©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Human Glucocorticoid Receptor Isoform
EXPRESSION, BIOCHEMICAL PROPERTIES, AND PUTATIVE FUNCTION (*)

(Received for publication, August 24, 1995; and in revised form, January 23, 1996)

Robert H. Oakley (1) (2)(§) Madhabananda Sar (2) John A. Cidlowski (1)(¶)

From the  (1)Laboratory of Integrative Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 and the (2)Department of Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two receptor isoforms, hGRalpha and hGRbeta, which differ at their carboxyl termini. The hGRalpha isoform conveys endocrine information to target tissues by altering patterns of gene expression in a hormone-dependent fashion. In contrast to hGRalpha, very little is known about the hGRbeta splice variant. Using hGRalpha- and hGRbeta-specific riboprobes on human multiple tissue Northern blots, we show that the hGRbeta 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 hGRbeta message occurs in these tissues. Because the hGRbeta protein differs from hGRalpha at the extreme COOH terminus, we investigated several of the biochemical properties of hGRbeta expressed in transfected cells. hGRbeta does not bind the glucocorticoid agonist dexamethasone nor the glucocorticoid antagonist RU38486 in vivo. Moreover, in contrast to hGRalpha, hGRbeta is located primarily in the nucleus of transfected cells independent of hormone administration. Finally, in the absence of hGRalpha, hGRbeta is transcriptionally inactive on a glucocorticoid-responsive enhancer. However, when both isoforms are expressed in the same cell, hGRbeta inhibits the hormone-induced, hGRalpha-mediated stimulation of gene expression. Thus, hGRbeta potentially functions as a dominant negative inhibitor of hGRalpha activity.


INTRODUCTION

Two human glucocorticoid receptor (hGR) (^1)cDNA clones, termed hGRalpha and hGRbeta, 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 hGRalpha and hGRbeta isoforms were identical through amino acid 727 but diverged beyond this position with hGRalpha having an additional 50 amino acids and hGRbeta an additional, nonhomologous 15 amino acids. Exons 1-8 of the hGR gene contain the 5` noncoding and coding sequences common to the hGRalpha and hGRbeta cDNAs, and exons 9alpha and 9beta contain the coding and 3` noncoding sequences specific to the hGRalpha and hGRbeta cDNAs(2) . Because the hGRalpha- and hGRbeta-specific sequences are located on the same gene, alternative splicing of exons 9alpha and 9beta was speculated to be the mechanism responsible for generating the two receptor isoforms. However, initial Western blot analysis detected only the larger 94-kDa hGRalpha isoform, and only hGRalpha 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, hGRalpha became the primary focus of subsequent research. As a result, its expression, biochemical properties, and physiological function have been well characterized.

hGRalpha 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, hGRalpha is comprised of a unique amino-terminal variable region that includes a transactivation domain that is important for regulation of gene expression. hGRalpha 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 hGRalpha 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, hGRalpha 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 hGRalpha 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. hGRalpha can also modulate gene expression by physically interacting with other nuclear proteins such as AP-1 (13, 14, 15) and NF-kappaB(16) .

In contrast to hGRalpha, very little is known about the physiological significance of hGRbeta. We demonstrate here that a mRNA transcript consistent in size with the hGRbeta 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 hGRbeta message occurs in these tissues. In addition, we show that the unique COOH-terminal end of hGRbeta influences several key biochemical properties of this isoform that distinguishes it from hGRalpha. We demonstrate that hGRbeta does not bind glucocorticoids or antiglucocorticoids in vivo, resides in the nucleus independent of hormone administration, and in the absence of hGRalpha is transcriptionally inactive on a glucocorticoid-responsive enhancer. It was recently reported that transfected hGRbeta inhibits transfected hGRalpha-mediated induction of the mouse mammary tumor virus (MMTV) promoter(17) . We extend these findings by demonstrating that hGRbeta represses the activity of endogenous hGRalpha and that this hGRbeta-mediated repression is a general phenomenon of glucocorticoid-responsive promoters. Thus, the physiological significance of hGRbeta may reside in its ability to antagonize the function of hGRalpha.


EXPERIMENTAL PROCEDURES

Materials

Dexamethasone (DEX) was obtained from Steraloids (Wilton, NH). [^3H]DEX (48.2 Ci/mmol) and [^14C]chloramphenicol (40-60 mCi/mmol) were obtained from DuPont NEN. RU38486 (RU486) and [^3H]RU486 (50.6 Ci/mmol) were kindly provided by Dr. R. Deraedt, Roussel-UCLAF (Romainville, France). [alpha-P]UTP (3000 Ci/mmol) was purchased from ICN Radiochemical. The murine leukemia virus reverse transcriptase, AmpliTaq DNA polymerase, deoxynucleotide triphosphates, MgCl(2), and the RT-PCR buffers were purchased from Perkin-Elmer. Acetyl coenzyme A was obtained from Boehringer Mannheim, and thin layer chromatography sheets were from EM Separations.

