Molecular Identification and Characterization of A and B Forms of the Glucocorticoid Receptor

Matthew R. Yudt and John A. Cidlowski

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human glucocorticoid receptor (hGR{alpha}) is a ligand-activated transcription factor that mediates the physiological effects of corticosteroid hormones and is essential for life. Originally cloned in 1986, the transcriptionally active hGR{alpha} was reported to be a single protein species of 777 amino acids (molecular mass = 94 kDa). Biochemical data, obtained using various mammalian tissues and cell lines, however, have consistently revealed an additional, slightly smaller, second hGR protein (molecular mass = 91 kDa) that is not recognized by antibodies specific for the transcriptionally inactive and dominant negative, non-hormone-binding hGRß isoform. We report here that when a single GR cDNA is transfected in COS-1 cells, or transcribed and translated in vitro, two forms of the receptor are observed, similar to those seen in cells that contain endogenous GR. These data suggest that two forms of the hGR{alpha} are produced by alternative translation of the same gene and are henceforth termed GR-A and GR-B. To test this hypothesis, we have investigated the role of an internal ATG codon corresponding to methionine 27 (M27) as a potential alternative translation initiation site for the GR. Mutagenesis of this ATG codon to ACG in human, rat, and mouse GR cDNA results in generation of a single 94-kDa protein species, GR-A. Moreover, mutagenesis of the initial ATG codon to ACG (Met 1 to Thr) also resulted in production of single, shorter protein species (91 kDa), GR-B. Mutagenesis of the Kozak translation initiation sequence strongly indicates that a leaky ribosomal scanning mechanism is responsible for generating the GR-A and -B isoforms. Western blot analysis using peptide-specific antibodies show both the A and B receptor forms are present in human cell lines. Both receptors exhibit similar subcellular localization and nuclear translocation after ligand activation. Functional analyses of hGR-A and hGR-B under various glucocorticoid-responsive promoters reveal the shorter hGR-B to be nearly twice as effective as the longer hGR-A species in gene transactivation, but not in transrepression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glucocorticoid receptor (GR) mediates the physiological effects of corticosteroid hormones in species from fish to mammals. GR is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors (1) and is essential for life (2). The activity of the GR, as well as the progesterone (PR), androgen (AR), and mineralocorticoid receptors (MR) is partially mediated through a palindromic response element termed the glucocorticoid response element (GRE) located in the promoter regions of target genes (3). Unlike most members of the steroid receptor superfamily, the GR is primarily cytosolic in the absence of ligand. Activation and nuclear translocation of the GR after ligand binding proceeds through a complex mechanism involving a loss of energy-dependent protein interactions with hsp90, hsp70, and several other proteins (4). Although the classical view of steroid action involves an increase in gene transcription in response to receptor activation, in fact several glucocorticoid target genes undergo a hormone-dependent repression (5). Furthermore, in addition to GRE-dependent processes, a growing body of literature indicates that many glucocorticoid responses involve protein interactions with other transcription factors and likely proceed through a mutual inhibitory antagonism involving direct protein-protein interactions with other transcription factors including, for example, nuclear factor-{kappa}B (NF-{kappa}B) and AP1 (6).

Our understanding of the complexity of nuclear receptor signaling mechanisms has advanced significantly in recent years. The discovery and characterization of receptor coactivators and corepressors bridge the gap between the DNA-bound receptors and the general transcription machinery (7, 8, 9). Similarly, our knowledge regarding the role of chromatin structure in steroid receptor signaling has been enhanced in recent years (10, 11). The three-dimensional structure of many nuclear receptor ligand binding domains has not only revealed a common protein fold and ligand binding symmetry among superfamily members, but exposed the subtle ligand interactions and associated conformational changes necessary for a mechanistic understanding of steroid action (reviewed in Ref. 12). Furthermore, examples of ligand-independent activation mechanisms in nuclear receptor signaling continue to multiply (13).

An additional level of complexity of steroid hormone receptor action is the existence of multiple receptor subtypes and isoforms with unique biological roles (14, 15, 16). For example, multiple genes encode different forms of the estrogen, retinoid, and thyroid hormone receptors. Alternative splicing of progesterone, glucocorticoid, and retinoid receptor mRNA gives rise to multiple forms of these proteins. The progesterone receptor (PR) exists as a mixture of A and B forms, generated from the same gene by alternative translation initiation. Although both PR isoforms can arise from a single mRNA (17), it appears that specific promoters may also regulate mRNA production specific for each PR isoform (18). Both forms of PRs are well known to display distinct biochemical and physiological properties (19). This extensive multiplicity within the nuclear receptor superfamily suggests that the diversity of receptor expression may be an important component mediating the various physiological actions of steroid hormones.

