Specific modulation of estrogen receptor mRNA isoforms in rat pituitary throughout the estrous cycle and in response to steroid hormones

Molecular and Cellular Endocrinology 131 (1997) 147 – 155

Specific modulation of estrogen receptor mRNA isoforms in rat pituitary throughout the estrous cycle and in response to steroid hormones Keith E. Friend 1,a, Eileen M. Resnick b, Le W. Ang a

1,a

, Margaret A. Shupnik a,*

Di6ision of Endocrinology and Metabolism and Department of Internal Medicine, Uni6ersity of Virginia Health Sciences Center, Charlottes6ille, VA 22903, USA b Department of Pharmacology, Uni6ersity of Virginia Health Sciences Center, Charlottes6ille, VA 22903, USA Received 17 February 1997; accepted 9 May 1997

Abstract We have identified several estrogen receptor (ER) mRNA isoforms in rat pituitary and characterized their regulation by gonadal steroids. The ER mRNAs correspond to splice variants in which either exon 4, exons 3 and 4, or exons 5 and 6 are deleted. A previously isolated pituitary-specific truncated mRNA, TERP-1, containing a unique 5%-end and exons 5 through 8 of the full-length ER, was also studied. The exon deletion variants were expressed in males and females, in pituitary, uterus, testes, heart, hypothalamus, and liver. An antibody to the ER C-terminus bound to full-length (64 kDa) and smaller (50 – 55 kDa and 40–45 kDa) ER proteins in uterus and pituitary and a pituitary-specific ER of 20 – 24 kDa corresponding to TERP-1. Estrogen (E) treatment in vivo stimulated full-length ER 2–3-fold, and TERP-1 7 – 10-fold, but had no effect on any exon deletion variant. Progesterone treatment, alone or with E, had no consistent effect on any ER mRNA form. TERP-1 mRNA was also dramatically and specifically modulated during the estrous cycle, increasing :500-fold between the morning of diestrous and the afternoon of proestrus. Thus, ER mRNA variants exist in estrogen-responsive tissues; the pituitary contains at least one tissue-specific ER which is regulated by steroids and which may contribute to changes in regulated biological activity. © 1997 Elsevier Science Ireland Ltd. Keywords: Estrogen receptor; mRNA isoforms; RNase protection assays; Estrous cycle

1. Introduction Estrogen plays a critical role in sexual differentiation and fertility, and in the growth and development of reproductive tissues. Like other steroid hormones, estrogen (E) exerts its biological actions by binding to specific nuclear proteins called estrogen receptors (ERs) (Evans, 1988; Fuller, 1991). ERs are ligand-activated regulatory proteins which act as dimers on specific * Corresponding author. Present address: Box 578 HSC, University of Virginia, Charlottesville, VA 22908, USA. Tel.: +1 804 9820010; fax: + 1 804 9820088. 1 Present address: Endocrine Section, M.D. Anderson Hospital, Houston, TX, USA.

target genes containing defined DNA sequences called estrogen regulatory regions, to stimulate or suppress the transcription of E-sensitive genes. ER proteins contain several discrete functional domains. These include an N-terminal ligand-independent transactivation function (AF-1), a central DNA binding domain, and a C-terminal region which contains sequences for hormone binding and a second transactivation function (AF-2) which is ligand-dependent. Sequences necessary for nuclear localization and dimerization are contained in both the DNA-binding and hormone-binding domains. The two transactivation functions have activities which vary with promoter and cell context, and many cellular proteins including various cofactors, corepres-

