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Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (T.K., G.P.C.), Bethesda, Maryland 20892-1583; and Human Retrovirus Section, Center for Cancer Research, National Cancer Institute-Frederick (R.H.S., J.H.R., G.N.P.), Frederick, Maryland 21702-1201
Address all correspondence and requests for reprints to: Tomoshige Kino, M.D., Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 9D42, 10 Center Drive, MSC 1583, Bethesda, Maryland 20892-1583.
Abstract
The syndrome of familial or sporadic glucocorticoid resistance is
characterized by hypercortisolism without the clinical stigmata of
Cushing syndrome. This condition is usually caused by mutations of the
human GR, a ligand-activated transcription factor that shuttles between
the cytoplasm and the nucleus. A pathological human mutant receptor, in
which Ile was replaced by Asn at position 559, had negligible ligand
binding, was transcriptionally extremely weak, and exerted a
transdominant negative effect on the transactivational activity of the
wild-type GR, causing severe glucocorticoid resistance in the
heterozygous state. To understand the mechanism of this mutant’s
trans-dominance, we constructed several N-terminal GR fusion chimeras
to green fluorescent protein (GFP) and demonstrated that their
transactivational activities were similar to those of the original
proteins. The GFP-human (h) GR
I559N chimera was predominantly
localized in the cytoplasm, and only high doses or prolonged
glucocorticoid treatment triggered complete nuclear import that took
180 vs. 12 min for GFP-hGR. Furthermore, hGRI559N
inhibited nuclear import of the wild-type GFP-hGR, suggesting that
its trans-dominant activity on the wild-type receptor is probably
exerted at the process of nuclear translocation. As the ligand-binding
domain (LBD) of the GR appears to play an important role in its
nucleocytoplasmic shuttling, we also examined two additional GR-related
fusion proteins. The natural hGR isoform ß (GFP-hGRß), containing a
unique LBD, was transactivation-inactive, moderately trans-dominant,
and localized instantaneously and predominantly in the nucleus;
glucocorticoid addition did not change its localization. Similarly,
GFP-hGR514, lacking the entire LBD, was instantaneously and
predominantly localized in the nucleus regardless of presence of
glucocorticoids. Using a cell fusion system we demonstrated that
nuclear export of GFP-hGRI559N (250 min) and GFP-hGRß (300 min)
was drastically impaired compared with that of GFP-hGR (50 min) and
GFP-hGR514 (50 min), suggesting that an altered LBD may impede the exit
of the GR from the nucleus. We conclude that the trans-dominant
negative effect of the pathological mutant is exerted primarily at the
translocation step, whereas that of the natural isoform ß is exerted
at the level of transcription.
GLUCOCORTICOIDS EXERT profound influences
on many physiological functions by virtue of their diverse roles in
growth, development, and maintenance of resting and stress-related
homeostasis (1, 2). They also exert antiinflammatory and
immunosuppressive effects, which have made them invaluable therapeutic
agents in numerous diseases (3). Glucocorticoids exert
their effects through the ubiquitously expressed GR isoform , a
ligand-dependent 777-amino acid transcription factor. In addition to
GR, alternative splicing of the human (h) GR gene produces a second
isoform of the receptor, GRß. GR and GRß are identical through
amino acid 727 (4). GR encodes an additional 50 amino
acids at its carboxyl-terminal, whereas GRß contains an additional 15
nonhomologous amino acids. In contrast to GR, GRß cannot bind
glucocorticoids and has dominant negative activity on the
transcriptional effects of GR, and its physiological and
pathological roles are as yet unclear (5).
