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Molecular Endocrinology 13 (1): 117-128
Copyright © 1999 by The Endocrine Society

Interaction of the Putative Androgen Receptor-Specific Coactivator ARA70/ELE1{alpha} with Multiple Steroid Receptors and Identification of an Internally Deleted ELE1ß Isoform

Philippe Alen, Frank Claessens, Erik Schoenmakers, Johannes V. Swinnen, Guido Verhoeven, Wilfried Rombauts and Ben Peeters

Division of Biochemistry (P.A., F.C., E.S., W.R., B.P.) and Laboratory for Experimental Medicine and Endocrinology (J.V.S., G.V.) Faculty of Medicine, Campus Gasthuisberg University of Leuven B-3000 Leuven, Belgium


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid-regulated gene transcription requires the coordinate physical and functional interaction of hormone receptors, basal transcription factors, and transcriptional coactivators. In this context ARA70, previously called RFG and ELE1, has been described as a putative coactivator that specifically enhances the activity of the androgen receptor (AR) but not that of the glucocorticoid receptor (GR), the progesterone receptor, or the estrogen receptor (ER). Here we describe the cloning of the cDNA for ELE1/ARA70 by RT-PCR from RNA derived from different cell lines (HeLa, DU-145, and LNCaP). In accordance with the previously described sequence, we obtained a 1845-bp PCR product for the HeLa and the LNCaP RNA. Starting from T-47D RNA, however, an 860-bp PCR product was obtained. This shorter variant results from an internal 985-bp deletion and is called ELE1ß; accordingly, the longer isoform is referred to as ELE1{alpha}. The deduced amino acid sequence of ELE1{alpha}, but not that of ELE1ß, differs at specific positions from the one previously published by others, suggesting that these two proteins are encoded by different nonallelic genes. ELE1{alpha} is expressed in the three prostate-derived cell lines examined (PC-3, DU-145, and LNCaP), and this expression is not altered by androgen treatment. Of all rat tissues examined, ELE1{alpha} expression is highest in the testis. This is also the only tissue in which we could demonstrate ELE1ß expression. Both ELE1{alpha} and ELE1ß interact in vitro with the AR, but also with the GR and the ER, in a ligand-independent way. Overexpression of either ELE1 isoform in DU-145, HeLa, or COS cells had only minor effects on the transcriptional activity of the human AR. ELE1{alpha} has no intrinsic transcription activation domain or histone acetyltransferase activity, but it does interact with another histone acetyltransferase, p/CAF, and the basal transcription factor TFIIB. The interaction with the AR occurs through the ligand-binding domain and involves the region corresponding to the predicted helix 3. Mutation in this domain of leucine 712 to arginine greatly reduces the affinity of the AR for ELE1{alpha} but has only moderate effects on its transcriptional activity. Taken together, we have identified two isoforms of the putative coactivator ARA70/ELE1 that may act as a bridging factor between steroid receptors and components of the transcription initiation complex but which lack some fundamental properties of a classic nuclear receptor coactivator. Further experiments will be required to highlight the in vivo role of ELE1 in nuclear receptor functioning.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The androgen receptor (AR) is a member of the superfamily of nuclear receptors (NR), which includes the receptors for the other steroid hormones, for thyroid hormones, retinoids, and vitamin D (1, 2, 3, 4). The NRs constitute a large group of evolutionarily related proteins with a common structural organization, yet diverse physiological functions. Apart from their role in embryonic development, metabolic homeostasis, sex determination and differentiation, and fertility, they are also implicated in a variety of pathologies, including cancer.

NRs are ligand-inducible transcription factors with a modular structure, encompassing an N-terminal transactivating domain, a centrally located DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD). They have two transcription activation functions, AF1 and AF2 (3, 4). AF1 is located in the N-terminal domain and can activate transcription constitutively (5, 6), whereas the AF2 function colocalizes with the LBD and requires the addition of ligand (7). The crystal structures of the LBDs of unliganded human retinoid X receptor-{alpha} (hRXR{alpha}) (8), of ligand-bound human retinoic acid receptor-{gamma} (hRAR{gamma}) (9), thyroid hormone receptor (TR) (10), and progesterone receptor (PR) (11) and of agonist and antagonist-bound estrogen receptor (ER) (12) revealed a common overall layered structure consisting of 12 {alpha}-helices. The most remarkable difference between free and ligand-bound receptors is the position of the most C-terminally located helix 12 (H12), which contains the core AF2-domain. In the absence of ligand, H12 is projected away from the hydrophobic ligand-binding pocket. Binding of ligand repositions this helix, thereby closing the hydrophobic core as a lid (13).