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sors, and integrator proteins can act within cells to modify the physiological activities of the steroid receptors (Tora et al., 1989; Horwitz et al., 1996; Onate et al., 1995). Other sources of biological diversity include ER isoforms or variants in a variety of cells and tissues, particularly in breast cancer cell lines and tumors (Wang and Miksicek, 1991; Fuqua et al., 1991; Garcia et al., 1988; McGuire et al., 1991). Many variants arise from alternative splicing and exon deletions, resulting in ER proteins lacking specific functional domains which have dominant negative or constitutively active effects in transfected cell lines (Wang and Miksicek, 1991; Fuqua et al., 1991). ERs with single nucleotide mutations with both unchanged and altered hormonal sensitivity also occur (Garcia et al., 1988; McGuire et al., 1991). Although the physiological significance of these ERs is unclear, variant ERs have been proposed to explain some ER positive, progesterone receptor (PR) negative and ER negative, PR positive breast cancers, as well as inappropriate responses to antiestrogen therapy (McGuire et al., 1991). Additional genomic alleles, and alternative transcriptional start sites have also recently been identified for human and rodent ER. A second functional ER was isolated from rat prostate and found in other rat and human reproductive tissues (Kuiper et al., 1996). This ERb has only moderate sequence homology with the previously described ER, and would not be identified by most RNA detection methods with probes for the previously identified rat ER (Koike et al., 1987). Alternative transcriptional start sites with distinctive ER exon 1 sequences for the ER have been identified in normal human uterus; the resultant proteins are identical (Keaveney et al., 1991). Our laboratory previously described a novel ER mRNA from female rat pituitaries (Shupnik et al., 1989), TERP-1 (truncated estrogen receptor product), which is transcribed from a start site separate from the full-length ER, encodes an N-terminal truncated ER protein, and is stimulated by E (Friend et al., 1995). In this report, we have identified and characterized three splice variants of ER mRNA from rat pituitary which have the capability to encode ER proteins of potentially altered biological function. These variants are all more widely expressed than TERP-1 mRNA. There is only moderate steroid regulation of the splice variant mRNAs, but TERP-1 mRNA is dramatically regulated by estrogen and throughout the estrous cycle. Immunopositive ER proteins of several sizes occur in uterine and pituitary tissue. These studies demonstrate that several variant forms of ER, some regulated by steroids, exist in reproductive tissues. Such modulation could result in a changing profile of ER with different biological functions.

2. Materials and methods

2.1. RNA isolation and ER cDNA cloning Total RNA was isolated from rat tissues by homogenization in guanidinium thiocyanate-containing buffers and centrifugation through cesium chloride gradients (Shupnik et al., 1989). The cloning of the TERP-1 cDNA by 5% rapid amplification of cDNA ends (RACE) anchored polymerase chain reaction (PCR) techniques has previously been described (Friend et al., 1995). Cloning of the ER mRNA splice variants was performed by reverse transcriptase (RT)-PCR techniques using 2 mg of total RNA from female rat pituitary and random primers for first strand cDNA synthesis. ER-specific primers were then used to amplify and isolate specific ER regions in two sets of nested reactions. The exon 4 and 3,4 deletion clones were obtained by performing a 35 cycle amplification reaction with the first primer pair (Upstream primer 5%-GTCTGGTCCTGTGAAGGCTGCAA-3%, aa 204– 213 and downstream primer 5%-AGGAGCAAACAGGAGCTTCCC-3%, aa 405–411). Products from this reaction were diluted 1/2000 and used in the second set of amplifications using the next primer set (Upstream primer 5%-CTGCAAGGCTTTCTTTAAGAG-3%, aa 209–217, and down stream primer 5%-TCATCAGGATCTCCAACC-3%, aa 388–394). For the exon 5,6 deletion mRNA the first round of synthesis was performed with the first primer set (upstream 5%GATCCTTCTAGACCCTTC-3%, aa 337–342 and downstream 5%-CTTCCAGAGACTTCAAGG-3%, 470– 476), and the products diluted as above for the second round of synthesis with the second primer set(upstream 5%-CTTATTGACCAACCTGGC-3%, aa 349– 355, and downstream 5%-GGTGCTGGATAGAAATGTG-3%, aa 464–470). The resultant PCR products were cloned into the vector pSPORT (GIBCO/BRL) and the DNA sequenced by the dideoxy termination method of Sanger (Sanger et al., 1997).