Binding of glucocorticoids to GR causes it to dissociate from a
cytoplasmic heterooligomer with heat shock proteins and induces its
translocation into the nucleus (6). The ligand-bound GR
is imported into the nucleus with the assistance of two nuclear
localization (NL) signals, NL1 and NL2 (7). NL1 is located
in the junction between the DNA-binding domain (DBD) and ligand-binding
domain (LBD), also called the hinge region, and belongs to a class of
basic-type nuclear localization signals that have a core basic sequence
adjacent to the DBD and two smaller clusters of basic amino acids
proximal to the core domain (8, 9, 10). NL1 catalyzes rapid
transport of the GR through the nuclear pore via the classic importin
pathway. On the other hand, NL2 encompasses the entire LBD of GR and
contributes to a slower traffic via an as yet unknown mechanism
(7). After entering the nucleus, GR rapidly binds as a
homo- or heterodimer with GRß to specific DNA enhancer elements, the
glucocorticoid response elements (GREs), to change the transcriptional
activity of glucocorticoid-dependent genes (5, 6, 11).
GR also influences the activities of other transcription factors,
such as activating protein-1, nuclear factor-
B, and several signal
transducers and activators of transcription, though protein-protein
interactions, possibly as a monomer (6). Withdrawal of
ligand allows export of GR from the nucleus and reincorporation into
the heat shock protein-containing heterooligomer, which reestablishes
its ligand-friendly state (6, 12).
The rare syndrome of familial or sporadic glucocorticoid resistance,
usually caused by mutations of the GR, is a disorder characterized by
biochemical hypercortisolism without the clinical stigmata of Cushing
syndrome (13). Over 10 kindreds and sporadic cases have
been reported to this day. As glucocorticoids possess a broad array of
life-sustaining functions, only partial or incomplete resistance has
been reported. Abnormalities of the intracellular GR concentration,
stability, and affinity for glucocorticoids, have been reported
(13, 14, 15, 16, 17, 18, 19, 20, 21, 22), and the molecular defects of 4 kindreds and 1
sporadic case have been elucidated (14, 16, 17, 20). All
were missense inactivating mutations of the LBD, resulting in decreased
affinity for the ligand, or splice junction changes, knocking out the
function of 1 of the hGR gene alleles, resulting in decreased
intracellular GR concentrations by 50%. We previously reported a
severely glucocorticoid-resistant man with a sporadic germline
de novo, heterozygous mutation of the GR gene in exon 4,
resulting in a nonconservative amino acid substitution at position 559
(Ile to Asn) in the hinge region of hGR and ß, very close to NL1
(17). The mutant receptor had undetectable affinity for
dexamethasone in a standard ligand binding assay and negligible
transactivational activity; we did not rule out interactions with other
steroids. The patient’s clinical and biochemical picture was more
severe than what would have been expected from the loss of 1 hGR allele
activity; indeed, the mutant receptor had a 30–50% trans-dominant
negative activity on the wild-type receptor, which resulted in about
10-fold compensatory elevations of serum cortisol with no Cushing
syndrome stigmata. Yet 2 yr after initiation of an effective
dexamethasone regimen, this patient developed full-blown Cushing’s
syndrome secondary to an ACTH-secreting pituitary tumor, with a
further 8-fold increase in his serum cortisol, a paradox that we could
not explain at the time of the report.
To further elucidate the mechanism of trans-dominance of this mutant
receptor and its clinical manifestations, and because of the location
of the mutation close to NL1, we examined its trafficking in living
cells, comparing its properties with those of the hGR and GRß as
well as an artificial mutant that lacks the entire LBD.
Materials and Methods
Plasmids
To generate the different green fluorescent protein (GFP)-hGR
expression plasmids, the coding regions of hGR, hGRß, or specific
mutants were amplified by PCR using appropriate primers containing
NarI and XbaI restriction sites and pRShGR,
pRShGRß, and pRShGRAsn559 as templates. The
PCR products were digested with NarI/XbaI and
cloned into the plasmid pF25GFP-Hyg (23), thereby
replacing the coding region for hygromycin resistance. In the resulting
plasmids (pF25hGR, pF25hGRß, pF25hGRI559N, and pF25hGR514; Fig. 1
), the expression of the chimeric gene
is under control of the cytomegalovirus (CMV) early promoter and the
bovine GH polyadenylation signal. The coding regions of all constructs
were confirmed by sequence analysis. pMMTV-luc, expressing luciferase
under the control of the mouse mammary tumor virus (MMTV) long terminal
repeat promoter, and the plasmid pSV40-ß-Gal were described
previously (24). pRSV-erbA-1, which
possesses the thyroid hormone receptor cDNA in the reverse orientation
instead of hGR cDNA, but is otherwise the same as pRShGR or
pRShGRAsn559, was used to keep the same amount
of DNA.
