(Received for publication, August 24, 1995; and in revised form, January 23, 1996)
From the
Alternative splicing of the human glucocorticoid receptor (hGR)
primary transcript produces two receptor isoforms, hGR and
hGR
, which differ at their carboxyl termini. The hGR
isoform
conveys endocrine information to target tissues by altering patterns of
gene expression in a hormone-dependent fashion. In contrast to
hGR
, very little is known about the hGR
splice variant. Using
hGR
- and hGR
-specific riboprobes on human multiple tissue
Northern blots, we show that the hGR
message has a widespread
tissue distribution. We also prove by reverse transcriptase-polymerase
chain reaction that the alternative splicing event underlying the
formation of the hGR
message occurs in these tissues. Because the
hGR
protein differs from hGR
at the extreme COOH terminus, we
investigated several of the biochemical properties of hGR
expressed in transfected cells. hGR
does not bind the
glucocorticoid agonist dexamethasone nor the glucocorticoid antagonist
RU38486 in vivo. Moreover, in contrast to hGR
, hGR
is located primarily in the nucleus of transfected cells independent of
hormone administration. Finally, in the absence of hGR
, hGR
is transcriptionally inactive on a glucocorticoid-responsive enhancer.
However, when both isoforms are expressed in the same cell, hGR
inhibits the hormone-induced, hGR
-mediated stimulation of gene
expression. Thus, hGR
potentially functions as a dominant negative
inhibitor of hGR
activity.
Two human glucocorticoid receptor (hGR) ()cDNA
clones, termed hGR
and hGR
, were isolated in 1985 that
predicted the existence of two receptor isoforms differing at their
carboxyl termini(1) . Amino acid sequence analysis revealed
that the hGR
and hGR
isoforms were identical through amino
acid 727 but diverged beyond this position with hGR
having an
additional 50 amino acids and hGR
an additional, nonhomologous 15
amino acids. Exons 1-8 of the hGR gene contain the 5` noncoding
and coding sequences common to the hGR
and hGR
cDNAs, and
exons 9
and 9
contain the coding and 3` noncoding sequences
specific to the hGR
and hGR
cDNAs(2) . Because the
hGR
- and hGR
-specific sequences are located on the same gene,
alternative splicing of exons 9
and 9
was speculated to be
the mechanism responsible for generating the two receptor isoforms.
However, initial Western blot analysis detected only the larger 94-kDa
hGR
isoform, and only hGR
appeared to bind hormone and induce
expression of a glucocorticoid-responsive reporter plasmid in a
hormone-dependent manner(1, 3) . Because of its
predominant expression, ligand binding properties, and transcriptional
activity, hGR
became the primary focus of subsequent research. As
a result, its expression, biochemical properties, and physiological
function have been well characterized.
hGR is expressed in most
human tissues and cell lines and belongs to the superfamily of
steroid/thyroid/retinoic acid receptor proteins that function as
ligand-dependent transcription factors (for reviews see (4, 5, 6) ). Members of this family are
organized into structurally and functionally defined domains.
Specifically, hGR
is comprised of a unique amino-terminal variable
region that includes a transactivation domain that is important for
regulation of gene expression. hGR
also contains a central
DNA-binding domain crucial for specific interaction of the receptor
with DNA sequences containing glucocorticoid receptor responsive
elements (GRE). The carboxyl terminus of the hGR
protein contains
the hormone-binding domain as well as sequences important for
interaction with heat shock protein 90 (hsp90)(7) , nuclear
translocation(8) , receptor dimerization(9) , and
transactivation(10) .
In the absence of hormone, hGR
resides predominantly in the cytoplasm of cells, where it exists as a
large multiprotein complex (for reviews see (11) and (12) ). This complex appears to consist of the receptor
polypeptide, two molecules of hsp90, and several additional proteins.
The association of hsp90 with the receptor is believed to maintain the
receptor in a high affinity hormone binding state and sequester the
receptor in the cytoplasm by inactivating the nuclear localization
signals (NLS). Once hormone binds the receptor, a conformational change
ensues resulting in the dissociation of hsp90 and the other associated
proteins. In its new conformation hGR
translocates into the
nucleus, where it binds as a homodimer to GREs that are usually found
in the promoter regions of steroid-responsive genes. The receptor then
communicates with the basal transcription machinery to either enhance
or repress transcription of the linked gene. hGR
can also modulate
gene expression by physically interacting with other nuclear proteins
such as AP-1 (13, 14, 15) and
NF-
B(16) .