2.2. Variant ER mRNA detection by polymerase chain reaction assays To determine if specific ER mRNA variants were present in various tissues, total tissue mRNA (2 mg) was subjected to RT-PCR analysis as above, except that only 25 cycles of amplification were performed. The resultant PCR products were displayed on 1.5% agarose TBE (88 mM Tris, pH 8.0, 50 mM boric acid, 2 mM EDTA) gels containing ethidium bromide. Bands were visualized under ultraviolet light. In some cases, DNA was transferred to nitrocellulose and hybridized with 32P-labelled probes specific for ER coding sequence. A probe to the entire rat ER coding sequence cDNA, labeled by random priming (Feinberg and

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Vogelstein, 1983), or oligonucleotides corresponding to specific exon sequences were end-labeled by T4 polynucleotide kinase (Friend et al., 1995). Hybridizations were performed as previously described in Section 2. Negative controls included RNA samples without RT, lung RNA as an ER negative tissue, and RNA from a stable ER negative cell line stably transfected with a cDNA for the entire ER cDNA coding region. In the last case, splicing could not occur, and this RNA should only demonstrate the presence of full-length ER mRNA.

2.3. Variant ER mRNA quantitation by RNase protection assays Specific ER mRNA variant forms were quantitated in pituitary mRNA preparations by RNase protection analysis. Labeled single-stranded RNA probes were synthesized from linearized plasmids containing specific ER cDNA forms, using either SP6 or T7 RNA polymerase and [a-32P]UTP (400 – 800 Ci/mmol), using reagents from Ambion technologies (Friend et al., 1994). All probes were gel purified. RNA samples (25 mg) were hybridized overnight at 45°C with radiolabeled probe (25 – 100 000 cpm, : 4 × 108 cpm/mg), digested with a combination of ribonuclease T1 and A, and subjected to electrophoresis at 250 V for 3 h on 8 M urea, 5% polyacrylamide gels. Gels were dried and exposed to Kodak X-OMP-AR film (Eastman Kodak, Rochester, NY) at − 70°C with an intensifying screen for 4 – 7 days. Films were analyzed with a Molecular Dynamics densitometer, and Image Quant 3.3 software. In most experiments, a probe for rat b-actin (rat TRI-actin, Ambion) was included in each reaction, and ER mRNA values were normalized for the amount of actin mRNA detected in the 125 base protected band. To detect TERP-1 mRNA, a previously described probe (Section 2) containing 27 bases of TERP-1-specific sequence and 73 bases of exon 5 sequence was used, resulting in a protected product of 100 bases for TERP-1 and 73 bases for full-length ER mRNA. For deletion variants, RNA probes were transcribed from cDNA plasmids representing the exon deletion variants as described in PCR reactions above. In each case, the probes resulted in a larger protected fragment for the exon deleted mRNA and a smaller fragment representing the full-length or exon-containing mRNA in the same assay. For the exon 4 deletion probe, protected fragments of 219 bases (exon 4 deletion) and 152 bases (full-length were obtained). For the exon 3,4 deletion variant probe, protected fragments of :107 bases (exon 3,4 deletion) and 70 bases (full-length) were obtained. The exon 5,6 deletion probe protected fragments of :90 bases (exon 5,6 deletion) and 64 bases (full-length) ER mRNA. A separate probe containing portions of exons 4 and 5 protects a 196 base ER mRNA fragment which represents the full-length ER mRNA exclusively, as previously described in Section 2.