We report here that the GR{alpha} gene is subject to alternative translation initiation from a downstream, in-frame ATG codon. Our data suggest that a leaky ribosomal scanning mechanism (20) produces two GR protein products, with the second initiating at an ATG codon corresponding to methionine 27 in the hGR. We term the longer protein, initiated from the first ATG codon (Met 1) as hGR-A, and the shorter protein (751 amino acids) as hGR-B. We have constructed a GR-A-specific antibody that, when used in conjunction with an antibody that recognizes both protein species, permits the discrimination of endogenous expression of the two hGR{alpha} isoforms. Interestingly, the shorter hGR-B is twice as effective as the longer hGR-A isoform in activating transcription from a GRE but has a similar efficacy in repression of NF-{kappa}B/p65 transactivation. This discovery of an alternative initiation site within the GR gene, and the functional divergence observed, provides a new potential mechanism to explain the diversity of glucocorticoid responses in different tissues.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Recombinant hGR
The cloning of the hGR{alpha} into mammalian and in vitro expression vectors has allowed a direct study of this protein, independent of the alternatively spliced GRß variant (21, 22). The human hGR{alpha} and -ß variants differ by only 35 amino acids in length at the extreme carboxy terminus. Although quantitative measurement of their coexpression in human tissues or cell lines remains difficult because of the relative abundance of hGR{alpha} to ß, the two isoforms can be discriminated immunologically using specific antibodies (23, 24). Interestingly, the recombinant hGR{alpha} when expressed alone, either in vitro with 35S methionine or in COS-1 cells, known to be void of detectable endogenous GR, consistently appears as a doublet of approximately equal intensities (Fig. 1AGo).



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Figure 1. In Vitro and in Vivo Expression of Recombinant hGR{alpha}

A, The wild-type hGR{alpha} was prepared either by in vitro translation using 35S methionine and reticulocyte lysates (left panel) or by transient transfection of COS-1 cells (right panel). Approximately 25 µg of protein were electrophoresed on an 8% polyacrylamide gel. The 35S-labeled receptor was detected by autoradiography of the dried gel, while the COS-1-expressed proteins were transferred to nitrocellulose and detected by Western blotting. The positions of the molecular mass markers in kilodaltons are indicated. Electrophoresis was carried out for an extended period to resolve the protein doublet of approximately 94 and 91 kDa. B, Wild-type hGR (1–777) and two carboxy-terminal truncation mutants, hGR(1–742) and hGR(1–706), were expressed in vitro using reticulocyte lysates. Electrophoresis was carried out as in panel A to resolve the hGR protein doublet. Data shown are representative of at least three different experiments.

 
There are several possible explanations for the origin of the observed hGR{alpha} protein doublet. Although proteolysis could explain the appearance of such a doublet, inclusion of several protease inhibitors did not block production of the lower mol wt (Mr) product, arguing against degradation as the source of the doublet. Moreover, in vitro transcription and translation of two carboxy-terminal truncation mutants, hGR(1–742) and hGR(1–706), results in a similar doublet pattern (Fig. 1BGo), suggesting that carboxy-terminal degradation is not the source of the second band. Another possible source of the doublet is phosphorylation; however, phosphatase treatment of the reticulocyte or COS-1 lysate containing hGR{alpha} does not affect the doublet pattern (data not shown).

Alternative Translation Initiation of the GR
To investigate the possibility of alternative translation initiation of the GR as a source of the observed doublet, the hGR cDNA was examined for downstream ATG start codons. Only one in-frame ATG codon, corresponding to methionine 27, was found within the first 300 nucleotides of the initial hGR ATG translation start site. Translation initiation from this internal ATG site would yield a protein almost 3 kDa shorter (apparent molecular mass = 91 kDa) than the full-length hGR from residues 1–777 (apparent molecular mass = 94 kDa). To test the hypothesis of alternative initiation as a source of the protein doublet observed in GR expression systems, both the initial ATG start codon (methionine 1) and the internal ATG (methionine 27) were mutated to ACG (a threonine codon). Mutagenesis of the individual ATG codons in the hGR{alpha} cDNA in both the in vitro expression vector and the mammalian expression vector resulted in the expression of a single hGR{alpha} species (Fig. 2Go). We have termed the longer GR, generated from the first ATG codon, GR-A. The shorter GR species, translated from the internal ATG corresponding to methionine 27 (amino acid 28 in rat and mouse GR), is designated as GR-B. A proteolytic fragment common to both GR forms of approximately 83 kDa is consistently observed at higher levels in cells expressing GR-B.