In contrast to hGR, very little is
known about the physiological significance of hGR
. We demonstrate
here that a mRNA transcript consistent in size with the hGR
cDNA
is expressed in various human adult and fetal tissues and in several
transformed human cell lines. We also confirm that the alternative
splicing event underlying the formation of the hGR
message occurs
in these tissues. In addition, we show that the unique COOH-terminal
end of hGR
influences several key biochemical properties of this
isoform that distinguishes it from hGR
. We demonstrate that
hGR
does not bind glucocorticoids or antiglucocorticoids in
vivo, resides in the nucleus independent of hormone
administration, and in the absence of hGR
is transcriptionally
inactive on a glucocorticoid-responsive enhancer. It was recently
reported that transfected hGR
inhibits transfected
hGR
-mediated induction of the mouse mammary tumor virus (MMTV)
promoter(17) . We extend these findings by demonstrating that
hGR
represses the activity of endogenous hGR
and that this
hGR
-mediated repression is a general phenomenon of
glucocorticoid-responsive promoters. Thus, the physiological
significance of hGR
may reside in its ability to antagonize the
function of hGR
.
As shown in Fig. 1A (upper and lower panels), the
hGR probe hybridizes with an abundant message in all tissues
migrating slightly below the 7.5-kb RNA marker. Additionally, a faint
band slightly below the 4.4-kb RNA marker is observed in the adult
heart, brain, placenta, lung, liver, skeletal muscle, and pancreas RNA
samples and in the fetal brain, lung, and liver RNA samples. The length
of the hGR
cDNA predicts the hGR
message to be at least 4.1
kb in size, thus the lower hybridization signal (approximately 4.3 kb
in size) may correspond to the hGR
mRNA transcript. In contrast to
the lower hybridization signal, the abundant message approximately 7.0
kb in size cross-reacts with the hGR
riboprobe (Fig. 1B, upper and lower panels).
The hGR
riboprobe also hybridizes with a message approximately 5.5
kb in size in many tissues and with a less abundant 4.4 kb message in
several tissues, and these two transcripts do not appear to cross-react
with the hGR
probe (Fig. 1B, upper and lower panels). Finally, the hGR mRNA transcripts approximately
7.0, 5.5, and 4.3 kb in size are all recognized by a probe made to the
common coding region of the hGR
and hGR
cDNAs (data not
shown). Similar hybridization patterns are also observed on Northern
blots of RNA isolated from HeLa S
cells (a human cervical
carcinoma cell line) and CEM-C7 cells (a human lymphoid cell line)
(data not shown).
Figure 1:
Northern
blot analysis of hGR messages in human adult (upper panel) and
fetal (lower panel) tissues. Human adult and fetal multiple
tissue Northern blots containing 2.0 µg of poly(A) RNA were hybridized with the hGR
-specific riboprobe (A, both panels). The blots were then stripped and
rehybridized with the hGR
-specific riboprobe (B, both
panels). RNA size markers are indicated along the left
margin, and the approximate sizes of the hybridization signals are
indicated along the right margin.
Figure 2:
Comparison of hGR exons 9 and 9
and intron J with the rat GR cDNA (A) and RT-PCR analysis of
the 7.0 kb hGR message (B). A, using the sequence
comparison program BestFit (Sequence Analysis Software Package,
Genetics Computer Group, University of Wisconsin Biotechnology
Center)(34) , the hGR
cDNA sequences 2156-2313 (exon
8), 2314-2466 (exon 9
coding), and 2467-4788 (exon
9
3`UTR)(1) ; intron J sequences 1-155(2) ;
and the hGR
cDNA sequences 2314-2361 (exon 9
coding)
and 2362-3791 (exon 9
3`UTR) (1) were aligned with
the rat GR cDNA sequences 1-6322(35) . The regions of
greatest similarity and the percentage of identity between the two
aligned sequences are indicated. Triangles identify consensus
polyadenylation signals, and arrows indicate the location of
PCR primers utilized in B. B, total RNA (1.0 µg)
from HeLa S
cells was reverse transcribed using random
hexamers, and first strand cDNA was subsequently amplified with the
addition of an upstream primer specific to the distal 3`UTR of exon
9
and a downstream primer specific to the proximal 3`UTR of exon
9
. The resulting RT-PCR products were then analyzed by agarose gel
electrophoresis. The reverse transcriptase was omitted in lane 1 but included in lane 2. In lanes 3-5, the
PCR product was digested with restriction enzymes that cut specifically
in exon 9
(Acc65I), intron J (HpaII), or exon
9
(HaeII). Sizes (in bp) of DNA markers (M) are
indicated in the left margin.
