ligand relatively quickly (t1/2 5 min) yet GR that has been withdrawn from hormone redistributes into the cytoplasm only slowly (t1/2 4 h) even though nucleocytoplasmic shuttling continues and reestablishment of an 8S complex has occurred (25, 48). In light of these kinetics, it stands to reason that the addition of hormone to a chimeric receptor whose structure reduces hormone affinity because of a high off rate may cause some nuclear accumulation even though the receptors are only occupied a small fraction of the time. The chimeric receptor may not be able to assume a proper conformation to bind coactivators and thus fail to promote transactivation. Alternatively, a high ligand off rate may not allow sufficient time for the completion of the full set of receptor-promoted steps necessary to initiate a round of transcription initiation before the entire process is aborted by ligand dissociation.

The examination of the GFP-tagged receptor chimeras has narrowed the domain that determines the differential distribution of unoccupied GR and PR to the N-terminal 87 amino acids of the LBD (helices 1–5) and suggest that multiple determinants may be involved in specifying nuclear localization. This region of GR and/or PR has been implicated in binding of chaperone proteins and in ligand binding. The present work implies that this region may have yet another role in interacting with nuclear trafficking proteins to determine kinetics of the import and export steps and therefore the equilibrium distribution of the protein. The difference in nucleocytoplasmic trafficking between ligand-free GR and PR suggests that the helices 1–5 region of the GR LBD may have a distinct conformation compared with PR. As mentioned earlier, GR that has been withdrawn from hormone redistributes into the cytoplasm only slowly even though nucleocytoplasmic shuttling continues and reestablishment of an 8S complex has occurred (25, 48). It may be that this hormone-withdrawn state represents an LBD conformation that is more PR-like and that reverts slowly to a conformation that is more typical of the naive receptor. Binding of the antiprogestin RU486 to GR appears to entrain a conformation that favors nuclear localization as GR withdrawn from RU486 fails to redistribute to the cytoplasm (Refs. 25, 48 and P. R. Housley, unpublished). Data with RU486 and with the pure antiestrogen ICI182 780 (49) suggest that disruption of the normal nucleocytoplasmic trafficking of receptors may play a role in the mechanism of action of steroid antagonists. The widespread clinical use of steroid antagonists makes this an important question to understand more fully.

The functional consequences of the differential localization of PR and GR are not clear. However, there are indications that the localization of PR may be regulated in a developmental or tissue-specific fashion (50). This regulation suggests that there are indeed biological consequences of receptor localization. Preliminary findings that suggest a molecular target have come from yeast two-hybrid studies. These studies indicate that PR has a strong SH3 binding domain and can physically interact in vitro with cell signaling molecules, such as the tyrosine kinase src, and regulate activity (51). Since these cell-signaling molecules are present predominantly in compartments other than the nucleus, such findings highlight the importance of a thorough understanding of steroid receptor trafficking.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Construction
GFP-hGR{alpha} was constructed by cloning a BamHI-XhoI fragment from pI9 (52) into the GFP-C2 vector (CLONTECH Laboratories, Inc., Palo Alto, CA). GFP-hPR was constructed by cloning an EcoRI-ScaI fragment from hPR1 (35) into the GFP-C1 vector (CLONTECH Laboratories, Inc.). Receptor chimeras G/P, P/G, P/P1-9G, P/P1-7G, G/P1-3G, and P/GP4-12 were constructed by cloning fragments of hGR amplified by PCR using Pfu polymerase (Stratagene, La Jolla, CA). Appropriate restriction sites were incorporated into the primers to facilitate the in-frame junction with naturally occurring restriction sites within hPR (see Fig. 3AGo). The BstBI site in the PR LBD immediately follows the coding region encompassing LBD helix 3, HindIII follows helix 7, and Ecl136 II cuts near the C terminus of helix 9. All PCR-generated sequences were confirmed by sequencing. Receptor chimeras G/P1-9G and G/P1-7G were constructed by replacing the LBD in G/P with the chimeric LBD from P/P1-9G and P/P1-7G, respectively, utilizing the BclI site at the junction between the hinge and the LBD. SpeI sites were introduced into the LBD of GFP-GR{alpha} and GFP-PR-B by Quick-Change site-directed mutagenesis (Stratagene), and the products were confirmed by DNA sequencing. Receptor chimeras P/P1-speG and G/GPspe-12 were constructed by using these SpeI sites. Chimera G/P1-speG was constructed by replacing the LBD in G/P with the LBD fragment from P/P1-speG at the BclI site. Chimera G/GP3-12 was constructed from G/GPspe-12 by Quick-Change site-directed mutagenesis (Stratagene), changing the sequences encoding the four amino acids immediately N-terminal of the introduced SpeI site from GR-like (WRIM) to PR-like (SSLL). The changes were confirmed by DNA sequencing. All the double chimeric receptors were constructed using the gene Splicing by Overlapping Extension (SOE) procedure (53, 54). G/P1-3GP6-7G and G/P1-3GP7G were constructed using G/P1-3G and G/P1-7G as progenitors. G/P1-speGP6-7G and G/P1-speGP7G were constructed using G/P1-speG and G/P1-7G as progenitors. G/P1-5G was constructed using G/P1-7G and GFP-GR as progenitors. Additional details on plasmid constructions, including the oligonucleotides used for PCR and for site-directed mutagenesis, are available on request.