Recombinant Plasmids

The hGRalpha expression vector pCYGR (by Y. Itoh-Lindstrom and J. A. Cidlowski) served as the vector for the expression plasmids pCMVhGRalpha and pCMVhGRbeta utilized in this study. pCYGR was constructed by isolating the 3.0-kb KpnI-XhoI cDNA fragment of pRShGRalpha(3) . This fragment (which includes the entire hGRalpha coding region and the first 384 bp of the hGRalpha 3`UTR) was then cloned downstream of the human cytomegalovirus major intermediate early gene promoter region in the plasmid pCMV5(18) . The hGRalpha expression vector pCMVhGRalpha was constructed by isolating the 3.3-kb ClaI-BamHI cDNA fragment from the hGRalpha clone OB7(1) . This fragment (which contains the distal 940 bp of the hGRalpha coding region and the 2322 bp hGRalpha 3`UTR) was then cloned into the ClaI-BamHI sites in pCYGR. The hGRbeta expression vector pCMVhGRbeta was constructed by isolating the 2.3-kb ClaI-BamHI cDNA fragment from the hGRbeta clone OB10(1) . This fragment (which contains the hGRbeta-specific coding sequences as well as the 1430-bp hGRbeta 3`UTR) was then cloned into the ClaI-BamHI sites in pCYGR. Plasmids pGMCS(19) , pGRE2CAT(20) , and pBLCAT2 (21) have been previously described.

Cell Culture and Transfections

HeLa S(3) and COS-1 cells were grown as described previously(22, 23) . CV-1 cells were grown in Dulbecco's minimum essential medium supplemented with 2 mM glutamine and 10% (v/v) heat-inactivated fetal calf serum. CEM-C7 cells were grown in suspension in RPMI 1640 medium supplemented with 2 mM glutamine and 10% (v/v) heat-inactivated fetal calf serum. All cultures were maintained in a 5% CO(2) humidified atmosphere at 37 °C and were passaged every 3-4 days. HeLa S(3) and COS-1 cells were transfected essentially as described previously(22) . Briefly, 4 h before transfection, medium was replaced with fresh Dulbecco's minimum essential medium containing 3% serum. Plasmid DNA was prepared as a calcium phosphate precipitate and incubated with cells for 5 h followed by a 30-s shock with 15% glycerol. Cells were then refed supplemented medium.

Northern Blots

Human adult and fetal multiple tissue Northern blots were purchased from ClonTech (Palo Alto, CA). Membranes were prehybridized and hybridized at 65 °C in 50% formamide, 5 times SSPE, 5 times Denhardt's solution, 2% SDS, 200 µg/ml yeast RNA, 200 µg/ml denatured sheared salmon sperm DNA. P-Labeled antisense RNA probes (1 times 10^6 cpm/ml hybridization fluid) were generated from a T3/T7 promoter-containing vector containing the distal hGRalpha 3`UTR (537-bp PstI-KpnI fragment from the hGRalpha clone OB7), the hGRbeta coding and proximal 3`UTR (581-bp NsiI-SstI fragment from the hGRbeta clone OB10), or the entire hGRalpha coding region (3-kb KpnI-XhoI fragment from the hGRalpha expression vector pRShGRalpha). Following hybridization, blots were washed once at room temperature and four times at 65 °C in 0.1 times SSPE, 0.1% SDS. The blots were then exposed to x-ray film. Membranes were stripped of radioactivity for 30 min in 0.1 times SSPE, 0.1% SDS heated to 100 °C before reprobing.

RT-PCR

Human liver and lung total RNA were kindly provided by Dr. Darryl Zeldin (NIEHS). Human heart, brain, and skeletal muscle total RNA was purchased from ClonTech. Total RNA from HeLa S(3) and CEM-C7 cells was isolated using TRIzol Reagent (Life Technologies, Inc.) according to the manufacturer's instructions. cDNA was prepared in a buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 5 mM MgCl(2), 1 mM each dGTP, dATP, dTTP, and dCTP, 20 units RNase inhibitor, 50 units murine leukemia virus reverse transcriptase, 2.5 µM random hexamers, 1.0 µg of sample RNA, in a final volume of 20 µl. This mixture was incubated for 10 min at RT, 15 min at 42 °C, and 5 min at 99 °C and then used for amplification of specific cDNAs by PCR. The buffer for PCR contained 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl(2), 2.5 units AmpliTaq DNA polymerase, 0.2 µM upstream and downstream primers, in a final volume of 100 µl. After an initial incubation for 1 min at 95 °C, samples were subjected to 45 cycles of 30 s at 95 °C, 30 s at 62 °C, and 30 s at 72 °C. This was followed by a final extension step at 72 °C for 10 min. The primers used for the amplification of the 7.0-kb hGR message were as follows: 5`-GCATTCATACAGGCAGCGAT-3` (upstream) and 5`-CCACGTATCCTAAAAGGGCAC-3` (downstream) corresponding to nucleotides 4221-4240 and 2503-2523 of the hGRalpha and hGRbeta cDNAs, respectively(1) . The primers used for the amplification of the hGRalpha message were as follows: 5`-CCTAAGGACGGTCTGAAGAGC-3` (upstream) and 5`-GCCAAGTCTTGGCCCTCTAT-3` (downstream), corresponding to nucleotides 2158-2178 and 2616-2635 of the hGRalpha cDNA(1) . The primers used for the amplification of the hGRbeta message were as follows: 5`-CCTAAGGACGGTCTGAAGAGC-3` (upstream) and 5`-CCACGTATCCTAAAAGGGCAC-3` (downstream), corresponding to nucleotides 2158-2178 and 2503-2523 of the hGRbeta cDNA(1) . Amplified DNA fragments were electrophoretically fractionated on 1.75% agarose gels. Restriction enzyme analysis of the RT-PCR products amplified by the hGRalpha- and hGRbeta-specific primers confirmed that these fragments contained the appropriate hGRalpha and hGRbeta cDNA sequences. Restriction enzymes employed included NsiI (specific to exon 8), SspI (specific to exon 9alpha), and HaeII (specific to exon 9beta).