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2.4. Immunoblots Total cellular pituitary and uterine protein was obtained by the direct homogenization of tissue in 50 mM tris(hydroxymethyl)aminomethane (pH 7.6) solution containing 2% sodium dodecyl sulfate (SDS). Protein samples (20–50 mg) were denatured by boiling in the presence of 2% b-mercaptoethanol and separated by electrophoresis on 12% polyacrylamide gels containing 1% SDS, transferred to nitrocellulose membranes, and ER detected with an antibody raised against amino acids 586–600 of the rat estrogen receptor sequence (C1355) by a commercial supplier (Macromolecular Resources, Fort Collins, CO). The peptide was chemically synthesized, conjugated with keyhole limpet hemocyanin, and injected into rabbits. The resulting polyclonal antibody was tested for binding specificity by its ability to recognize authentic in vitro translated ER, binding to recombinant transfected ER, and binding to a 64–66 000 Dalton peptide from immature rat uterine cytosolic protein on polyacrylamide SDS gels that was also recognized by previously defined antiestrogen receptor antibody to the hinge regions of the rat ER (ER715) (Furlow et al., 1990). To detect ER protein from tissues, blots were first incubated in a blocking solution containing 5% nonfat dry milk, 0.1% polyoxyethylene sorbitan monolaurate (TWEEN-20) phosphate buffered solution (pH 7.6) for 1–2 h followed by 1 h incubations with primary antibody (1:7500 dilution) and secondary antibody (1:100 dilution of peroxidase labeled donkey antirabbit IgG; Amersham) with washing as described (Furlow et al., 1990). Enhanced chemiluminescence detection was used to visualize bound proteins (ECL: Amersham).

2.5. Experimental animals Male and female CD-1 rats, 200–225 g, were obtained from Charles River Breeding Laboratories in accordance with the guidelines established by the University of Virginia. For steroid treatments, male or female ovariectomized rats (14 day post-ovariectomy) were injected for 3 days with vehicle (sesame oil), 17b-estradiol(20 mg/100 g body weight), progesterone (2 mg/100 g body weight, or a combination of both steroids (ten animals per group)). For estrous cycle experiments, intact female rats were maintained on a 14:10 h light:dark cycle, and estrous cycle stage was determined by vaginal lavage. Animals were required to have two normal consecutive cycles prior to use in the study, and each group contained 10–14 rats. Animals were killed at 09.00 and 17.00 h of each day, of estrus and proestrus. Total pituitary RNA was prepared from pooled tissue (five to eight rats) and used for RNase protection studies or RT-PCR. Quantitative data was obtained from normalized values for ER mRNA from two to six separate

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Fig. 1. (Left) Schematic representation of estrogen receptor mRNAs identified from female rat pituitary cDNAs. A portion of exons 1 and 8 contain untranslated mRNA at the 5% and 3% ends, respectively. The stippled box at the 5% end of the truncated ER mRNA, TERP-1, represents mRNA transcribed from a different transcriptional start site encoding an alternative first exon. (Right) Expression of ER mRNA splice variants in tissues of male (M) and female (F) rats. RT-PCR and electrophoresis of amplification products on ethidium bromide-containing agarose gels were performed as in Section 2. Tissue abbreviations are: P, pituitary; B, brain; Y, hypothalamus; L, liver; U, uterus; T, testes; H, heart. PCR products representing ER mRNA of full-length (FL), exon 4 deletion (d 4), exon 3,4 deletions (d 3,4), and exon 5,6 deletions (d 5,6) of ER mRNA are indicated. M1, or marker 1 is DNA from bacteriophage l digested with BsteI restriction endonuclease. M2, or marker 2, is a ligation ladder of progressive lengths of 123 bp DNA. The numbers of the left axis indicate the lengths of the amplification products.

experiments, with two to four samples in each group in each experiment. When calculating steroid effects on mRNA isoforms, normalized densitometric mRNA values from treated groups were compared with control groups by the Student’s t-test, comparing the mean 9 S.E.M. for at least six independent samples. For RTPCR studies, total RNA was prepared from individual tissues from male and female rats, and analysis was performed on individual RNA samples from four to six separate animals for each tissue and ER mRNA isoform.

3. Results

3.1. Isolation of 6ariant ER cDNAs ER mRNA splice variants were identified and cDNAs isolated from female rat pituitary tissue by RTPCR cloning techniques. Variants, depicted in Fig. 1, included mRNAs representing precise deletions of exon 4, exons 3 and 4, and exons 5 and 6. In each case, the mRNAs represented splicing within or between glycine residues and encoded potential ER proteins which

maintained the in-phase reading frame of the ER. RTPCR reactions using primers to the entire coding regions verified these deletion variants at very low levels, and did not identify additional variants. The exon 4 deletion variant appeared in every ER positive tissue examined from both male and female rats, including pituitary, brain, hypothalamus, liver, heart, uterus, ovary, and testes, while no PCR product for either full-length or exon 4 deletion mRNA was present in the ER negative tissue, lung, or in an ER negative cell line stably transfected with full-length ER cDNA (not shown). The exon 5,6 deletion appeared in all tissues but at very low levels in hypothalamus, and the exon 3,4 deletion variant was present in all tissues except heart. Estrogen treatment had no qualitative effect on the appearance of ER mRNA isoforms.

