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.

EXPERIMENTAL PROCEDURES

Materials

Recombinant Plasmids

Cell Culture and Transfections

Northern Blots

RT-PCR

Quantitative RT-PCR

Sucrose Density Gradients and Western Blots

Immunohistochemistry

CAT Activity

RESULTS

Northern Blot Analysis of the hGR Message in Human Tissues and Cell Lines

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.

Characterization of the 7.0-kb hGR Message

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) .

RT-PCR Analysis of the hGR Message in Human Tissues and Cell Lines

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.

Ligand Binding Analysis of hGR

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.

Subcellular Distribution of hGR

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).

Transcriptional Activity of hGR in the Absence of hGR

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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.

Transcriptional Activity of hGR in the Presence of hGR

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.

DISCUSSION

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, TR1 and TR2, that differ at the carboxyl terminus(36) . The TR2 isoform does not bind thyroid hormones, but it represses the transcriptional activity of TR1 by competing with TR1 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.

FOOTNOTES

*

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported in part by a National Science Foundation Graduate Fellowship.

¶

To whom correspondence should be addressed: National Institute of Environmental Health Sciences, P.O. Box 12233, MD E2-02, Research Triangle Park, NC 27709. Tel.: 919-541-1564. Fax: 919-541-1367.

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The abbreviations used are: hGR, human glucocorticoid receptor; GRE, glucocorticoid receptor responsive elements; hsp90, heat shock protein 90; NLS, nuclear localization signal; MMTV, mouse mammary tumor virus; DEX, dexamethasone; RU486, RU38486; CAT, chloramphenicol acetyltransferase; 3`UTR, 3`-untranslated region; TR, thyroid hormone receptor ; RT, reverse transcriptase; PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair(s).

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R. H. Oakley and J. A. Cidlowski, unpublished observations.

ACKNOWLEDGEMENTS

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