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Laboratory of Signal Transduction National Institute of Environmental Health Sciences National Institutes of Health Research Triangle Park, North Carolina 27709
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) 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 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 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 |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B (NF-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 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 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-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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 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 to ß, the two isoforms
can be discriminated immunologically using specific antibodies (23, 24). Interestingly, the recombinant hGR 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. 1A
).
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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. 1B
), 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 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 cDNA in both the in vitro
expression vector and the mammalian expression vector resulted in the
expression of a single hGR species (Fig. 2). 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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). 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. 3, 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. 3, 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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). 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. 4B, 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. As observed with the
recombinant protein, the Ab57 detects the GR doublet while the
hGR-A-specific antibody detects only the top band (Fig. 4C). 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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).
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isoforms. As shown
in Fig. 6A, 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. 6A). 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. 6B).
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. 6C).
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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). 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. 6E
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. 6E). 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. 6, 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-B (29). The ability of hGR-A and hGR-B to
repress transactivation of the NF-B p65 subunit was evaluated.
Interestingly, both receptor isoforms appeared to antagonize p65
reporter activity to the same degree (Fig. 7) 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-B.
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.
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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). These data conclusively demonstrate that leaky ribosomal
scanning of a weak initiation sequence generates a second, B form, of
the GR. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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-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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Use of the hGR mammalian (pCMV-hGR) and in vitro
(pT7-hGR) 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
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-B/p65
GR-mediated repression of NF-B/p65 transactivation was
studied as reported previously (44). A constant amount (12.5 ng) of the
NF-B-p65 subunit expression vector (pCMVp65) was used, in
conjunction with the NF-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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication November 22, 2000. Revision received March 15, 2001. Accepted for publication April 2, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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isoform
(GR) using GR-specific antibody. Steroids 64:742–751[CrossRef][Medline]
B
and steroid receptor-signaling pathways. Endocr Rev 20:435–59B and the steroid hormone receptors: mechanisms of mutual
antagonism. Mol Endocrinol 12:45–56This article has been cited by other articles:
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S. Saito, Y. Sidis, A. Mukherjee, Y. Xia, and A. Schneyer Differential Biosynthesis and Intracellular Transport of Follistatin Isoforms and Follistatin-Like-3 Endocrinology, December 1, 2005; 146(12): 5052 - 5062. [Abstract] [Full Text] [PDF]
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 T. Rhen and J. A. Cidlowski Antiinflammatory action of glucocorticoids--new mechanisms for old drugs. N. Engl. J. Med., October 20, 2005; 353(16): 1711 - 1723. [Full Text] [PDF]