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For microscopic and functional reporter assay studies, HLtat (25) and CV-1 cells were employed. The cells were transfected with CaPO4 or lipofectin (Life Technologies, Inc., Gaithersburg, MD), as described previously (24, 26).
Reporter assay
To examine the transactivational and trans-dominant activities
of each of the GR-related proteins, CV-1 cells were transfected with
0.05 µg/well pF25hGR plasmids together with 0.5 µg/well pMMTV-luc
and 0.1 µg/well pSV40-Gal. Twenty-four hours later the cells were
exposed to graded concentrations of dexamethasone over 24 h, and
cell lysates were collected. To address trans-dominant activity, 0.03
µg/well pF25hGR was cotransfected with 0.03–0.3 µg/well
pF25hGRI559N, pF25hGRß, or pF25hGR514, respectively, and the cells
were stimulated with graded concentrations of dexamethasone. Luciferase
and ß-galactosidase activities were determined in the cell lysates as
described previously (24). All measurements of the
reporter gene activity were conducted in triplicate.
Detection and localization of GFP-fused hGR-related receptors
HLtat cells were plated on coated 20-mm glass bottom dishes (MatTek Corp., Ashland, MA) in phenol red-free DMEM containing 10% charcoal-treated FCS (Sigma, St. Louis, MO), and the cells were transfected with the indicated plasmids 24 h later. After an additional 24 h, the cells were analyzed by an LSM 410 Micro System (Carl Zeiss, Thornwood, NY) or observed with an inverted fluorescence microscope (Carl Zeiss Axiovert 135) as described previously (25). Twelve-bit black and white images were captured using a digital CCD camera (Photometrix, Tucson, AZ). Image analysis and presentation were performed using IPLab Spectrum software (Scanalytics, Vienna, VA).
Cell fusion system
HLtat cells transfected with the plasmids expressing different GFP-fused hGR-related proteins were harvested 24 h posttransfection, mixed, and replated with 5-fold amounts of untransfected cells. After an additional 24 h, polyethylene glycol (PEG)-mediated cell fusion was performed as described previously (25). To inhibit de novo protein synthesis, 25 µg/ml cycloheximide were added 30 min before fusion and remained during the experiment. Cells were observed under phase contrast and fluorescent illumination and scanned for fusions involving one or two donor cells.
Results
Subcellular localization of the GFP-fused hGR-related proteins
Tagging with GFP allowed us to study the subcellular localization
of the different GFP-fused hGR-related proteins in HLtat cells in the
absence or presence of glucocorticoids. In the absence of
dexamethasone, GFP-hGR was primarily localized in the cytoplasm
(Fig. 2). Addition of
10-6 M dexamethasone triggered a
fast (within 12 min) translocation of the wild-type receptor from the
cytoplasm into the nucleus, which could be monitored continuously in
the cells (Fig. 2 and Table 1). The
pathological mutant GFP-hGRI559N was predominantly observed in the
cytoplasm in the absence or presence of dexamethasone (Fig. 3A); high doses of dexamethasone
(10-5 M) and/or prolonged exposure
to dexamethasone (>2 h), however, induced slow (within 180 min)
translocation of this mutant receptor into the nucleus (Fig. 3B and
Table 1). On the other hand, GFP-hGRß or GFP-hGR514 was observed
predominantly in the nucleus, independently of dexamethasone (Fig. 3, C
and D).