Quantitative RT-PCR

Human RNA (0.5 µg) was reverse transcribed, and the resulting cDNA was amplified as described above. 5 µl of the PCR reaction was removed at 2-cycle intervals and electrophoresed on 1.75% agarose gels stained with ethidium bromide. The intensity of the ethidium bromide fluorescence was measured densitometrically and plotted as a function of cycle number to generate amplification curves for the hGRalpha and hGRbeta PCR fragments. Regression equations of the form y = a times b^n, where y is the intensity and n is the number of cycles, were fitted to the data in the linear portion of the semi-logarithmic graphs. The amplification efficiencies (E) for the hGRalpha and hGRbeta fragments, while nearly identical within tissues, varied slightly across tissues: Ealpha = 0.71 and Ebeta = 0.71 for CEM-C7 cells but Ealpha = 0.82 and Ebeta = 0.81 for human lung. Amplification curves were also generated (and regression equations fitted) for external standards containing linearized pCMVhGRbeta and pCMVhGRalpha at ratios of 1:1000, 10:1000, 100:1000, and 1000:1000, respectively. A standard curve was generated by plotting the difference in the number of cycles required to amplify (during the exponential phase of each reaction) an identical amount of the hGRalpha and hGRbeta PCR products (3000 densitometric units) as a function of the pCMVhGRbeta/pCMVhGRalpha ratio. Similarly, the difference in the number of cycles required to amplify (during the exponential phase of each reaction) the same amount of hGRalpha and hGRbeta PCR products (3000 densitometric units) for each human sample was calculated. Using the standard curve regression equation, the approximate hGRbeta/hGRalpha cDNA (and hence mRNA) ratio was determined for each human tissue and cell line.

Sucrose Density Gradients and Western Blots

Subconfluent COS-1 cells (4 times 10^6) were transfected by the calcium phosphate method as described above with equimolar amounts of pCMVhGRalpha (40 µg), pCMVhGRbeta (36.4 µg), or pCMV5 (20.4 µg). Each transfection was standardized to 40 µg of DNA using pBR322. Cells were harvested 48 h post-transfection, resuspended in unsupplemented Dulbecco's minimum essential medium containing 100 nM [^3H]DEX or 20 nM [^3H]RU486, and incubated for 2 h on ice with gentle agitation. Whole cell extracts were prepared and processed on sucrose density gradients essentially as described by Tully and Cidlowski(24) . To verify equivalent expression of the hGRalpha and hGRbeta proteins, an aliquot of each extract was analyzed by Western blotting. Proteins (125 µg) were resolved by electrophoresis through 7.5% polyacryalmide gels and electrophoretically transferred to nitrocellulose. After incubating the membrane with epitope-purified polyclonal anti-hGR antiserum #57 (25) at a dilution of 1:2000, immunoreactivity was visualized using enhanced chemiluminescence according to the manufacturer's instructions (ECL, Amersham Corp.).

Immunohistochemistry

Subconfluent COS-1 cells (2 times 10^5) were transfected with equimolar amounts of pCMVhGRalpha (20 µg) or pCMVhGRbeta (18.2 µg) by the calcium phosphate method as described above or by the DEAE method of Sompayrac and Danna (26) as modified by Gorman(27) . Each transfection was standardized to 20 µg of DNA using pBR322. Following transfection, the cells were incubated for 24 h in Dulbecco's minimum essential medium supplemented with 10% dextran-coated charcoal-treated serum before plating in two-chamber glass slides. After an additional 24-h incubation, transfected cells were treated for 2 h with DEX (100 nM) or control vehicle and processed for immunohistochemical staining as described previously (25) . Immunoreactivity was visualized by staining with avidin-biotin-peroxidase or Texas red fluorescent dye.

CAT Activity

Subconfluent COS-1, CV-1, and HeLa S(3) cells were transfected as indicated in the appropriate figure legends by the calcium phosphate method described above. 16 h post-transfection, medium was removed and replaced with control medium or medium containing DEX or RU486. Cells were harvested 24 h later and CAT assays were performed as described previously(22) .