To test
whether the 3` end of the 7.0-kb hGR message is organized in this
fashion, we performed RT-PCR using a sense 5` primer specific to the
distal 3`UTR of exon 9 and an antisense 3` primer specific to the
proximal 3`UTR of exon 9
. If the 3` end of the 7.0-kb hGR message
consists of sequences from exon 9
, intron J, and exon 9
,
these primers will amplify a 933-bp PCR product. When total RNA
extracted from HeLa S
cells is used for RT-PCR, a PCR
product of this size is generated (Fig. 2B, lane
2). The PCR fragment is not produced when the reverse
transcriptase is omitted from the reaction, demonstrating that
contaminating DNA is not present (Fig. 2B, lane 1). In
addition, restriction enzymes that cleave sites specific to exon
9
, intron J, and exon 9
were used to confirm the sequence of
the 933-bp PCR fragment (Fig. 2B, lanes
3-5). Therefore, exon 9
makes up the distal coding and
proximal 3` noncoding regions of the 7.0-kb hGR message and both intron
J and exon 9
form the distal 3` noncoding region of the 7.0-kb hGR
message. This message would be expected to encode the hGR
isoform.
These RT-PCR results also demonstrate that sequences previously
identified as intron J (2) are actually exonic sequences
separating the 9 and 9
exonic sequences. In agreement with
this finding, an oligonucleotide probe specific to intron J hybridizes
on Northern blots with the 7.0-kb hGR
message (data not shown).
Therefore, we propose that the hGR sequences formerly identified as
exon 9
, intron J, and exon 9
comprise one large terminal exon
(exon 9) approximately 4.1-kb in size and that the hGR gene is
organized into nine exons rather than the previously reported
ten(2) .
A sense 5` primer specific to exon 8
and an antisense 3` primer specific to the 9 sequences were
utilized in the PCR reaction. If the alternative splicing event
underlying the formation of the 4.3-kb hGR
message occurs (in
which the end of exon 8 is linked to the 9
sequences located in
the distal portion of exon 9) the hGR
-specific primers will
produce a PCR product 366-bp in length. If these primers hybridize with
the 7.0-kb hGR
message (which also contains the 9
sequences
at its far 3` end), they will generate a PCR product approximately 3000
bp in length. Conditions of our PCR amplification reaction did not
favor production of this large PCR fragment, and it was never observed.
For parallel analysis of the hGR
mRNA transcripts, an antisense 3`
primer specific to the 9
sequences was used in combination with
the same sense 5` primer. If the default splicing event underlying the
formation of the hGR
messages occurs (in which the end of exon 8
is linked to the 9
sequences at the beginning of exon 9) these
hGR
-specific primers will produce a PCR product 477 bp in length.
When total RNA extracted from human heart, brain, lung, liver, and
skeletal muscle is used for RT-PCR, a 366-bp PCR product is generated
by the hGR-specific primers, suggesting that the hGR
message
is present in these human tissues (Fig. 3A). In
addition, the hGR
-specific primers amplify the expected 477-bp PCR
product in each tissue (Fig. 3B). When the reverse
transcriptase is omitted from the RT-PCR reaction, the expected PCR
fragments are not produced, indicating that only cDNA produced by the
RT step is serving as template for the correctly sized PCR product.
RT-PCR analysis was also performed on RNA isolated from HeLa S
and CEM-C7 cells (Fig. 3, A and B).