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The equilibrium localization and trafficking of the GFP-fused
GR-related proteins were subsequently studied in HLtat cells using
PEG-mediated cell fusion. After fusion of the plasma membranes of donor
and acceptor cells, the export from the donor nuclei of GFP-fused
hGR-related proteins to acceptor cytosol was investigated over
time. GFP-hGR, which was localized in the nucleus after
dexamethasone treatment, and GFP-hGR514, which was found in the
nucleus, were exported from the nucleus with similar kinetics (within
50 min; Fig. 4, a and b, and Table 1
). On the other
hand,
GFP-hGRI559N and GFP-hGRß behaved entirely differently (Fig. 4, c
and d, and Table 1). After cell fusion, these chimeric peptides were
exported slowly (250 and 300 min, respectively) from the donor nuclei
into the acceptor cytosol (Table 1).
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To address the transcriptional activity of the GFP-fused
hGR-related proteins, we transfected pF25-derived plasmids, which
express these fusion receptors, with pMMTV-luc in CV-1 cells (Fig. 5). GFP-hGR stimulated MMTV promoter
activity in a dexamethasone-dependent fashion, whereas GFP-hGRß did
not stimulate the MMTV promoter. GFP-hGRI559N transactivated weakly
the MMTV promoter only at high concentrations of dexamethasone, whereas
GFP-hGR514 stimulated weakly the MMTV promoter in a
dexamethasone-independent fashion.
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We tested the trans-dominant activity of GFP-hGR-related proteins
on dexamethasone-stimulated GR-directed transactivation of the MMTV
promoter in CV-1 cells. Figure 6a shows
the profile of the wild-type GR-induced transactivation of the MMTV
promoter and the effect of overexpression of GFP-hGRI559N,
GFP-hGRß, or GFP-hGR514 on GFP-hGR-directed MMTV promoter
transactivation in absolute luciferase activity values. Figure 6b shows
the same results expressed as a percentage of the wild-type GFP-GR
transactivation effect. All three GR-related proteins, GFP-hGRI559N,
GFP-hGRß, and GFP-hGR514, inhibited GFP-hGR-directed MMTV promoter
transactivation.
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I559N delays import of GFP-hGR into the nucleus
As hGRI559N exerts a strong trans-dominant negative effect on
hGR, we examined the subcellular localization of GFP-hGR in the
presence of hGRI559N by cotransfecting the two plasmids at 1:1 and
1:3 ratios. In the absence of hGRI559N and at
10-9 M dexamethasone, GFP-hGR
entered the nucleus completely by 80 min; in the presence of
hGRhI559N at both the 1:1 and 1:3 ratios, on the other hand, it took
160 min to obtain a lesser proportion of nuclear amounts of GFP-hGR
(Fig. 7). In contrast, coexpression of
hGRß with GFP-hGR did not affect the latter’s nuclear import
(data not shown).
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N-Terminal fusion of GFP to different hGR mutants appeared functionally neutral, as it did not change their properties regarding GRE-mediated transcriptional activation (24, 27). This allowed us to extrapolate the localization and trafficking data of these proteins in living cells to conclusions regarding the behavior of the nonchimeric original proteins.
GFP-hGR was completely transported from the cytoplasm into the
nucleus within 12 min after the addition of dexamethasone and shuttled
between the nucleus and the cytoplasm in a cell fusion assay.
GFP-hGR514, which does not possess a LBD and does not bind to heat
shock proteins, entered the nucleus instantaneously, as did GFP-hGRß,
with its own unique LBD and its low affinity for heat shock proteins.
As hGR514 contains only NL1, this signal is probably responsible for
the fast translocation. We can conclude that hGR, hGR514, and hGRß
through their NL1 region interact properly with importin and enter
the nucleus through the nuclear pores using the classic importin
pathway. The binding of heat shock proteins to hGR is known to
partially inactivate NL1 activity, and this may explain the difference
in the time required for entry between hGR and hGR514 or hGRß
(28, 29, 30).