RESULTS

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

Northern blot analysis of RNA isolated from various human cell lines routinely shows multiple hGR mRNA transcripts approximately 7.0 and 5.0 kb in size(1, 28, 29, 30, 31, 32, 33) . These transcripts are thought to arise from the use of alternative polyadenylation signals within the 3`UTR of exon 9alpha and are presumed to encode the hGRalpha isoform. Because early studies utilized probes that recognized a region common to the hGRalpha and hGRbeta messages, one or more of these transcripts might actually be the hGRbeta splice variant. In order to discriminate between hGRalpha and hGRbeta messages and to assess the relative amounts of these transcripts in a given tissue, we sequentially hybridized poly(A) RNA isolated from various human adult and fetal tissues with hGRbeta- and hGRalpha-specific cRNA riboprobes. The hGRbeta riboprobe was designed to recognize a 578-nucleotide segment spanning the coding and proximal 3` noncoding regions of exon 9beta. The hGRalpha riboprobe was designed to recognize a 534-nucleotide segment in the distal 3`UTR of exon 9alpha.

As shown in Fig. 1A (upper and lower panels), the hGRbeta 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 hGRbeta cDNA predicts the hGRbeta 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 hGRbeta mRNA transcript. In contrast to the lower hybridization signal, the abundant message approximately 7.0 kb in size cross-reacts with the hGRalpha riboprobe (Fig. 1B, upper and lower panels). The hGRalpha 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 hGRbeta 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 hGRalpha and hGRbeta cDNAs (data not shown). Similar hybridization patterns are also observed on Northern blots of RNA isolated from HeLa S(3) 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 hGRbeta-specific riboprobe (A, both panels). The blots were then stripped and rehybridized with the hGRalpha-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

Because the 7.0-kb hGR message is recognized by both the hGRalpha and hGRbeta riboprobes, it must contain information from both the 9alpha and 9beta exons. These exons, therefore, do not appear to be mutually exclusive as previously reported(2) . This observation made it important to determine whether the 7.0-kb message encodes the hGRalpha or hGRbeta isoform. Various data suggested that it would encode hGRalpha. Exon 9alpha precedes exon 9beta in the linear organization of the hGR gene(2) . If this order is maintained in the mature message, exon 9alpha would form the distal coding and proximal 3` noncoding regions, and exon 9beta would comprise the distal 3` noncoding region. A hGR message with this arrangement at its 3` end would be expected to encode the hGRalpha protein. Sequence conservation between hGR exons 9alpha and 9beta (as well as the 155-bp intron separating these two exons) and the distal coding and 3` noncoding regions of the 6.5-kb rat GR message (Fig. 2A) further suggested that the 7.0-kb hGR mRNA transcript contains (moving 5` to 3`) exon 9alpha, intron J, and exon 9beta sequences at its distal 3` end. If all 10 exons and intron J of the hGR gene are represented in a mature hGR message, this message would be approximately 6.8 kb in size, consistent with the size of the hybridization signal observed on our Northern blots.


Figure 2: Comparison of hGR exons 9alpha and 9beta 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 hGRalpha cDNA sequences 2156-2313 (exon 8), 2314-2466 (exon 9alpha coding), and 2467-4788 (exon 9alpha 3`UTR)(1) ; intron J sequences 1-155(2) ; and the hGRbeta cDNA sequences 2314-2361 (exon 9beta coding) and 2362-3791 (exon 9beta 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(3) 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 9alpha and a downstream primer specific to the proximal 3`UTR of exon 9beta. 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 9alpha (Acc65I), intron J (HpaII), or exon 9beta (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 9alpha and an antisense 3` primer specific to the proximal 3`UTR of exon 9beta. If the 3` end of the 7.0-kb hGR message consists of sequences from exon 9alpha, intron J, and exon 9beta, these primers will amplify a 933-bp PCR product. When total RNA extracted from HeLa S(3) 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 9alpha, intron J, and exon 9beta were used to confirm the sequence of the 933-bp PCR fragment (Fig. 2B, lanes 3-5). Therefore, exon 9alpha makes up the distal coding and proximal 3` noncoding regions of the 7.0-kb hGR message and both intron J and exon 9beta form the distal 3` noncoding region of the 7.0-kb hGR message. This message would be expected to encode the hGRalpha isoform.