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Figure 2. Mutation Analyses of Potential hGR Start Sites

Plasmids containing the hGR{alpha} cDNA used for in vitro translation (T7 promoter) and for in vivo transient expression [cytomegalovirus (CMV) promoter] were independently subjected to site-directed mutagenesis, changing potential translation initiation codons (ATG) 1 and 27 individually to threonine codons (ACG). The wild-type and start site mutants were prepared as both in vitro translated, 35S-labeled protein, as well as transiently expressed in COS-1 cells, and detected as described in Fig. 1Go. The synthesis of hGR{alpha} from methionine 1 (in mutant M27T) is referred to as hGR-A while the protein synthesized from methionine 27 (in mutant M1T) is referred to as hGR-B. The 83-kDa band detected in transfected COS-1 cells is a common GR degradation product. Western blots shown are representative of more than five separate experiments.

 
In every mammalian species in which GR has been cloned and sequenced, except the guinea pig, an internal ATG was found 27 or 28 codons from the initial ATG (Fig. 3AGo). Interestingly, the guinea pig has been shown to be relatively glucocorticoid resistant in comparison to other mammals (25). In addition, neither the African frog nor rainbow trout GR contain a second potential translational start site near this position. Since doublets are detected in both mouse (mGR) and rat GR (rGR) expression systems, a similar start site mutagenesis analysis was carried out on these receptor species. As observed for the hGR, mutagenesis of the potential start sites of the mGR and rGR resulted in expression of single protein species in vivo and in vitro (Fig. 3Go, B and D). To compare the native GR with the recombinant forms, mouse and rat liver samples were analyzed for GR expression. Both mouse and rat liver GR are expressed as doublets, directly corresponding to the A and B forms observed with the recombinant proteins (Fig. 3Go, C and E). This is a particularly important observation considering neither mouse nor rat contain the ß form of the GR (Bofetiado, D. M., and J. A. Cidlowski, unpublished observations). These results suggest that the human, rat, and mouse GR genes can produce two proteins via alternative initiation of the same gene transcript.



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Figure 3. Species Comparison of GR Translation Initiation Sites

A, Sequence alignments of residues 1–40 of GRs from various species. The indicated sequences were compiled from NCBI databases (SWISS-PROT accession numbers: human, P04150; squirrel monkey P79686; rat, P06536; mouse, P06537; xenopus, P49844; rainbow trout, P49843). A dot indicates a conserved residue, while a dash indicates a single residue gap. B, The mouse GR expression vector (pCMV-mGR) was mutated as in Fig. 2Go for the hGR, changing potential translation initiation codons (ATG) 1 and 28 individually to threonine codons (ACG) to yield M1T and M28T, respectively. Protein expression in transfected COS-1 cells was measured by Western blot with Ab57. A similar band pattern was observed with in vitro translated wt and mutant mGR (not shown). C, Fifty micrograms of mouse liver protein extract were subjected to Western blot analysis with Ab57. The two major immunoreactive bands at 94 and 91 kDa correspond with the mGR-A and -B bands detected in panel B. D, The rat GR expression vector (pSG5-rGR) was also mutated at the potential rGR start sites (Met1 and Met 28) and subjected to in vitro expression using reticulocyte lysates and 35S-methionine followed by autoradiography. An identical pattern is observed when the same plasmids are expressed in COS-1 cells (not shown). E, Fifty micrograms of rat liver protein were analyzed for GR content by Western blotting with Ab57. The two major immunoreactive bands at 94 and 91 kDa correspond directly with the rGR-A and -B isoforms detected in panel D. All Western blots are representative of at least three separate experiments.

 
Detection of Endogenous hGR-A Using an hGR-A-Specific Antibody
To evaluate the endogenous expression pattern of the GR-A and -B isoforms, an antibody was generated that is specific to the longer, hGR-A isoform (Fig. 4AGo). A dual Western blot analysis was then carried out on COS-1-expressed hGR wild type (wt) and the start site mutants, hGR-A (M27T) and hGR-B (M1T), using both the hGR-A-specific antibody and Ab57, a polyclonal epitope-purified antibody generated against a peptide sequence common to both GR-A and GR-B (26). As seen in Fig. 4BGo, the hGR-A-specific antibody recognizes only the longer hGR-A species, while the Ab57 recognizes both isoforms. A similar immunoreactive evaluation was carried out using various human cell culture lines (HeLa, CEM-C7, and HEK-293) known to express endogenous hGR{alpha}. As observed with the recombinant protein, the Ab57 detects the GR doublet while the hGR-A-specific antibody detects only the top band (Fig. 4CGo). The 83-kDa band detected in HeLa cell extracts is a common GR degradation product observed in this cell line. These data suggest both hGR-A and hGR-B are endogenously expressed in several human cell lines.