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J. D Turner and C. P Muller Structure of the glucocorticoid receptor (NR3C1) gene 5' untranslated region: identification, and tissue distribution of multiple new human exon 1 J. Mol. Endocrinol., October 1, 2005; 35(2): 283 - 292. [Abstract] [Full Text] [PDF]
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H. Russcher, P. Smit, E. L. T. van den Akker, E. F. C. van Rossum, A. O. Brinkmann, F. H. de Jong, S. W. J. Lamberts, and J. W. Koper Two Polymorphisms in the Glucocorticoid Receptor Gene Directly Affect Glucocorticoid-Regulated Gene Expression J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5804 - 5810. [Abstract] [Full Text] [PDF]
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L. Pascual-Le Tallec and M. Lombes The Mineralocorticoid Receptor: A Journey Exploring Its Diversity and Specificity of Action Mol. Endocrinol., September 1, 2005; 19(9): 2211 - 2221. [Abstract] [Full Text] [PDF]
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H. Russcher, E. F. C. van Rossum, F. H. de Jong, A. O. Brinkmann, S. W. J. Lamberts, and J. W. Koper Increased Expression of the Glucocorticoid Receptor-A Translational Isoform as a Result of the ER22/23EK Polymorphism Mol. Endocrinol., July 1, 2005; 19(7): 1687 - 1696. [Abstract] [Full Text] [PDF]
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 E. Smith, T. E. Meyerrose, T. Kohler, M. Namdar-Attar, N. Bab, O. Lahat, T. Noh, J. Li, M. W. Karaman, J. G. Hacia, et al. Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo Nucleic Acids Res., March 1, 2005; 33(4): 1298 - 1308. [Abstract] [Full Text] [PDF]
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 B. M. Necela and J. A. Cidlowski Mechanisms of Glucocorticoid Receptor Action in Noninflammatory and Inflammatory Cells Proceedings of the ATS, November 1, 2004; 1(3): 239 - 246. [Full Text] [PDF]
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E. F. C. van Rossum, P. G. Voorhoeve, S. J. te Velde, J. W. Koper, H. A. Delemarre-van de Waal, H. C. G. Kemper, and S. W. J. Lamberts The ER22/23EK Polymorphism in the Glucocorticoid Receptor Gene Is Associated with a Beneficial Body Composition and Muscle Strength in Young Adults J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 4004 - 4009. [Abstract] [Full Text] [PDF]
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 C. L. Smith and B. W. O'Malley Coregulator Function: A Key to Understanding Tissue Specificity of Selective Receptor Modulators Endocr. Rev., February 1, 2004; 25(1): 45 - 71. [Abstract] [Full Text] [PDF]
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 E. F.C. van Rossum and S. W.J. Lamberts Polymorphisms in the Glucocorticoid Receptor Gene and Their Associations with Metabolic Parameters and Body Composition Recent Prog. Horm. Res., January 1, 2004; 59(1): 333 - 357. [Abstract] [Full Text]
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H. K. Kinyamu and T. K. Archer Estrogen Receptor-Dependent Proteasomal Degradation of the Glucocorticoid Receptor Is Coupled to an Increase in Mdm2 Protein Expression Mol. Cell. Biol., August 15, 2003; 23(16): 5867 - 5881. [Abstract] [Full Text] [PDF]
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K. De Bosscher, W. Vanden Berghe, and G. Haegeman The Interplay between the Glucocorticoid Receptor and Nuclear Factor-{kappa}B or Activator Protein-1: Molecular Mechanisms for Gene Repression Endocr. Rev., August 1, 2003; 24(4): 488 - 522. [Abstract] [Full Text] [PDF]
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V. Gupta and B. J. Wagner Expression of the Functional Glucocorticoid Receptor in Mouse and Human Lens Epithelial Cells Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 2041 - 2046. [Abstract] [Full Text] [PDF]
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 L. Cherbas, X. Hu, I. Zhimulev, E. Belyaeva, and P. Cherbas EcR isoforms in Drosophila: testing tissue-specific requirements by targeted blockade and rescue Development, March 2, 2003; 130(2): 271 - 284. [Abstract] [Full Text] [PDF]
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G. Ozisik, G. Mantovani, J. C. Achermann, L. Persani, A. Spada, J. Weiss, P. Beck-Peccoz, and J. L. Jameson An Alternate Translation Initiation Site Circumvents an Amino-Terminal DAX1 Nonsense Mutation Leading to a Mild Form of X-Linked Adrenal Hypoplasia Congenita J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 417 - 423. [Abstract] [Full Text] [PDF]
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M. R. Yudt and J. A. Cidlowski The Glucocorticoid Receptor: Coding a Diversity of Proteins and Responses through a Single Gene Mol. Endocrinol., August 1, 2002; 16(8): 1719 - 1726. [Abstract] [Full Text] [PDF]
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 S. D. Chessler, W. T. Simonson, I. R. Sweet, and L. P. Hammerle Expression of the Vesicular Inhibitory Amino Acid Transporter in Pancreatic Islet Cells : Distribution of the Transporter Within Rat Islets Diabetes, June 1, 2002; 51(6): 1763 - 1771. [Abstract] [Full Text] [PDF]
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