GFP-hGRI559N was located predominantly in the cytoplasm, and only
high doses or prolonged exposure to dexamethasone treatment allowed
transport of this mutant into the nucleus, which took about 180 min for
complete transport. Therefore, the mutation replacing Ile with Asn at
amino acid 559, which is located very close to NL1, changed this
mutant’s nucleocytoplasmic shuttling activity, probably through
impairment of NL1 function. This impairment could be due to an altered
NL1 as a primary cause and/or could be secondary to the diminished
ligand binding, preventing exposure and interaction of the NL1 site
with components of the importin system (7). Some effect of
the mutation on the NL2 subdomain is possible. However, as the
contribution of this subdomain to the nuclear translocation of GR is
small and delayed, no major negative effect would be expected. Yet this
site could be responsible to some extent for the delayed nuclear entry
of the mutant receptor at high dexamethasone concentrations.
Active export of GFP-fused hGR-related molecules through the nuclear
pore complex must also take place, granted that the mol wt of all
proteins employed in this study exceeds 60 kDa, which is the upper
limit for passive diffusion (31, 32). The exportin-1/CRM1
system is responsible for nuclear export of many proteins that contain
a nuclear export signal (31, 33), but it is still unclear
whether this system is involved in the nuclear export of the GR
(7, 34). As GFP-hGR514 was exported with kinetics similar
to those of GFP-hGR, we suggest that the immunogenic domain and/or
DBD possess one or more functional motifs that are involved in the
active export of these molecules. On the other hand, as GFP-hGRß,
which contains the same domains, was exported very slowly from the
nucleus, we suggest that the LBD of the hGRß may contain a motif(s)
that suppresses active nuclear export or increases the retention
of the receptor in the nucleus via other interactions. This possibility
might explain the nuclear localization of this receptor isoform.
GFP-hGRI559N was also exported slowly from the nucleus. As the
hGR LBD is not necessary for nuclear export, we suggest that the
mutation at amino acid 559 suppresses nuclear export functions of the
immunogenic domain and/or DBD, an effect that might be similar to the
nuclear retention mechanism of hGRß by an altered LBD.
GFP-hGR was fully active in its stimulation of the MMTV promoter in
the presence of dexamethasone. On the other hand, GFP-hGR514, which was
located in the nucleus, was constitutively mildly active in a
dexamethasone-independent fashion, possibly through an intact
activation function-1 (AF1) residing in its immunogenic domain.
GFP-hGRß, as expected, was transcriptionally inactive. As hGRß
contains AF1 and can bind to GREs (6, 35), its unique LBD
may somehow suppress AF1 as well as AF2. GFP-hGRI559N was inactive
in the transactivation of the MMTV promoter at low or moderate
concentrations of dexamethasone, but was able to submaximally (<5% of
maximum) stimulate it at very high concentrations of dexamethasone
(10-4 M). As its transcriptional
activity was parallel to its translocation into the nucleus, we suggest
that the major defect of this mutant in transactivation is most likely
due to a dysfunction in the nuclear translocation step.
We tested the ability of GFP-hGR514, GFP-hGRß and GFP-hGRI559N to
influence the capacity of GFP-hGR to transactivate the MMTV
promoter. The mild trans-dominant activity of GFP-hGR514 was
proportional to its concentration at all concentrations of
dexamethasone used. As GFP-hGR514 dominates over the transactivational
activity of hGR on the MMTV promoter at a 10:1 ratio of
transfection, at which level the luciferase activity is similar to that
obtained with GFP-hGR514 alone, it is likely that the trans-dominant
activity of this constitutively weakly active mutant is due to
competition with GFP-hGR for binding to rate-limiting
coregulator molecules and/or GREs. On the other hand, the
trans-dominant negative activity of GFP-hGRI559N and GFP-hGRß
depended on the concentrations of dexamethasone employed. This suggests
that another mechanism(s) may contribute to the trans-dominance of
these GR-related molecules. GFP-hGRI559N especially showed
proportionally higher trans-dominant activity at lower concentrations
of dexamethasone.