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Figure 4. Western Analysis of hGR-A and hGR-B

A, hGR-A-specific antibody production. A polyclonal antibody was prepared from rabbits, using a synthetic peptide antigen corresponding to residues 3–22 of the hGR. The location of the Ab57 binding site (residues 346–357 of the mouse GR), which is present in both hGR-A and hGR-B, is shown for comparison. B, The hGR-A-specific antibody recognizes the hGR initiated from the methionine 1 codon but not the GR-B form initiated from methionine 27 codon. COS-1 cells were transfected with the wt hGR{alpha} and the two start site mutants described in Fig. 2Go. As observed in Fig. 2Go, Ab57 detects both forms of the hGR as illustrated by Western blotting (left panel). In contrast, the hGR-A specific antibody only detects the longer, hGR-A form (right panel). C, Endogenous hGR production is a mixture of the A and B GR isoforms. Endogenously produced hGR from several cell lines (HeLa, HEK293, CEM-C7) were analyzed for hGR-A and -B production by Western blotting with the GR-A-specific antibody. Approximately 50 µg of total protein extract from the human cell lines indicated, were probed with the Ab57 (left panel). The same samples were probed with the GR-Aspecific antibody, in which case only the longer, GR-A, form was detected. The 83-kDa band detected in HeLa extracts is a commonly observed GR degradation product. Blots shown are representative of three or more experiments.

 
Unlike the majority of steroid hormone receptors, the GR is primarily localized in the cytoplasm in the absence of ligand (27). However, in response to hormone signal, the functional GR undergoes nuclear translocation. To examine potential functional differences between the GR-A and -B isoforms, immunocytochemistry was performed to evaluate the nuclear and cytoplasmic localization of the two proteins in the absence and presence of hormone. Using this method, no difference is observed between the hGR-A (M27T) and hGR-B (M1T) forms in nuclear translocation in response to dexamethasone (Dex) (Fig. 5Go).



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Figure 5. Immunocytochemical Analysis of hGR-A and hGR-B

The nuclear translocation of the GR-A and -B isoforms in response to Dex was compared by immunocytochemistry. COS-1 cells were transfected with either hGR-A (top panels) or hGR-B (bottom panels) and plated on chamber slides the following day. Approximately 48 h after transfection, cells in chamber slides were treated for 2 h with 100 mM Dex (+) or vehicle (-) as indicated. Cells were fixed on slides and analyzed by immunostaining as previously described (22 ). Both the Ab57 and GR-A-specific antibodies were used in these experiments. Data shown are representative of immunostained cells from one experiment, which was reproduced three separate times.

 
Gene Activation by hGR-A and -B
To compare the cellular functions of hGR-A and hGR-B, transactivation studies were carried out on both receptor forms using transient transfections of hGR-A and hGR-B in COS-1 cells. Utilizing three GRE-driven reporter genes, a striking difference in transactivation was observed between the two hGR{alpha} isoforms. As shown in Fig. 6AGo, the hGR-B form is more than 1.5-fold as effective in transactivation from a single GRE-driven reporter gene (GRE1-CAT) than the longer hGR-A form (Fig. 6AGo). When two GREs are found in tandem (GRE2-luc), the maximal transactivation activity of hGR-B is enhanced to nearly 2-fold that of hGR-A (Fig. 6BGo). Finally, this functional difference is also observed using a mouse mammary tumor virus (MMTV) promoter reporter gene, where the GR-B transactivation is at least 1.4-fold greater than in hGR-A (Fig. 6CGo). These data suggest that GR-B, lacking the first 27 residues of GR-A, exhibits enhanced transcriptional activity in a variety of promoter contexts and argue for a general mechanism, not strictly dependent on promoter sequences. Although the EC50 values vary depending on the promoter used, none of our transactivation assays are sensitive enough to detect a difference between GR-A and GR-B within 5 nM.



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Figure 6. Functional Comparison of hGR-A and hGR-B

The transactivation function of hGR-A and -B in response to Dex was measured using three glucocorticoid responsive reporter genes containing single or multiple copies of GRE-binding sites. A schematic of each reporter gene is shown at the top of the data set in which it was used. A, COS-1 cells were transfected with a constant amount of expression vector (50 ng) for either hGR-A or hGR-B as well as a constant amount of the GRE1-CAT reporter (0.5 µg). After transfection, cells were treated with increasing concentrations of Dex ranging from 10 pM to 100 nM and harvested approximately 16–20 h later for CAT activity assay. Data shown are an average of three separate experiments in triplicate with the indicated SEM. B, Cells were transfected as in panel A, but with a constant amount of the GRE2-luc reporter (0.5 µg). Cells were treated with Dex as indicated and harvested approximately 16–20 h later for luciferase activity assay. Data shown are an average of five separate experiments with the indicated SEM. C, COS-1 cells transfected as in panels A and B, but with a constant amount of the MMTV-CAT reporter (0.5 µg). Cells were treated and harvested for CAT assay as in panel A. Data shown are an average of triplicate samples from a representative of three separate experiments, with the indicated SEM. D, To contrast the hormone titration experiments, cells were transfected with increasing amounts of either hGR-A or hGR-B together with the GRE2-luc reporter and treated with a constant amount of Dex (100 µM). Cells were harvested and analyzed for luciferase production as in panel B. Data shown are an average of two individual experiments with the indicated SEM. E, To measure relative expression levels of hGR-A and hGR-B, COS-1 cells were transfected with 2.5 µg of wt hGR as a control (lane 1), and hGR-A (lanes 2–4), or hGR-B (lanes 5–7) in 10-cm plates for 20 h. Cells were harvested in ice-cold RIPA buffer containing 5 mM DTT and protease inhibitors and immediately subjected to SDS-PAGE and Western blot analysis with Ab57. The approximate molecular masses of detected bands are shown to the right. The 83-kDa band, commonly observed in both transient and endogenous receptor expression systems, is likely a product of degradation.