3.2. Detection of immunopositi6e ER forms Immunoblot analysis (Fig. 2) with a C-terminal specific ER antibody detected several proteins in homogenates of female rat uterus (64–66, 50–55, and 40–45 kDa apparent molecular weight) and pituitary (64–44, 40–45, and 20–24 kDa). Binding to all im-

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munopositive bands was eliminated when antibody was preincubated with antigen peptide (not shown). The heterogeneity of immunopositive proteins is unlikely to result from random proteolytic digestion, as tissue was homogenized in buffer containing detergent and protease inhibitors. The pituitary-specific band is of the predicted size and the expected tissue specificity for the TERP-1 protein (Friend et al., 1995). The 20 – 24 kDa protein is detected only with C-terminal, and not N-terminal or hinge region antibodies (Friend et al., 1995).

Fig. 3. RNase protection analysis of pituitary RNA from male and ovariectomized female rats injected with sesame oil control vehicle (C), estrogen (E), progesterone (P), or estrogen plus progesterone (E+ P). Each reaction contained total RNA and labeled RNA probes for actin and the ER exon 4 deletion variant. Samples of undigested probe are also shown. The actin probe protected a product of 125 bases, while the ER RNA protected products for the exon 4 deletion variant of 219 bases, and exon 4 containing mRNA of 152 bases (F/L ER).

3.3. Steroid regulation of ER mRNA forms

Fig. 2. Immunoblotting of ER proteins in tissue homogenates from female rat uterus (Ut, 20 mg) and pituitary (pit, 100 mg). Proteins were subjected to electrophoresis on a 12% SDS-containing polyacrylamide gel, and ER immunopositive proteins detected by binding to a C-terminal antibody to rat ER.

Possible steroid modulation of ER mRNA isoforms, including the previously identified truncated ER form TERP-1, was examined in pituitary tissue from both male and ovariectomized female rats treated with E, P, or E plus P. There was no significant consistent effect of steroid treatment on the exon 4 (Fig. 3), exon 5,6 (Fig. 4), or exon 3,4 (Fig. 5) deletion ER mRNA forms

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the estrous cycle (Fig. 6). No TERP-1 mRNA was detected on the evening of estrus, although the fulllength ER mRNA is present at this time. A small amount of TERP-1 mRNA is observed the morning of metestrus, with higher levels as the cycle progresses. Relative levels of TERP-1 mRNA to full-length ER mRNA also changes as a function of cycle stage. From the evening of estrus, when there is no detectable TERP-1, the ratio of TERP-1/ER rises until the morning of proestrus, when TERP-1 becomes the predominant receptor mRNA form.

4. Discussion

Fig. 4. RNase protection analysis of pituitary RNA from male and female rats treated with steroids as in Fig. 3. Each reaction contained total RNA and probes specific for actin (protected fragment of 125 bases) and ER mRNA exon 5,6 deletion (90 base protected fragment), with a 70 base protected fragment for full-length (F/L) ER mRNA.

in either males or females (Table 1). In contrast, E treatment stimulated levels of the full-length ER mRNA 2–3-fold (Table 1), and TERP-1 mRNA 7–10fold (Fig. 6 and Friend et al., 1995). P treatment had no significant effect, either alone or in combination with E, on full-length ER or TERP-1 mRNA levels. TERP-1 mRNA could also be modulated by changes in physiological steroid levels, as its expression varies throughout

Fig. 5. RNase protection analysis of pituitary RNA from steroid treated male and female rats hybridized with probes for actin (125 bases protected fragment) and a probe for the ER mRNA exon 3,4 deletion (107 base protected fragment), which also protects a fragment of 64 bases corresponding to full-length (F/L) ER mRNA.