These RT-PCR results also demonstrate that sequences previously identified as intron J (2) are actually exonic sequences separating the 9alpha and 9beta exonic sequences. In agreement with this finding, an oligonucleotide probe specific to intron J hybridizes on Northern blots with the 7.0-kb hGRalpha message (data not shown). Therefore, we propose that the hGR sequences formerly identified as exon 9alpha, intron J, and exon 9beta 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 hGRbeta Message in Human Tissues and Cell Lines

The hGRbeta-specific riboprobe also hybridizes with a faint message approximately 4.3 kb in size on the Northern blots shown in Fig. 1. The size of this message suggests that it might be the hGRbeta mRNA transcript. However, because the 7.0-kb hGRalpha message contains the 9beta sequences at its 3` end, the 4.3-kb signal might instead be a degradation product of the 7.0-kb hGRalpha message. Alternatively, the 4.3-kb signal might represent nonspecific hybridization of the hGRbeta probe with 28 S rRNA. Recently, it was reported using RT-PCR that the alternative splicing event underlying the formation of the hGRbeta mRNA transcript occurs in many different human tissues(17) . However, these experiments employed a very large number of PCR cycles, and these workers apparently did not control for potential amplification of contaminating DNA. Therefore, to confirm that the hGRbeta message is expressed, we performed RT-PCR on RNA isolated from various human tissues and cell lines using primers that hybridize on either side of the alternatively spliced region of the 4.3-kb hGRbeta message.

A sense 5` primer specific to exon 8 and an antisense 3` primer specific to the 9beta sequences were utilized in the PCR reaction. If the alternative splicing event underlying the formation of the 4.3-kb hGRbeta message occurs (in which the end of exon 8 is linked to the 9beta sequences located in the distal portion of exon 9) the hGRbeta-specific primers will produce a PCR product 366-bp in length. If these primers hybridize with the 7.0-kb hGRalpha message (which also contains the 9beta 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 hGRalpha mRNA transcripts, an antisense 3` primer specific to the 9alpha sequences was used in combination with the same sense 5` primer. If the default splicing event underlying the formation of the hGRalpha messages occurs (in which the end of exon 8 is linked to the 9alpha sequences at the beginning of exon 9) these hGRalpha-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 hGRbeta-specific primers, suggesting that the hGRbeta message is present in these human tissues (Fig. 3A). In addition, the hGRalpha-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(3) and CEM-C7 cells (Fig. 3, A and B). Again, a 366-bp PCR fragment is produced by the hGRbeta-specific primers, suggesting that the hGRbeta message is present in these transformed human cell lines. Consistent with our Northern blot data, the hGRbeta message appears to have a widespread tissue distribution.


Figure 3: RT-PCR analysis of RNA isolated from human tissues and cell lines using hGRbeta- and hGRalpha-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(3) and CEM-C7 cells) was reverse transcribed using random hexamers. The resulting cDNA was amplified using either hGRbeta-specific (A) or hGRalpha-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 hGRalpha and hGRbeta messages. The more abundant transcripts are approximately 7.0 and 5.5 kb in size and are expected to encode the hGRalpha isoform. Consensus polyadenylation signals are located at the end of the 9alpha sequences in exon 9, and use of these signals would generate a hGRalpha message approximately 1.6 kb shorter than the full-length 7.0-kb hGRalpha message. These consensus signals are functional because they terminate transcription of the hGRalpha cDNA cloned into an expression vector lacking other polyadenylation signals (data not shown). Therefore, the 5.5-kb hGRalpha message appears to originate from alternative polyadenylation at these consensus sites. The less abundant hGR message recognized by the hGRbeta-specific probe and approximately 4.3 kb in size is expected to encode the hGRbeta isoform. This mRNA transcript results from alternative splicing in which a 3` acceptor site preceding the 9beta sequences in exon 9 is utilized by the splicing machinery rather than the normal 3` acceptor site preceding the 9alpha 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 hGRalpha and hGRbeta 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 9alpha, intron J, and exon 9beta 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 hGRalpha 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 9beta sequences is predicted to generate the 4.3-kb hGRbeta message. Translation of the messages produces the hGRalpha and hGRbeta 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 hGRalpha- and hGRbeta-specific cRNA probes used in this study are indicated by solid lines.



Based on the intensity of the Northern blot signals, the two hGRalpha messages (7.0 and 5.5 kb) are much more abundant than the 4.3-kb hGRbeta message (see Fig. 1). To more accurately assess the relative levels of the hGRalpha and hGRbeta mRNA transcripts, we performed quantitative RT-PCR on RNA isolated from adult lung, adult liver, HeLa S(3) cells, and CEM-C7 cells. Reaction cycle intensity curves for the 477-bp hGRalpha and 366-bp hGRbeta PCR products are shown for each tissue and cell line in Fig. 5A. For estimation of the hGRbeta/hGRalpha 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 hGRalpha and hGRbeta PCR product was calculated. Similarly, the difference in cycle number required to amplify an equal amount of hGRalpha and hGRbeta PCR product was determined for a series of external standards containing known hGRbeta/hGRalpha cDNA ratios. Using the standard curve shown in Fig. 5B, the hGRbeta/hGRalpha 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(3) 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 hGRalpha fragment is derived from two hGRalpha messages (7.0 and 5.5 kb), whereas the amount of 366-bp hGRbeta fragment comes only from the 4.3-kb hGRbeta message. In addition, our approach assumes that the efficiency of the RT reaction is the same for both the hGRalpha and hGRbeta mRNA transcripts. This may not be the case.