 
To further address the functional differences in transactivation, experiments were carried out in which the expression vector concentrations were varied and hormone levels were kept constant. When the GR-A and B levels were varied under saturating concentration of Dex (100 nM), hGR-B was approximately twice as effective as the hGR-A in transactivating the GRE2-luc reporter (Fig. 6DGo). Similar results are observed when using a luciferase reporter construct containing the MMTV promoter. To test whether the functional differences can be attributed to differences in protein expression of hGR-A and hGR-B, equivalent amounts of the expression vectors were transfected in separate wells and subjected to Western blot analysis. Despite the higher transactivation capacity observed for hGR-B, Fig. 6EGo shows that transfection of equivalent amounts of expression vector consistently results in a greater accumulation of expressed hGR-A protein. These data suggest that the observed transactivation differences may be underestimates of the transcriptional potential of hGR-B when considering the actual levels of expressed protein. Interestingly, the level of the 83-kDa degradation product is also significantly higher when hGR-B vectors are used for receptor expression, suggesting that hGR-B may be more susceptible to proteolysis than is hGR-A (Fig. 6EGo). However, since we do not know the activity of the 83-kDa band, we cannot eliminate the possibility that it does contribute to the transactivation levels observed in Fig. 6Go, A–D.

Gene Repression by hGR-A and -B
A growing body of data suggests that many GRmediated effects occur independently of direct DNA (GRE) binding (28). These processes occur via cross-talk with other signaling pathways and through protein interactions independent of a GRE. One well studied cross-talk pathway is the mutual repression observed between hormone-activated GR and the transcription factor NF-{kappa}B (29). The ability of hGR-A and hGR-B to repress transactivation of the NF-{kappa}B p65 subunit was evaluated. Interestingly, both receptor isoforms appeared to antagonize p65 reporter activity to the same degree (Fig. 7Go) in contrast to the observed difference in GRE-dependent transactivation. These data support the hypothesis that hGR activation and repression functions are contained within separate regions of the protein and suggest a role for the first 27 residues in transactivation but not transrepression of NF-{kappa}B.



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Figure 7. Inhibition of NF-{kappa}B/p65 by GR-A and GR-B

The ability of hGR-A and hGR-B to repress the transactivation function of the p65 subunit of NF-{kappa}B was measured. A constant amount of p65 expression vector (12.5 ng) and a NF-{kappa}B luciferase reporter construct (3XMHC-luc) were cotransfected in COS-1 cells along with increasing amounts (10, 50, and 250 ng) of expression vector for either hGR-A (A) or hGR-B (B). In the presence of 100 nM Dex (+), both hGR-A and hGR-B repressed p65 transactivation with approximately the same efficiency over the entire range of hGR expression. The x-axis labels shown are identical for both panels A and B. Data shown are an average of three separate experiments with the indicated SEM.

 
Mechanism of the Alternative Translation Initiation of GR
The cause of alternative initiation of the GR transcript may lie within the sequence itself. Eukaryotic ribosomes appear to select the start site for translation initiation by a scanning mechanism (reviewed in Ref. 30). An AUG start codon is classified as strong or weak depending on the adherence to a specific surrounding consensus sequence (Kozak sequence). A purine at position -3 and a G at position +4 relative to the AUG (the A is considered as +1) are considered strong initiator sequences. However, leaky scanning, in which the first AUG is bypassed in favor of a nearby downstream AUG, is most predictably caused by deviations from the strong sequence context within the first AUG (31). The sequence contexts of both AUG start sites of human, mouse, and rat GR are shown in Fig. 8AGo. For all three species (human, rat, and mouse), the first AUG does not contain a purine at position -3, indicating it is a weak initiator sequence. However, the second AUG in all three species of GR does contain a purine at this position, indicating it is actually a stronger translation initiation site, and could explain the high degree of leaky ribosomal scanning and the production of two GR proteins from the same mRNA.