The mutant GR of our patient had undetectable affinity for
dexamethasone in a standard dexamethasone binding assay and virtually
no transactivational activity (<5% of the maximum exerted by the
wild-type receptor) at all levels of dexamethasone examined, ranging
from 10-11–10-5 (0%) to
10-4 (<5%) (Ref. 17 and this
study). If the assumption that the patient’s cells contained equal
amounts of mutant and wild-type GR is correct, we can conclude that
the maximum glucocorticoid effect sensed by the cells of this patient
would be 50% by the wild-type molecule minus 30–50% of this, as a
result of dominant negative activity by the mutant receptor at the
equivalent of physiological glucocorticoid concentrations
(10-9–10-7 M
dexamethasone). This means that at best the patient’s tissues receive
25–35% of the normal glucocorticoid effect. As 50% is sufficient to
be expressed as the glucocorticoid resistance syndrome (kindred with
one GR allele knocked out) (16), 25–35% of normal
sensitivity is bound to be worse. However, this effect was
progressively diminished with increasing concentrations of
dexamethasone.
Based on the above results, we examined the coexpression of hGRI559N
with GFP-hGR to address its ability to inhibit the nuclear
translocation of the latter. Figure 7 shows that hGRI559N caused
cytosolic retention of the wild-type receptor. The mechanism underlying
this effect is uncertain. One possibility is that the mutant interacts
with some cellular component(s) of the importin pathway necessary for
GR nuclear import and competes with ligand-bound, wild-type GR
for them. Alternatively, this mutant may heterodimerize with
ligand-bound hGR in the cytoplasm, interfering with its
trans-location, an effect that may be overcome as more wild-type GR is
bound to increasing concentrations of the steroid. Once most of hGR
molecules are activated by high doses of dexamethasone, a substantial
part of GR translocates to the nucleus as a GR-GR homodimer or
a GR-GRI559N heterodimer, where it fully activates transcription,
whereas GRI559N loses its trans-dominant activity, which is based on
its ability to stay in the cytoplasm and/or to impede the nuclear
translocation of the ligand-bound GR. We suggest that this behavior
of the mutant receptor explains its ability to cause glucocorticoid
resistance up to a certain concentration of circadially secreted
glucocorticoid, but allows expression of Cushing syndrome stigmata
at chronically higher levels of cortisol secreted in a
noncircadian fashion, as frequently observed in Cushing’s disease
(36).
Acknowledgments
We thank E. Hudson for help with confocal microscopy, Drs. R. M. Evans and G. Hager for providing plasmids, and Dr. B. K. Felber for helpful discussions.
Footnotes
This work was supported in part by NCI, DHHS, under contract with ABL and by the DKFZ AIDS-Stipendium Program (to R.S.).
1 T.K. and R.H.S. contributed equally to this work. Present address
for R.H.S.: Chemotherapeutisches Forschungs Institut,
Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, 60596 Frankfurt/Main,
Germany. 
2 Present address for J.H.R.: Van Andel Institute, 333
Bostwick NE, Grand Rapids, Michigan 49503.
3 G.N.P. and G.P.C. contributed equally to the supervision of this
study.
Abbreviations: AF, Activation function; CMV, cytomegalovirus; DBD, DNA-binding domain; GFP, green fluorescence protein; GRE, glucocorticoid response element; h, human; LBD; ligand-binding domain; MMTV, mouse mammary tumor virus; NL, nuclear localization; PEG, polyethylene glycol; RSV, Rous sarcoma virus.
Received March 8, 2001.
Accepted August 3, 2001.
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A. Vottero, T. Kino, H. Combe, P. Lecomte, and G. P. Chrousos A Novel, C-Terminal Dominant Negative Mutation of the GR Causes Familial Glucocorticoid Resistance through Abnormal Interactions with p160 Steroid Receptor Coactivators J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2658 - 2667. [Abstract] [Full Text] [PDF]
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),
GFP-hGRß (
), or GFP-hGR
) were
determined in CV-1 cells. GFP-hGR