Again, a 366-bp PCR fragment is produced by the hGR
-specific
primers, suggesting that the hGR
message is present in these
transformed human cell lines. Consistent with our Northern blot data,
the hGR
message appears to have a widespread tissue distribution.
Figure 3:
RT-PCR analysis of RNA isolated from human
tissues and cell lines using hGR- and hGR
-specific primers.
Total RNA (0.5 µg) isolated from various human adult tissues
(heart, brain, lung, liver, and skeletal muscle) and cell lines (HeLa
S
and CEM-C7 cells) was reverse transcribed using random
hexamers. The resulting cDNA was amplified using either
hGR
-specific (A) or hGR
-specific (B)
primers. For each set of primers, the reverse transcriptase was omitted
in lanes 2, 4, 6, 8, 10, 12, and 14 but included in lanes 3, 5, 7, 9, 11, 13, and 15. No RNA was added in lane 1. The RT-PCR products
were analyzed by agarose gel electrophoresis. The sizes (in bp) of the
DNA markers (M) are 603, 310, 281/271, 234, and
194).
Together, the Northern blot and RT-PCR analyses indicate that the
hGR mRNA heterogeneity observed in human tissues and cell lines
includes both hGR and hGR
messages. The more abundant
transcripts are approximately 7.0 and 5.5 kb in size and are expected
to encode the hGR
isoform. Consensus polyadenylation signals are
located at the end of the 9
sequences in exon 9, and use of these
signals would generate a hGR
message approximately 1.6 kb shorter
than the full-length 7.0-kb hGR
message. These consensus signals
are functional because they terminate transcription of the hGR
cDNA cloned into an expression vector lacking other polyadenylation
signals (data not shown). Therefore, the 5.5-kb hGR
message
appears to originate from alternative polyadenylation at these
consensus sites. The less abundant hGR message recognized by the
hGR
-specific probe and approximately 4.3 kb in size is expected to
encode the hGR
isoform. This mRNA transcript results from
alternative splicing in which a 3` acceptor site preceding the 9
sequences in exon 9 is utilized by the splicing machinery rather than
the normal 3` acceptor site preceding the 9
sequences in exon 9.
The model shown in Fig. 4summarizes the predicted structure at
the 3` end of the hGR gene, primary transcript, and mature hGR
and
hGR
mRNAs; the processing events underlying the formation of these
mature messages; and the predicted translation products of these
transcripts.
Figure 4:
Predicted structure of the hGR gene and
gene products. hGR sequences formerly identified as exon 9, intron
J, and exon 9
comprise one large exon (exon 9). Alternative
processing of exon 9 generates multiple hGR messages. Specifically,
splicing event #1 (default splicing pathway) in which the end of exon 8
is linked to beginning of exon 9 is predicted to generate the 7.0- and
5.5-kb hGR
messages, which differ in size due to the use of
alternative polyadenylation signals. Splicing event #2 (alternative
splicing pathway) in which the end of exon 8 is linked to the beginning
of the 9
sequences is predicted to generate the 4.3-kb hGR
message. Translation of the messages produces the hGR
and hGR
isoforms, which are identical through amino acid 727 but then diverge.
The functional domains and the putative site of hsp90 interaction are
indicated for each isoform. Exons and introns (not to scale) are
designated by boxes and lines, respectively. The arrows along the primary transcript identify the location of
consensus polyadenylation signals. Splicing of introns A-G is not shown. The hGR
- and hGR
-specific cRNA probes used
in this study are indicated by solid
lines.
Based on the intensity of the Northern blot signals,
the two hGR messages (7.0 and 5.5 kb) are much more abundant than
the 4.3-kb hGR
message (see Fig. 1). To more accurately
assess the relative levels of the hGR
and hGR
mRNA
transcripts, we performed quantitative RT-PCR on RNA isolated from
adult lung, adult liver, HeLa S
cells, and CEM-C7 cells.