Figure 5: Quantitative RT-PCR analysis of hGRalpha and hGRbeta messages. A, human RNA (0.5 µg) was reverse transcribed, and the resulting cDNA amplified using hGRalpha- or hGRbeta-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 hGRalpha and 366-bp hGRbeta fragments are from human lung (upper panel). By plotting ethidium bromide fluorescence as a function of cycle number, hGRalpha and hGRbeta amplification curves were generated for adult lung, adult liver, HeLa S(3) cells, and CEM-C7 cells (lower panel). B, standard curve showing the relationship between a known hGRbeta/hGRalpha cDNA ratio and the additional number of cycles required by the hGRbeta primers to amplify as much PCR product as the hGRalpha 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 hGRbeta/hGRalpha cDNA (and hence mRNA) ratio.



Ligand Binding Analysis of hGRbeta

Operating under the assumption that the endogenous hGRbeta message is translated into the endogenous hGRbeta protein, we began investigating the biochemical properties of hGRbeta when overexpressed in transfected COS-1 cells. COS-1 cells contain undetectable levels of endogenous GR (3) and therefore provide a model system for studying the biochemical properties of hGRbeta in the absence of hGRalpha. The carboxyl-terminal 50 amino acids of hGRalpha have been replaced in hGRbeta with 15 unique amino acids. To investigate the effect this has on the ability of hGRbeta to bind hormone or antihormone, we transfected COS-1 cells with a hGRalpha expression vector (pCMVhGRalpha), a hGRbeta expression vector (pCMVhGRbeta), or the expression vector backbone (pCMV5). The cells were incubated with radiolabeled DEX, a synthetic glucocorticoid agonist, or radiolabeled RU486, a synthetic glucocorticoid antagonist. [^3H]Steroid-receptor complexes formed in vivo were then analyzed by sucrose density gradients. Showing the standard sedimentation profile for the 8 S unactivated GR(24) , hGRalpha binds both [^3H]DEX and [^3H]RU486 (Fig. 6A). In contrast, hGRbeta does not appear to bind either of these ligands, because the sedimentation profiles for the pCMVhGRbeta- and pCMV5-transfected cells are superimposable. Western blot analysis of the extracts analyzed for hormone binding demonstrates that both the 94-kDa hGRalpha and 90-kDa hGRbeta isoforms were synthesized at similar levels (Fig. 6B). Therefore, lack of steroid binding by hGRbeta is not due to insufficient expression of the hGRbeta protein. Interestingly, the hGRalpha expression vector does not contain the hGRbeta-specific sequences, yet a 90-kDa protein is observed in the pCMVhGRalpha-transfected cells (Fig. 6B). This protein, which cannot be hGRbeta, appears to be a degradation product or post-translational modification of the 94-kDa hGRalpha isoform.


Figure 6: In vivo ligand binding analysis of hGRbeta expressed in transfected COS-1 cells. A, COS-1 cells were transfected with equimolar amounts of pCMVhGRalpha, pCMVhGRbeta, or pCMV5 (mock) as described under ``Experimental Procedures'' and incubated with 100 nM [^3H]dexamethasone (upper panel) or 50 nM [^3H]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 hGRalpha and 90-kDa hGRbeta proteins. Molecular mass standards are indicated in the left margin.



Subcellular Distribution of hGRbeta

Numerous studies have shown that hGRalpha resides in the cytoplasm of cells in the absence of hormone and translocates to the nucleus in a hormone-dependent manner(11) . To determine where hGRbeta is localized within a given cell, COS-1 cells were transiently transfected with the hGRalpha or hGRbeta expression vectors. The plasmids were delivered into the cells using either the DEAE-dextran or calcium phosphate transfection method to rule out potential artifacts due to the transfection procedure. After treating the cells for 2 h with or without DEX, immunohistochemistry was performed, and immunoreactivity was visualized by staining with Texas red fluorescent dye or avidin-biotin-peroxidase. In the absence of hormone hGRalpha is found in the cytoplasm of the transfected cells, but following hormone administration it translocates and becomes predominantly nuclear (Fig. 7, left panel). In contrast, the hGRbeta protein resides primarily in the nucleus of the transfected cells independent of hormone treatment (Fig. 7, right panel). These findings were consistent regardless of the transfection method or immunohistochemical staining procedure employed.


Figure 7: Subcellular distribution of hGRbeta expressed in transfected COS-1 cells. COS-1 cells were transfected with equimolar amounts of pCMVhGRalpha (left panel) or pCMVhGRbeta (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 hGRbeta in the Absence of hGRalpha

hGRbeta does not appear to bind hormone or antihormone, but it resides primarily in the nucleus of transfected COS-1 cells (and CV-1 cells) (^2)and has been reported to bind a consensus GRE in vitro(17) . Therefore, we investigated the ability of hGRbeta to activate or repress the glucocorticoid-responsive MMTV enhancer in the presence or the absence of steroid. For these experiments, COS-1 cells were cotransfected with an MMTV-CAT reporter plasmid (pGMCS) and either pCMV5, pCMVhGRalpha, or pCMVhGRbeta. In response to DEX, hGRalpha induces a 4-fold increase in CAT expression over that observed in the vehicle-treated cells, whereas RU486 alone has little or no effect (Fig. 8A, lanes 4-6). In contrast, CAT expression is unchanged in the hGRbeta-containing cells treated with steroid, which is consistent with the inability of hGRbeta to bind these ligands (Fig. 8A, lanes 7-9). Moreover, hGRbeta does not appear to be constitutively active as an enhancer or repressor because levels of CAT activity in the vehicle-treated cells are similar whether they contain the CMV-vector backbone or pCMVhGRbeta (Fig. 8A, compare lanes 1 and 7).