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Figure 8. Translation Scanning of GR Transcripts

A, Sequence analysis of GR cDNA surrounding the first two ATG codons. The consensus Kozak sequence, required for maximum efficiency of protein translation initiation, is shown above the sequence surrounding the first two ATG codons from human, mouse, and rat GR. The underlined ATG sequence represents bases +1, +2, and +3 respectively. The bases at position -3 and +4 have been found to signal either weak or strong initiator codons, as indicated. The second ATG in human corresponds to Met 27, while in rat and mouse it is Met 28. B, Mutagenesis of the Kozak sequences surrounding the two hGR ATG start sites. The weak Kozak sequence at the upstream (Met 1) ATG was changed to a strong Kozak consensus site (M1 Kozak mutant). COS-1 cells were transfected with this mutant along with WT hGR, and the two ATG mutants described elsewhere (M1T and M27T). Cells were harvested and analyzed by Western blotting for hGR with Ab 57.

 
To test the hypothesis that leaky ribosomal scanning is the cause for the production of GR A and B isoforms, we mutated the Kozak sequence of the first AUG start site. When the "weak" consensus site of the first AUG (Met 1) is changed to a "strong" one by a point mutation at the -3 position (C to G), production of GR-B is completely lost (Fig. 8BGo). These data conclusively demonstrate that leaky ribosomal scanning of a weak initiation sequence generates a second, B form, of the GR.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The data presented in this paper demonstrate that the GR is a product of alternative translation initiation, which results in the production of both a GR-A and GR-B form. Both the A and B forms are generated in approximately equivalent levels from a single cDNA when expressed in vitro using reticulocyte lysates. However, the GR-B form appears to be more susceptible to degradation, at least when expressed in mammalian cells. Translation of an hGR{alpha} message from an internal AUG codon, corresponding to methionine 27, results in a protein with twice the amount of maximal transactivation as the longer protein initiated from the first AUG codon. Interestingly, deletion of the first 25–30 residues of hPR-B also results in more effective transcriptional activation (Horwitz, K., and L. Tung, personal communication). These data suggest that the extreme amino termini of steroid receptors may be involved in regulating receptor function.

One possible explanation for the increased activity of the shorter GR-B is that the tertiary structure including the first 27 residues masks the activation function(s) associated with other regions of the receptor. A second possibility is that this region of the GR is essential for an important protein interaction responsible for either transcriptional silencing or repression. The first 27 residues of hGR, however, do not appear to have significant homology with recognized protein-protein interaction sites or other known functions. The fact that the GR-B is a more effective transactivator on three separate glucocorticoid-responsive promoters argues for a general mechanism involving GR interactions with additional cellular factors. It remains to be seen whether both isoforms homo- and heterodimerize equivalently, bind DNA with the same affinities, and respond to different hormone signals with the same relative potency. In addition, the tissue distribution of the two isoforms remains to be elucidated.

It is now established that other steroid receptors produce N-terminal truncation variants. For example, it is well known that two PRs are derived from the same gene in humans and chickens (32). It was originally suggested that the origin of the PR isoforms was alternative translation initiation from the same message (17, 33). However, additional studies with human breast cancer cell lines and hPR or cPR cDNA suggest that the PR-A and -B isoforms may also be generated from distinct promoters (18, 34, 35). The data presented in this paper clearly show the production of two GR protein products from a single cDNA source. In addition, Northern blot analysis of hGR{alpha}-transfected COS-1 mRNA shows only a single hGR-specific message, further supporting the one-message/twoprotein hypothesis (36). The androgen receptor (AR) has also been shown to exist as multiple forms, differing at the amino terminus, which are expressed in a tissue-specific manner (37). Alternative initiation has been implicated as the mechanism responsible for the generation of these two AR forms (38).

The mechanism of alternative initiation of the GR is shown to be under the translational direction exerted in the sequence surrounding the ATG codons (31). As would be expected, creation of a strong Kozak consensus site (-3 C to G) by a point mutation 3' of GR Met1 AUG completely abolished ribosomal read-through and production of GR-B. This new GR expression construct contains no coding region mutations and functions similarly to the M27T hGR used in these studies. That a point mutant in the noncoding, promoterless region of the GR cDNA had such a drastic effect on protein expression and function is remarkable and unprecedented in the nuclear receptor field. Recent interest in mRNA regulation mechanisms, such as splicing, stability, and the role of structure, are likely to lead to increased analysis of translational control mechanisms as identified here.