Cell Culture and Transfection
Mouse fibroblast E82.A3 and Ltk- cells were maintained in MEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% FBS (HyClone Laboratories, Inc.), 10 mM HEPES, and nonessential amino acids. Transient transfections of both lines were performed using a diethylaminoethyl (DEAE)/Dextran method as previously described (55). For fluorescence experiments, cells were plated on coverslips in culture dishes and transfected with 5 µg/ml of receptor expression vector. Cells were maintained in medium containing charcoal-stripped serum before fluorescence imaging. Fluorescence was assessed 24–30 h after transfection and 1 h after vehicle or hormone addition. For quantitation of reporter gene expression, cells were transfected with 1 µg/ml of receptor expression vector. Cells were cotransfected with 2 µg/ml of pAHluc and 0.1 µg/ml of pCMVß-gal. The response of the former was used to assess hormone response, and the latter served as an internal transfection control. The promoter of pAHluc is an AvaI-HpaII fragment spanning nearly the entire MMTV long terminal repeat. Cells were treated with hormone for 20–24 h beginning the second day after transfection. Extracts were prepared by first washing the cells, harvesting them in 0.5 ml of cell lysis buffer, and then pelleting debris (55). For luciferase assays, 25 µl of soluble lysate were used and for ß-galactosidase assays, 2.5 µl were used. Luciferase and ß-galactosidase assays were assessed using a Monolight 3010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) as previously described (55). Data are reported as luciferase activity normalized to ß-galactosidase activity in the same transfection.

COS-1 cells were maintained in DMEM (Life Technologies, Inc.) supplemented with 10% FBS (Life Technologies, Inc.). Transient transfection of COS cells was performed using a modified DEAE/Dextran method with 200 µg/ml DEAE/Dextran and 30 µM chloroquine. Cells were incubated in the DNA-DEAE/Dextran-chloroquine transfection mixture for 2 h at 37 C. The transfection mixture was aspirated and the cells subjected to a 1-min shock (55). Cells were then washed twice with PBS and refed with DMEM with charcoal-stripped serum. Hormone or vehicle were added 18–20 h after transfection and fluorescence was monitored 1 h thereafter.

Fluorescence Microscopy
Transfected cells on coverslips were analyzed by confocal scanning laser microscopy with a MRC instrument (Bio-Rad Laboratories, Inc., Hercules, CA). Living cells in medium were scanned at low laser power to avoid photobleaching and at a sufficient depth to correctly assess the presence of intranuclear signal. The figures show representative cells from each transfected DNA; at least 50–100 cells from each transfection were inspected and scored as described in the legend to Fig. 3Go.

Whole-Cell Hormone Binding Assays
For hormone binding assays, 5 µg/ml of each construct was transfected as described into COS-1 cells in six-well plates (R5020 assays) or a T150 flask (dexamethasone assay). For the latter, cells were replated into six-well dishes 24 h after transfection so that they would be 60–70% confluent at the time of harvest. Forty eight hours after transfection, three wells were treated with 3H-labeled hormone for total hormone binding, three wells were treated with 3H-labeled hormone plus excess unlabeled hormone for nonspecific binding, and three wells were left untreated for protein determination. For R5020 binding assay, cells were treated with 1 nM 3H-R5020 ± 100 nM unlabeled R5020. For dexamethasone binding assay, cells were treated with 20 nM 3H-dexamethasone ± 20 µM unlabeled dexamethasone. After incubation for 4 h at 37 C, the cells were washed five times with cold PBS. Hormone was then extracted with ethanol at room temperature for 30 min. Ethanol was transferred into scintillation vials for quantitation of bound hormone. To obtain specific binding (picomoles/mg protein) the following formula was used: total binding - nonspecific binding - specific binding from empty vector transfected cells. The latter term is included to subtract the small amount of specific dexamethasone binding seen in COS-1 cells (a few percent or less of that seen in transfected cells).


    ACKNOWLEDGMENTS
 
The authors would like to acknowledge the assistance of the DNA sequencing core and the tissue culture core of the University of Colorado Cancer Center, and the Instrumentation Resource Facility at the University of South Carolina School of Medicine.


    FOOTNOTES
 
Address requests for reprints to: Paul R. Housley, Department of Pharmacology and Physiology, University of South Carolina School of Medicine, Columbia, South Carolina 29208. E-mail: Housley{at}dcsmserver.med.sc.edu; or Steven K. Nordeen, Department of

This work has been supported by NIH Grants DK-37061 and DK-47951 to S.K.N. and to P.R.H., respectively.

1 In the naming system that will be followed for all remaining GFP-tagged receptor chimeras, the first letter designates the origin of the receptor sequences comprising the Nterminal domain through the hinge. The slash denotes the hinge-LBD border. The source of each segment of the LBD is indicated by P or G. For receptors with chimeric LBDs, subscripts denote the helices or other sequence features encompassed by the PR segments in the chimeric LBD. The GR-derived segments can be inferred. In all cases GFP is present at the N terminus of the protein. See schematics of each receptor chimera in the figures for exact boundaries of the junctions and other details. Back

Received for publication July 7, 2000. Revision received October 3, 2000. Accepted for publication October 9, 2000.


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