Figure 8: Transcriptional activity of hGRbeta in the absence of hGRalpha. COS-1 (A) or CV-1 (B) cells were cotransfected with pGMCS (5.0 µg) and equimolar amounts of pCMV5 (2.8 µg), pCMVhGRalpha (5.5 µg), or pCMVhGRbeta (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 hGRalpha-mediated induction of CAT activity in transfected COS-1 cells was only 4-fold. In cell lines where hGRalpha inductions are much greater, hGRbeta might display partial transcriptional activity. Therefore, we investigated the transcriptional activity of hGRbeta on the MMTV enhancer in receptor negative CV-1 cells. In response to DEX, hGRalpha induces a 74-fold increase in CAT expression (Fig. 8B, lanes 4 and 5). Interestingly, hGRalpha 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 hGRbeta and treated with steroid (Fig. 8B, lanes 7-9). In addition, hGRbeta does not appear to be constitutively active (Fig. 8B, compare lanes 1 and 7). Thus, we conclude that in the absence of hGRalpha, hGRbeta is transcriptionally inactive on the glucocorticoid-responsive MMTV enhancer.

Transcriptional Activity of hGRbeta in the Presence of hGRalpha

In the absence of hGRalpha, hGRbeta is unable to directly enhance or repress transcription of the MMTV-CAT reporter plasmid. To determine if hGRbeta influences gene expression indirectly by modulating the transactivation capacity of hGRalpha, HeLa S(3) cells (which contain approximately 20,000 hGRalpha receptors per cell) were cotransfected with pGMCS and various amounts of pCMVhGRbeta (Fig. 9A). When pGMCS alone is transfected into the cells, DEX treatment results in a 50-fold induction of CAT activity. However, as increasing amounts of pCMVhGRbeta are transfected into the cells, the glucocorticoid-induced, hGRalpha-mediated activation of the MMTV enhancer is repressed in a dose-dependent manner. We observe a 78 and 96% inhibition of CAT expression when 1.0 and 3.0 µg, respectively, of pCMVhGRbeta is used in the transfection mixture. Transfection of a 2-fold molar excess of the CMV-vector backbone (pCMV5) has no effect on hGRalpha-mediated induction of CAT expression, indicating that hGRbeta is responsible for the repression.


Figure 9: Transcriptional activity of hGRbeta in the presence of hGRalpha. HeLa S(3) cells were cotransfected with 5.0 µg of pGMCS (A) or pGRE2CAT (B) and various amounts of pCMV5 and pCMVhGRbeta 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 hGRbeta on hGRalpha-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(3) cells are cotransfected with pGRE2CAT and various amounts of pCMVhGRbeta (Fig. 9B), the hGRalpha-mediated stimulation of CAT expression is inhibited in a dose-dependent manner, and the repression of hGRalpha activity is similar to that observed on the MMTV promoter. The inhibitory action of hGRbeta is restricted to glucocorticoid-responsive promoters because the hGRbeta 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 hGRbeta represses the function of hGRalpha by specifically inhibiting GRE-mediated transcription.


DISCUSSION

Alternative splicing of the hGR primary transcript produces two highly homologous isoforms, termed hGRalpha and hGRbeta, which differ at their carboxyl termini(1, 2) . In contrast to the well characterized hGRalpha isoform, very little is known about the hGRbeta splice variant. In this report, we examine the expression, biochemical properties, and physiological function of hGRbeta. Northern blot analysis with a hGRbeta-specific riboprobe demonstrates the presence of a message approximately 4.3 kb in size (consistent with the length of the hGRbeta cDNA) in many different human tissues. We subsequently confirmed by RT-PCR that the alternative splicing event proposed to underlie the formation of the hGRbeta mRNA transcript occurs in these tissues as well as in several transformed human cell lines. Together, these results indicate that the hGRbeta message is endogenous to a variety of cells. Because both the hGRalpha and hGRbeta messages are co-expressed in many of the same tissues, previous studies investigating hGRalpha expression with probes that did not discriminate between hGRalpha and hGRbeta may be in error. The hGRbeta mRNA transcript generated in vivo from the pCMVhGRbeta expression vector is efficiently translated into the 90-kDa hGRbeta protein in transfected COS-1, CV-1, and HeLa S(3) cells, suggesting that the endogenous hGRbeta message can also serve as a template for protein synthesis. However, whether the endogenous hGRbeta message is actually translated into the hGRbeta isoform is unknown. Anti-hGR antibodies made to date in several different laboratories have epitopes in the amino terminus and thus recognize both the hGRalpha and hGRbeta proteins. The small difference in size between the two isoforms and the potential for hGRalpha to be post-translationally modified or degraded into a smaller protein make this cross-reactivity undesirable. To test directly for the expression of the hGRbeta protein, we are presently making a hGRbeta-specific antibody.