Alternative translation initiation produces two functionally distinct forms of the GR. The presence of both forms in several human cell lines and rodent tissues suggests that the generation of these two protein species may be a general phenomenon. However, the ultimate physiological significance of these receptor isoforms remains to be established. Although our data, utilizing both in vitro translation and transient expression systems, suggest both the GR-A and -B are being expressed via leaky ribosomal scanning from a single cDNA and corresponding mRNA, we cannot rule out the existence of alternative promoters regulating expression in vivo, as has been shown for the PR (35). We have presented evidence suggesting that both GR products are generated in vivo and in a variety of mammalian cell lines and that a significant functional difference exists between the two. It is intriguing to speculate whether differences exist between GR-A and GR-B in tissue distribution and/or expression during development, aging, or cell death. Such studies, however, will require currently unavailable antibodies that selectively recognize the shorter GR-B form in the presence of GR-A. For example, it is now known that the PR-A and -B isoforms function in a tissue-specific fashion (39) and that both isoforms regulate a distinct subset of genes (40). Regulated expression of either GR isoform in favor of the other would suggest a physiological role of alternative translation initiation of the GR. Indeed, there are several reports that present evidence for physiological regulation of alternative translation initiation of critical transcription factors and cell cycle regulators (41, 42). The potential for differential regulation of functionally distinct GR isoforms, at the level of translation, is an area that clearly needs further inquiry.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials, Antibody Production, and Plasmids
trans-35S-label (1,108 Ci/mmol) was purchased from ICN Biochemicals, Inc. (Irvine CA). [14C]Chloramphenicol (40–60 mCi/mmol) was obtained from NEN Life Science Products (Boston, MA). Dex was supplied by Steraloids (Wilton, NH). Acetyl-coenzyme A and protease inhibitors were purchased from Roche Molecular Biochemicals (Indianapolis, IN). Oligonucleotide primers for mutagenesis and PCR were synthesized by Oligo’s Etc. (Bethel, ME). The hGR-A-specific antibody was produced by Covance Laboratories, Inc.(Denver, PA) using a synthetic peptide antigen corresponding to residues 3–22 of the hGR synthesized at the University of North Carolina at Chapel Hill. The GR-A- specific antibody was purified on a peptide-linked sepharose affinity column as previously described for antibody purification (24). Characterization of this antibody is described below. The peroxidase-labeled secondary antibodies and enhanced chemiluminescence (ECL) reagents were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).

Use of the hGR mammalian (pCMV-hGR{alpha}) and in vitro (pT7-hGR{alpha}) expression vectors was described in a previous publication (23). The mouse GR mammalian and in vitro expression vectors were also used as previously described (43). The rat GR expression vector, pRSV-rGR, was a gift of Dr. Trevor Archer and was used for both in vitro and COS-1 expression systems. The reporter plasmids GRE1- and GRE2-CAT (44), and pGCMS-CAT (45) have been described elsewhere. All site-specific mutagenesis was done with the Quick Change Mutagenesis kit (Promega Corp., Madison WI), following their protocol for primer design. The carboxyterminal hGR{alpha} truncation mutants, hGR(1–742) and hGR(1–706), were created by mutating the codons at positions 743 and 707 to TGA stop codons. All mutants were verified by DNA sequencing.

Cell Culture, Transfections, Luciferase, and Chloramphenicol Acetyltransferase (CAT) Assays
COS-1 and HEK293 cells were maintained in DMEM with high glucose containing 2 mM glutamine and 10% (vol/vol) mixture of heat-inactivated FCS/calf serum (1:1). For transactivation assays, cells were incubated for 1–2 days in media containing dextran-coated charcoal-stripped sera to remove endogenous steroids. HeLa cells were maintained in Eagle’s MEM supplemented with glutamine and 10% FCS/calf serum. CEM-C7 cells were grown in suspension in RPMI 1640 medium supplemented with 2 mM glutamine, 10% (vol/vol) heat-inactivated FCS, and 0.1 M HCl. All cell culture media contained 100 IU/ml penicillin and 100 mg/ml streptomycin. Cell cultures were maintained in a 5% CO2 humidified incubator at 37 C and passaged every 3–4 days. All transfections were carried out with Mirus TransIT LT-1 reagent according to the manufacture’s protocol (Pan Vera, Madison WI). An appropriate amount of TransIT reagent (3 µl per µg of transfected plasmid) was added to OPTIMEM (Life Sciences, Inc., St. Petersburg, FL) for 5 min. Purified plasmid DNA was then added and allowed to complex for 30 min at room temperature, before being added to cells with media containing stripped serum. Six to eight hours after transfection, the media were replaced with fresh serum-stripped media containing vehicle or Dex.

Transfected cells were incubated in the presence or absence of the indicated amount of Dex for 18–24 h before harvesting. Cells for luciferase assays were harvested in 1x Reporter Lysis Buffer (Promega Corp.). Total protein was measured using the Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA) according to the manufacture’s protocol, and equivalent amounts of total protein were used for luciferase activity assays. The luciferase activity was measured using the 96-well plate format with an MLX automated microtiter plate luminometer from Dynex.