These data demonstrate that several mRNAs encoding the rat ER appear in pituitary tissues in significant levels relative to the full-length mRNA. TERP-1 mRNA levels vary, but can be up to 2-fold above (at proestrus) that of the full-length ER mRNA. Estimated steady-state levels of mRNA exon deletion splice variants are : 10–25% that for the full-length mRNA. Previous blot hybridization studies had identified two hybridizing species of ER mRNA in female rat pituitaries (Shupnik et al., 1989; Friend et al., 1995). These included a broad band of : 6.2 kb which also appeared in RNA from male rat pituitaries, uteri, and liver. Because of the relatively small mRNA regions deleted in the variants, ranging from 273 to 453 bases, and the high degree of heterogeneity in polyadenylation at the 3% end of mRNA, this 6.2 kb band likely contains all the ER mRNA exon deletion variants as well as the full-length species. The second ER mRNA band was :5.2–5.5 kb in size and specific for female pituitary RNA (Shupnik et al., 1989; Friend et al., 1995). Our laboratory subsequently cloned and characterized the major mRNA transcript in this band as TERP-1 (truncated estrogen receptor product), and found it contains a unique 5% region indicating an alternate transcriptional start site and exon 1, and sequence corresponding to exons 5 through 8 of the full-length ER mRNA (Friend et al., 1995). We previously hypothesized that an internal in-phase initiation site of translation in TERP-1 mRNA could result in a truncated ER protein of :20 kDa molecular weight, and the presence of a strong immunopositive protein band at 20–24 kDa in female pituitary but not uterine tissue extracts in Fig. 3 suggests that TERP mRNA can be translated to make significant levels of protein in rat pituitary tissue. Because TERP-1 mRNA has a unique 5% end, indicating a transcriptional start site distinct from that of the fulllength ER mRNA, the promoter for the TERP-1 transcript may also contain distinct regulatory elements which may account for its dramatic and divergent regulation throughout the estrous cycle.

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Table 1 Steroid regulation of ER mRNA variants in male and female rat pituitaries ER form

Exon 4 deletion Exon 3,4 deletion Exon 5,6 deletion Full length

Males (Treatment)

Females (Treatment)

Con

E

P

E+P

Con

E

P

E+P

2.829 0.58 0.689 0.16 0.99 0.20 1.00

2.079 0.50 0.999 0.34 0.939 0.24 1.969 0.27b

2.2790.50 1.19 0.41 1.09 0.28 1.519 0.36

2.44 90.44 0.99 90.33 0.92 90.16 1.49 9 0.18a

3.6 9 1.7 0.799 0.18 0.96 9 0.29 1.0

6.6 9 2.9 0.90 9 0.25 1.35 91.0 2.75 9 1.0b

2.9 90.7 0.46 90.10a 1.41 9 0.80 1.89 9 1.41

2.0 9 0.7 1.1 9 0.39 1.70 90.24a 3.31 91.4b

Data are presented as the mean9 S.E.M. of densitometric values for each mRNA form normalized for actin controls from 4 – 5 gels. For the full length ER mRNA, values are expressed relative to control values of 1.0 to compare results from all experiments. a PB0.05 versus control. b PB0.01 versus control.