During our investigation of the expression of the hGRbeta message, we made several observations that provide new insights both into the structure of the hGR gene and hGRalpha mRNAs and into the expression of the hGRalpha 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, 9alpha and 9beta, 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 hGRalpha 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 hGRalpha- and hGRbeta-specific riboprobes with the 7.0- and 5.5-kb hGRalpha 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 hGRalpha messages may differ in one or more of these properties. Both hGRalpha messages have a widespread tissue distribution, although the 5.5-kb hGRalpha 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 hGRalpha messages are much more abundant than the 4.3-kb hGRbeta 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 hGRalpha. However, because alternative splicing is often regulated in a spatial and/or temporal fashion, the hGRbeta message may be expressed at higher levels in a tissue-specific and/or developmental stage-specific manner. In addition, the hGRalpha/hGRbeta mRNA ratio may or may not reflect the hGRalpha/hGRbeta protein ratio due to potential differences in stability and/or translation efficiency of the hGRalpha and hGRbeta messages and/or due to differences in protein half-life. Furthermore, because the 7.0-kb hGRalpha message has the 9beta sequences at its far 3`UTR, we cannot exclude the possibility that it also encodes the hGRbeta protein. Development of hGRalpha- and hGRbeta-specific antibodies will provide insight into this issue. Interestingly, the 9beta sequences are well conserved in the rat GR cDNA 3`UTR, suggesting that an hGRbeta 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.^2 Thus, although the hGRbeta message is expressed at low levels relative to the hGRalpha 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 hGRalpha have been replaced in hGRbeta 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 hGRbeta 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 hGRbeta to bind RU486 but found no evidence of binding. The 15 amino acids at the end of hGRbeta 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, hGRbeta is more aptly described as an orphan receptor whose natural ligand, if any, is unknown.

The hGRalpha 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 hGRalpha appears to inactivate the NLS(52, 53) . Once hormone binds hGRalpha, hsp90 dissociates from the receptor resulting in the activation of the NLS and subsequent nuclear import of hGRalpha(8) . In contrast to hGRalpha, we demonstrate that hGRbeta resides primarily in the nucleus of transfected cells independent of hormone treatment. The amino acids necessary for interacting with hsp90 (7) are present in hGRbeta, suggesting that hGRbeta may be in the nucleus in spite of its association with hsp90. Perhaps the unique COOH-terminal amino acids of hGRbeta delete sequences that inhibit the NLS or slightly alter the tertiary structure of the hGRbeta/hsp90 complex such that the NLS are partially activated. This might account for our observation that most, but not all, of the hGRbeta protein is located in the nucleus. Further studies will be required to elucidate the precise mechanism(s) underlying the nuclear distribution of hGRbeta.

In the absence of hGRalpha, the hGRbeta isoform is transcriptionally inactive on the MMTV enhancer independent of steroid treatment. However, when hGRalpha and hGRbeta are expressed in the same cell, hGRbeta inhibits the glucocorticoid-induced, hGRalpha-mediated activation of the MMTV promoter. Although this dominant negative effect was first reported in COS-7 cells cotransfected with hGRalpha and hGRbeta 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 hGRalpha receptors. In addition, we show that the repression of hGRalpha 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 hGRbeta-mediated repression of hGRalpha activity are unknown. The hGRbeta 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 hGRalpha for binding to the GRE. Another possibility is that hGRbeta forms a heterodimer with hGRalpha that is transcriptionally inactive or less active than an hGRalpha homodimer. Alternatively, the hGRbeta isoform may inhibit the function of hGRalpha by interacting with and titrating out an essential cofactor needed by hGRalpha for full transcriptional activity. We are currently trying to identify the mechanism(s) responsible for the dominant negative activity of hGRbeta and to determine whether hGRbeta can inhibit hGRalpha-mediated repression of gene expression. Moreover, we hope to discern if hGRbeta 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 hGRalpha and hGRbeta is the processing that occurs at the thyroid hormone receptor alpha subunit (TRalpha) locus. Alternative splicing of the last exon generates two receptor isoforms, TRalpha1 and TRalpha2, that differ at the carboxyl terminus(36) . The TRalpha2 isoform does not bind thyroid hormones, but it represses the transcriptional activity of TRalpha1 by competing with TRalpha1 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.

(^1)
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; TRalpha, thyroid hormone receptor alpha; RT, reverse transcriptase; PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair(s).

(^2)
R. H. Oakley and J. A. Cidlowski, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Ron Evans for the hGR clones OB7 and OB10 and Dr. Darryl Zeldin for human liver and lung total RNA. We also appreciate the technical assistance of Roger Componovo.


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