CAT assays were carried out essentially as described previously (45). Approximately 0.1–0.2 mg of protein extracts were incubated overnight at 37 C with 1 mM acetyl-coenzyme A and 0.1 µCi of [14C]chloramphenicol in Tris-EDTA (TE). Samples were extracted in mixed xylenes and then back extracted one time with TE, pH 8.0, before liquid scintillation counting. A standard curve was generated using commercially available, purified CAT as described by the manufacturer (Promega Corp.). All experiments were conducted under conditions in which substrate was in excess and the relationship of counts per min to CAT activity was linear. Data are expressed as counts per min per microgram of total protein.

Animals
Male Sprague Dawley rats (2–3 months old) and C57BL mice (6 months old) were used in all experiments. All animals were maintained under controlled conditions of temperature (25 C) and lighting and allowed free access to food and saline. All experimental protocols were approved by the animal review committee at the institute and were performed in accordance with the guidelines set forth in the NIH Guide for the Care and Use of Laboratory Animals published by the USPHS. Rats were killed by decapitation and mice were asphyxiated under CO2, before removal of liver tissue. Liver tissue fragments were homogenized on ice for 30 sec at maximum speed with a Tekmar Tissuemizer in a radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton x-100, 0.5% sodium deoxycholate, 2 mM EDTA, and 150 mM NaCl) containing 5 mM dithiothreitol (DTT) and protease inhibitors. After a brief low-speed centrifugation to remove tissue debris, extracts were incubated for an additional 20 min on ice before centrifugation at 20,000 x g for 20 min. The resulting supernatants were measured for total protein concentration (typically 10–20 mg/ml) and subjected to Western blotting as described below.

Immunocytochemistry and Western Blotting
Immunocytochemistry was carried out essentially as previously described (22). The day after transfection, cells were split on two-chamber glass slides. Approximately 48 h after transfection, the cells were treated with 100 nM Dex or vehicle for 2 h. The cells were fixed in 2% paraformaldehyde, washed in PBS, and permeabilized with 0.2% Triton X-100. Cells were again washed in PBS, treated with 2% normal goat serum, washed in PBS, and incubated with epitope-purified Ab57 (1:7500) for 20 h at 4 C. The cells were washed in PBS and incubated with biotinylated goat antirabbit IgF (1:400) for 1 h at room temperature. Immunoreactivity was visualized by staining with avidin-biotin-peroxidase.

For Western blotting, cells were lysed for 20 min on ice in RIPA buffer containing 150 mM NaCl, 5 mM DTT, and protease inhibitors (0.1 mM Pefa Block, 1 µM leupeptin, and 1 µM pepstatin). After a high-speed spin to remove cellular debris, total protein was measured using the Bio-Rad Laboratories, Inc. detection kit. Unless indicated otherwise, 50 µg of protein extract were then separated on precast 8% Tris-glycine gels (Novex, San Diego CA) and transferred to nitrocellulose. The membranes were washed in TBST (Tris-buffered saline with 0.1% Tween-20) and blocked in TBST containing 5% nonfat milk for a minimum of 2 h at room temperature. Blots were next incubated in the same solution supplemented with affinity-purified primary antibodies, Ab57 (1:2,500) and GR-A specific (1:5,000), overnight at 4 C. After extensive rinsing and washing in TBST (three times, 10–15 min), the blots were probed with peroxidase-conjugated goat antirabbit secondary antibody (1:10,000) for 2 h at room temperature. Bands were visualized using ECL reagents (Amersham Pharmacia Biotech).

Repression of NF-{kappa}B/p65
GR-mediated repression of NF-{kappa}B/p65 transactivation was studied as reported previously (44). A constant amount (12.5 ng) of the NF-{kappa}B-p65 subunit expression vector (pCMVp65) was used, in conjunction with the NF-{kappa}B-luciferase reporter, 3XMHC-luc (0.5 µg), to measure p65-mediated transactivation. The repression of p65 activity by GRs was measured by cotransfecting increasing amounts (10, 50, and 250 ng) of the hGR-A or hGR-B expression vectors. After transfections, cells were treated without or with 100 nM Dex. Approximately 16–20 h after hormone treatment, cells were harvested and luciferase activity was determined as described above. Equivalent amounts of total protein were assayed for luciferase activity as described above, and data from individual experiments were averaged and presented along with the SEM.


    ACKNOWLEDGMENTS
 
We wish to thank Dr. Trevor Archer for the rGR plasmid, Dr. Turk E. Rhen for supplying the rat liver tissue, and Dr. Ester Carballo-Jane for the mice. The authors would also like to thank Chris Jewell for technical assistance and discussions, and Drs. J. Hall and H. Kinyamu for reviewing the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. John Cidlowski, Laboratory of Integrative Biology, National Institute of Environmental Health Sciences, National Institutes of Health, 111 TW Alexander Drive NC F307, Research Triangle Park, North Carolina 27709-2233. E-mail: cidlowski{at}niehs.nih.gov

Received for publication November 22, 2000. Revision received March 15, 2001. Accepted for publication April 2, 2001.


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