Other investigators have verified the presence of the 5.2 – 5.5 kb ER mRNA encoding TERP-1 in female rat pituitaries by blot hybridization analysis (Demay et al., 1996). They found detectable levels of TERP-1 mRNA only in pituitaries of female rats who were in the proestrus (Demay et al., 1996), in general agreement with our finding of the highest levels of TERP-1 mRNA on this day. The difference in detection on other days may lie in the greater sensitivity of solution hybridization and RNAse protection assays compared with blot hybridization. These investigations also examined pituitary cells separated by gradient elutriation and found that the TERP-1 (i.e. 55 kb ER mRNA)/full length ER mRNA ratio was much greater in enriched lactotropes relative to either gonadotropes or to the general pituitary cell population (Demay et al., 1996). At present, we have not verified which cell type contains TERP-1 mRNA. However, several studies suggest that the lactotropes and gonadotropes both contain significant levels of ER (Friend et al., 1994; Demay et al., 1996; Geffroy-Roisne et al., 1992; Keefer et al., 1976; Zafar et al., 1995). Heterogeneity in ER levels, size of ER protein (Geffroy-Roisne et al., 1992; 1993), and response of various cell types to estrogen have also been noted. For example, progesterone receptor is contained primarily in gonadotrope cells, and appears to be stimulated by E only in gonadotropes (Fox et al., 1990; Sprangers et al., 1991). Although the full biological actions of TERP-1 have not been fully defined, we have preliminary information which suggests that TERP-1 can augment the stimulatory transcriptional effects of the full-length ER in transfection studies (Schreihofer, D.A., Pace, C., Friend, K.E. and Shupnik, M.A.; 10th International Congress on Endocrinology, San Francisco, CA, 1996, Abstract OR51-7). Similar transcriptional stimulation has been noted with a C-terminal portion of the progesterone receptor (Wei et al., 1996). Since both full-length ER and TERP-1 are stimulated by E-treatment, this

may represent a mechanism for pituitary cells to increase their sensitivity to E. The splice variant mRNAs do not display gender or tissue specificity or significant regulation by gonadal steroids. All three splice variant ER mRNAs have in-frame deletions of the coding region that would result in proteins translated in phase. Because of the modular nature of the ER, deletions would be expected to have defined effects on protein biological activity. Exon 3 (117 bases) encodes a portion of the DNA binding domain, exon 4 (336 bases) encodes portions of the DNA binding, hinge and hormone binding domains, and exons 5 and 6 (273 bases total) portions of the hormone binding and dimerization domains. An exon 3,4 deletion protein is not likely to bind DNA or influence gene transcription directly. Recently, ER mRNA lacking exons 3 and 4 has been identified in both normal uterine tissue and uterine cancer tissue (Hu et al., 1996), but biological activity of the protein was not examined. Other investigators have identified the exon 4 deletion mRNA in rat and lizard brain (Skipper et al., 1993) and in MCF7 cells (Koehorst et al., 1994). While the exon 5,6 deletion protein could not bind hormone, it could bind DNA. ER proteins lacking exon 5 have been reported to have constitutive activity (Fuqua et al., 1991), but the exon 5,6 deletion variant has not been studied. Other groups have demonstrated the existence of smaller ER proteins in rat pituitaries, including a 50 kDa species which binds E from male pituitaries (Geffroy-Roisne et al., 1992; 1993) and an : 50–55 kDa species containing exon 4 (Furlow et al., 1990). We have observed 55 and 40–45 kDa ER species in uterine and pituitary extracts, but have not conclusively identified these proteins as splice variants. Although the exon deletion ER mRNA forms do not exhibit steroid regulation, it is possible that under other physiological conditions they may contribute to the response to E. ER actions can differ dramatically between cell types (Webb et al., 1995; Montano et al.,

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Fig. 6. RNase protection analysis of pituitary RNA from intact female rats as a function of estrous cycle stage (left panel) or in ovariectomized female rats treated with steroids (right panel). The TERP-l-specific probe protects a 100 base fragment corresponding to TERP-1 mRNA and a 73 base fragment corresponding to full-length ER mRNA.

1995), at least in part due to different complements of accessory proteins which interact with separate and distinct functional domains of the receptors (Evans, 1988; Fuller, 1991; Tora et al., 1989). ER proteins which have differential ability to interact with these factors may contribute to the specificity and variation in the biological response.

Acknowledgements This work was supported by the National Institutes of Health grants RO1-HD25719 (M.A.S.) and K08HD00982 (K.E.F), training grant T32-GM07055 (E.M.R) and the Center for Cellular and Molecular Studies in Reproduction at the University of Virginia (P30-HD28934).

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