Reaction cycle intensity curves for the 477-bp hGR
and 366-bp
hGR
PCR products are shown for each tissue and cell line in Fig. 5A. For estimation of the hGR
/hGR
mRNA
ratio, regression equations were fitted to the linear portion of each
amplification curve, and the difference in the number of cycles
required to amplify an equal amount of hGR
and hGR
PCR
product was calculated. Similarly, the difference in cycle number
required to amplify an equal amount of hGR
and hGR
PCR
product was determined for a series of external standards containing
known hGR
/hGR
cDNA ratios. Using the standard curve shown in Fig. 5B, the hGR
/hGR
cDNA (and hence mRNA)
ratio for each human sample was calculated and is as follows: 0.34% for
lung, 0.21% for liver, 0.21% for HeLa S
cells, and 0.22%
for CEM-C7 cells. Although these values reflect a large difference in
expression levels, one should bear in mind that the amount of the
477-bp hGR
fragment is derived from two hGR
messages (7.0 and
5.5 kb), whereas the amount of 366-bp hGR
fragment comes only from
the 4.3-kb hGR
message. In addition, our approach assumes that the
efficiency of the RT reaction is the same for both the hGR
and
hGR
mRNA transcripts. This may not be the case.
Figure 5:
Quantitative RT-PCR analysis of hGR
and hGR
messages. A, human RNA (0.5 µg) was reverse
transcribed, and the resulting cDNA amplified using hGR
- or
hGR
-specific primers. Aliquots of the PCR reaction were removed at
2-cycle intervals and electrophoresed on agarose gels stained with
ethidium bromide. Representative gels showing amplification of the
477-bp hGR
and 366-bp hGR
fragments are from human lung (upper panel). By plotting ethidium bromide fluorescence as a
function of cycle number, hGR
and hGR
amplification curves
were generated for adult lung, adult liver, HeLa S
cells,
and CEM-C7 cells (lower panel). B, standard curve
showing the relationship between a known hGR
/hGR
cDNA ratio
and the additional number of cycles required by the hGR
primers to
amplify as much PCR product as the hGR
primers. ``Cycle
number difference'' calculations are from the exponential phase of
each PCR reaction and are described under ``Experimental
Procedures.'' For each human tissue and cell line, the standard
curve regression equation y = -4.427LOG(x) +
0.297 (r = 0.994) was used to determine the
hGR
/hGR
cDNA (and hence mRNA)
ratio.
Figure 6:
In vivo ligand binding analysis
of hGR expressed in transfected COS-1 cells. A, COS-1
cells were transfected with equimolar amounts of pCMVhGR
,
pCMVhGR
, or pCMV5 (mock) as described under ``Experimental
Procedures'' and incubated with 100 nM [
H]dexamethasone (upper panel) or
50 nM [
H]RU486 (lower panel)
for 2 h on ice. Whole cell extracts were prepared and loaded on
5-20% sucrose gradients. Following centrifugation, the gradients
were fractionated, and radioactivity was determined. B refers
to the bottom of the gradient, and T refers to the top. B, a portion of each whole cell extract was analyzed by
Western blotting using the anti-hGR antibody #57 (25) that
recognizes an epitope common to the 94-kDa hGR
and 90-kDa hGR
proteins. Molecular mass standards are indicated in the left
margin.
Figure 7:
Subcellular distribution of hGR
expressed in transfected COS-1 cells. COS-1 cells were transfected with
equimolar amounts of pCMVhGR
(left panel) or pCMVhGR
(right panel) using either the DEAE or calcium phosphate (CaPO
) transfection methods as described
under ``Experimental Procedures.'' 36 h post-transfection,
cells were treated for 2 h with vehicle (-Dex) or with
100 nM DEX (+Dex). Immunohistochemistry was then
performed using the anti-hGR antibody #57(25) , and
immunoreactivity was visualized by staining with Texas red fluorescent
dye (left side, each subpanel) or avidin-biotin-peroxidase (right side, each subpanel).
Figure 8:
Transcriptional activity of hGR in
the absence of hGR
. COS-1 (A) or CV-1 (B) cells
were cotransfected with pGMCS (5.0 µg) and equimolar amounts of
pCMV5 (2.8 µg), pCMVhGR
(5.5 µg), or pCMVhGR
(5.0
µg). Each transfection was standardized to 10.5 µg of DNA using
pBR322. 16 h post-transfection, medium containing vehicle (CON), 100 nM DEX, or 1 µM RU486 was
added to the cells, which were then incubated an additional 24 h. Cells
were then harvested, and CAT activity was determined. The data are
plotted as fold change from basal activation (pCMV5, CON). A shows the average of three independent experiments, and B is representative of three independent
experiments.
The
hGR-mediated induction of CAT activity in transfected COS-1 cells
was only 4-fold. In cell lines where hGR
inductions are much
greater, hGR
might display partial transcriptional activity.
Therefore, we investigated the transcriptional activity of hGR
on
the MMTV enhancer in receptor negative CV-1 cells. In response to DEX,
hGR
induces a 74-fold increase in CAT expression (Fig. 8B, lanes 4 and 5). Interestingly,
hGR
induces a 29-fold increase in CAT expression in response to
RU486 (Fig. 8B, lanes 4 and 6). This partial
agonist activity of RU486 has been reported previously and appears to
be cell type-specific(37) . Consistent with our findings in
COS-1 cells, CAT expression is unchanged in CV-1 cells transfected with
hGR
and treated with steroid (Fig. 8B, lanes
7-9). In addition, hGR
does not appear to be
constitutively active (Fig. 8B, compare lanes 1 and
7). Thus, we conclude that in the absence of hGR
, hGR
is
transcriptionally inactive on the glucocorticoid-responsive MMTV
enhancer.
Figure 9:
Transcriptional activity of hGR in
the presence of hGR
. HeLa S
cells were cotransfected
with 5.0 µg of pGMCS (A) or pGRE2CAT (B) and
various amounts of pCMV5 and pCMVhGR
as indicated. 16 h
post-transfection, medium containing vehicle (CON) or 100
nM DEX was added to the cells, which were then incubated an
additional 24 h. Cells were then harvested, and CAT activity was
determined. The data are plotted as fold change from basal activation
and are representative of three independent
experiments.
We next
evaluated whether the dominant negative activity of hGR on
hGR
-mediated transcription is restricted to the MMTV promoter or
is a general property of glucocorticoid-responsive promoters. The
pGRE2CAT reporter plasmid contains two copies of the GRE consensus
sequence derived from the tyrosine aminotransferase gene and a TATA box
element upstream of the CAT gene(20) . In contrast to MMTV,
this ``minimal promoter'' does not contain binding sites for
other ancillary transcription factors. When HeLa S
cells
are cotransfected with pGRE2CAT and various amounts of pCMVhGR
(Fig. 9B), the hGR
-mediated stimulation of CAT
expression is inhibited in a dose-dependent manner, and the repression
of hGR
activity is similar to that observed on the MMTV promoter.
The inhibitory action of hGR
is restricted to
glucocorticoid-responsive promoters because the hGR
protein has no
effect on the constitutively active, nonglucocorticoid-responsive
thymidine kinase CAT reporter plasmid (pBLCAT2) (data not shown). In
sum, these results suggest that hGR
represses the function of
hGR
by specifically inhibiting GRE-mediated transcription.
Alternative splicing of the hGR primary transcript produces
two highly homologous isoforms, termed hGR and hGR
, which
differ at their carboxyl termini(1, 2) . In contrast
to the well characterized hGR
isoform, very little is known about
the hGR
splice variant. In this report, we examine the expression,
biochemical properties, and physiological function of hGR
.
Northern blot analysis with a hGR
-specific riboprobe demonstrates
the presence of a message approximately 4.3 kb in size (consistent with
the length of the hGR
cDNA) in many different human tissues. We
subsequently confirmed by RT-PCR that the alternative splicing event
proposed to underlie the formation of the hGR
mRNA transcript
occurs in these tissues as well as in several transformed human cell
lines. Together, these results indicate that the hGR
message is
endogenous to a variety of cells. Because both the hGR
and
hGR
messages are co-expressed in many of the same tissues,
previous studies investigating hGR
expression with probes that did
not discriminate between hGR
and hGR
may be in error. The
hGR
mRNA transcript generated in vivo from the
pCMVhGR
expression vector is efficiently translated into the
90-kDa hGR
protein in transfected COS-1, CV-1, and HeLa S
cells, suggesting that the endogenous hGR
message can also
serve as a template for protein synthesis. However, whether the
endogenous hGR
message is actually translated into the hGR
isoform is unknown. Anti-hGR antibodies made to date in several
different laboratories have epitopes in the amino terminus and thus
recognize both the hGR
and hGR
proteins. The small difference
in size between the two isoforms and the potential for hGR
to be
post-translationally modified or degraded into a smaller protein make
this cross-reactivity undesirable. To test directly for the expression
of the hGR
protein, we are presently making a hGR
-specific
antibody.
During our investigation of the expression of the hGR
message, we made several observations that provide new insights both
into the structure of the hGR gene and hGR
mRNAs and into the
expression of the hGR
messages. The hGR gene has been previously
reported to consist of 10 exons(2) . Results from our Northern
blot and RT-PCR analyses suggest that the last two exons, 9
and
9
, and the intronic sequences separating these two exons (intron
J) together form one large terminal exon (exon 9). Exon 9 encodes the
3` end of the hormone-binding domain of the hGR
protein (under
normal splicing conditions) and contains approximately 4.0 kb of 3`UTR.
It is interesting to note that the genes for the human androgen
receptor, human estrogen receptor, and chicken progesterone receptor
show a similar organization to that we have proposed here for the hGR.
In each case, the most 3` exon (exon 8) encodes the COOH-terminal
portion of the hormone-binding domain and specifies a very large
3`UTR(38, 39, 40, 41, 42) .
The differential hybridization of the hGR
- and hGR
-specific
riboprobes with the 7.0- and 5.5-kb hGR
messages suggests that
they originate from the use of alternative polyadenylation signals
located in the 4.0-kb 3`UTR of exon 9. Similar alternative
polyadenylation events have been proposed to explain the GR mRNA
heterogeneity observed in rat tissues(35) . Within the 3`UTR of
eukaryotic mRNA reside signals that influence mRNA localization, mRNA
stability, and translation initiation(43, 44) . The
7.0- and 5.5-kb hGR
messages may differ in one or more of these
properties. Both hGR
messages have a widespread tissue
distribution, although the 5.5-kb hGR
message does not appear to
be expressed at high levels in the brain. In addition, the 7.0-kb
message is consistently more abundant than the 5.5-kb message.
The
7.0- and 5.5-kb hGR messages are much more abundant than the
4.3-kb hGR
mRNA transcript. Quantitative RT-PCR analysis of RNA
isolated from two adult tissues and two human cell lines suggests that
there is 200-500-fold more hGR
. However, because alternative
splicing is often regulated in a spatial and/or temporal fashion, the
hGR
message may be expressed at higher levels in a tissue-specific
and/or developmental stage-specific manner. In addition, the
hGR
/hGR
mRNA ratio may or may not reflect the
hGR
/hGR
protein ratio due to potential differences in
stability and/or translation efficiency of the hGR
and hGR
messages and/or due to differences in protein half-life. Furthermore,
because the 7.0-kb hGR
message has the 9
sequences at its far
3`UTR, we cannot exclude the possibility that it also encodes the
hGR
protein. Development of hGR
- and hGR
-specific
antibodies will provide insight into this issue. Interestingly, the
9
sequences are well conserved in the rat GR cDNA 3`UTR,
suggesting that an hGR
homolog may exist in rat. RT-PCR analysis
of rat liver (as well as a mouse lymphoma cell line) does indeed
produce a PCR product that comigrates with the 366-bp PCR fragment
derived from human cells.
Thus, although the hGR
message is expressed at low levels relative to the hGR
mRNA
transcripts in the human tissues so far examined, its conservation
across species suggests that it plays an important physiological role.
With few exceptions(45, 46) , modification of the
GR hormone-binding domain results in a reduction or complete loss of
hormone
binding(3, 47, 48, 49, 50) .
The COOH-terminal 50 amino acids of hGR have been replaced in
hGR
with 15 unique amino acids. In agreement with previous
reports(1, 3, 17) , we show that this natural
COOH-terminal modification prevents agonist binding to the hGR
protein. Recently, it was reported that a truncated version of the
human progesterone receptor B form missing the COOH-terminal 42 amino
acids did not bind progesterone or the synthetic agonist R5020 but
still bound the antiprogestin RU486(51) . This finding
suggested that amino acids at the extreme COOH terminus of the human
progesterone receptor are critical for agonist but not antagonist
binding. Because members of the steroid hormone receptor superfamily
share many of the same properties, we tested the ability of hGR
to
bind RU486 but found no evidence of binding. The 15 amino acids at the
end of hGR
may prevent the association of RU486 with this isoform.
Alternatively, the observation made for human progesterone receptor B
form may not be conserved among other family members. At this time,
hGR
is more aptly described as an orphan receptor whose natural
ligand, if any, is unknown.
The hGR receptor isoform
translocates from the cytoplasm to the nucleus in a hormone-dependent
manner(11) . In the absence of hormone, the association of
hsp90 with hGR
appears to inactivate the
NLS(52, 53) . Once hormone binds hGR
, hsp90
dissociates from the receptor resulting in the activation of the NLS
and subsequent nuclear import of hGR
(8) . In contrast to
hGR
, we demonstrate that hGR
resides primarily in the nucleus
of transfected cells independent of hormone treatment. The amino acids
necessary for interacting with hsp90 (7) are present in
hGR
, suggesting that hGR
may be in the nucleus in spite of
its association with hsp90. Perhaps the unique COOH-terminal amino
acids of hGR
delete sequences that inhibit the NLS or slightly
alter the tertiary structure of the hGR
/hsp90 complex such that
the NLS are partially activated. This might account for our observation
that most, but not all, of the hGR
protein is located in the
nucleus. Further studies will be required to elucidate the precise
mechanism(s) underlying the nuclear distribution of hGR
.
In the
absence of hGR, the hGR
isoform is transcriptionally inactive
on the MMTV enhancer independent of steroid treatment. However, when
hGR
and hGR
are expressed in the same cell, hGR
inhibits
the glucocorticoid-induced, hGR
-mediated activation of the MMTV
promoter. Although this dominant negative effect was first reported in
COS-7 cells cotransfected with hGR
and hGR
expression
vectors(17) , we have extended this initial observation in
several respects. First, we demonstrate that this dominant negative
activity occurs in cells that have endogenous hGR
receptors. In
addition, we show that the repression of hGR
activity occurs with
the simple promoter pGRE2CAT. This indicates that the repression is a
general phenomenon of glucocorticoid-responsive promoters and that it
is GRE-mediated transcription that is actually inhibited.
The
mechanisms responsible for the hGR-mediated repression of hGR
activity are unknown. The hGR
protein is primarily located in the
nucleus of transfected cells, has an intact DNA-binding domain and has
been reported to bind a consensus GRE in vitro(17) .
Therefore, it may compete with hGR
for binding to the GRE. Another
possibility is that hGR
forms a heterodimer with hGR
that is
transcriptionally inactive or less active than an hGR
homodimer.
Alternatively, the hGR
isoform may inhibit the function of
hGR
by interacting with and titrating out an essential cofactor
needed by hGR
for full transcriptional activity. We are currently
trying to identify the mechanism(s) responsible for the dominant
negative activity of hGR
and to determine whether hGR
can
inhibit hGR
-mediated repression of gene expression. Moreover, we
hope to discern if hGR
exhibits its dominant repressive effect on
other members of the closely related subgroup of nuclear receptors that
includes the progesterone, androgen, and mineralocorticoid receptors.
We have demonstrated that an alternatively spliced form of the hGR
is present in many different tissues and is able to antagonize the
physiological function of its predominant gene product. Alternative
splicing plays a critical role in regulating the activity of several
other members of the steroid/thyroid/retinoic acid receptor
superfamily. Most closely resembling that observed for hGR and
hGR
is the processing that occurs at the thyroid hormone receptor
subunit (TR
) locus. Alternative splicing of the last exon
generates two receptor isoforms, TR
1 and TR
2, that differ at
the carboxyl terminus(36) . The TR
2 isoform does not bind
thyroid hormones, but it represses the transcriptional activity of
TR
1 by competing with TR
1 for binding to the thyroid hormone
receptor responsive elements(36) . Clearly, the ability of
steroid/thyroid/retinoic acid receptor genes to encode transcription
factors with opposing biological activities adds another level of
complexity to the regulation of the function of these receptors.