Although direct interactions between NRs and components of the basal transcription machinery have been described (14, 15, 16, 17), they cannot fully explain the ligand-dependent transactivation of AF2. Therefore, another class of proteins called coactivators must be taken into account (18, 19). A number of candidate NR coactivators have been described, including RIP140 (20, 21), TIF1 (22), TRIP-1/SUG1 (23, 24), and a family of related 160-kDa proteins comprising SRC-1 (25, 26, 27), GRIP1/TIF2 (28, 29, 30), and RAC3/AIB1/ACTR/TRAM-1/p/CIP (31, 32, 33, 34, 35). While the functions of RIP140, TIF1, and TRIP-1/SUG1 remain to be established, the p160 proteins exhibit all the characteristics expected for NR coactivators: 1) they interact with the LBD of NRs in a ligand-dependent and AF2 integrity-dependent way both in vivo and in vitro through a conserved {alpha}-helical LXXLL-motif (27, 36, 37, 38, 39, 40); 2) upon cotransfection in mammalian cells they potentiate the ligand-dependent transcriptional activity of several NRs; 3) they harbor autonomous transcription activation functions; and 4) they have intrinsic histone acetyltransferase (HAT) activity and/or interact with yet other HATs such as CREB-binding protein (CBP) and p300/CBP-associated factor (p/CAF). Thus, apart from the recruitment of basal transcription factors to the promoter and the formation of a stable preinitiation complex, steroid receptor transactivation of target genes in vivo also involves chromatin remodeling, probably through targeted histone acetylation by the recruited coactivators (41).

Yeh and Chang (42) reported the first receptor-specific coactivator, ARA70 (70 kDa AR-activator), which stimulates the transcriptional activity of the AR but not that of the glucocorticoid receptor (GR), the PR, or the ER. This protein had originally been identified by others in thyroid cancer cells and named RFG (43) and ELE1 (44, 45). In many cases of thyroid papillary carcinoma, the RET protooncogene, a transmembrane receptor of the tyrosine kinase family, is found activated, due to chromosomal rearrangements resulting in recombinant genes. One such recombination, caused by a paracentric inversion within band q11.2 of chromosome 10 (45), leads to the fusion of the genomic region encoding the RET tyrosine kinase (TK) domain with the 5'-terminal region of the ele1 gene (43, 44). The resulting protein, called RET/PTC3, is expressed under the control of the ele1 gene promoter and consists of the first 238 amino acids (aa) of ELE1 fused to the RET TK-domain.

The goal of our study was to characterize the putative AR-specific coactivator properties of ARA70/RFG/ELE1. Since our results indicate that it lacks some of the fundamental properties of a bona fide NR coactivator, we prefer the nomenclature ELE1 to ARA70.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
cDNA Cloning
To obtain the ELE1 cDNA, we performed RT-PCR on total RNA from HeLa cells, LNCaP cells, and T-47D cells using primers based on the sequence published by Yeh and Chang (42). For the HeLa and LNCaP RNA, a cDNA of the expected 1845 bp was obtained, but surprisingly, the T-47D RNA generates a 860-bp cDNA (Fig. 1AGo). The full-length cDNA will further be referred to as ELE1{alpha}; accordingly, the shorter variant will be called ELE1ß. Sequence analysis revealed that ELE1ß results from an internal 985-bp deletion from nucleotides 790-1674 (the A of the ATG translation startcodon was arbitrarily given the number +1). This deletion does not cause a frame shift but results in a cDNA encoding a protein of approximately 35 kDa, and the 238 aa before the deletion correspond to the ELE1-derived fragment in the RET/PTC3 fusion protein (43). Moreover, Q238 is the last residue encoded by exon 5 of the ELE1 gene (46) (Fig. 1BGo). Remarkably, the ELE1{alpha} cDNA clones derived from HeLa RNA, as well as those from LNCaP RNA, contain five base substitutions (see Table 1Go) as compared with the sequence published by Yeh and Chang (42) and by Santoro and co-workers (43). We have analyzed several ELE1{alpha} clones from different PCR reactions and consistently found the same mutations, irrespective of the RNA or the polymerase used (Taq or Pfu). Furthermore, in some clones amplified with Taq polymerase but not in those obtained with the Pfu polymerase, which has a lower error rate, we found additional random mutations that only occurred in single clones and are therefore likely due to misreading by the polymerase. In the ELE1ß cDNA, however, we did not find the mutations listed in Table 1Go.



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  		Figure 1. ELE1 Is Expressed as at Least Two Different Isoforms, ELE1{alpha} and ELE1ß

A, Total RNA from T-47D cells, HeLa cells, and LNCaP cells was reverse transcribed as indicated in Materials and Methods. The first strand cDNA was subsequently PCR-amplified with two ELE1-specific primers. The reaction products were separated by electrophoresis in a 1% agarose gel and visualized by ethidium bromide staining. The length (in basepairs) of the bands in the 1-kb DNA ladder (lane M) is depicted on the right. Arrowheads indicate the positions of the 1845-bp ELE1{alpha} and the 860-bp ELE1ß PCR products. B, Schematic representation of ELE1{alpha} and ELE1ß and of the chimeric protein RET/PTC 3. RET/PTC 3 consists of the first 238 aa of ELE1, fused in frame to the tyrosine kinase domain of the RET protooncogene. ELE1ß lacks aa 239–565 from ELE1{alpha}. The glutamine at position 238 is the last residue encoded by exon 5 of the ele1 gene.

 

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  		Table 1. Point Mutations Detected in the ELE1{alpha} but Not in the ELE1ß cDNA

 
Expression of ELE1
Because of its putative role in androgen physiology and the possible implications for the development of prostate cancer, we examined the expression of ELE1 at the RNA level in three different human prostate cancer-derived cell lines (DU-145, LNCaP, and PC-3), grown in the absence or the presence of the synthetic androgen R1881. As shown in Fig. 2AGo, all cell lines express ELE1 and only contain the ELE1{alpha} mRNA as can be concluded from the size of the radioactive band. Of the three cell types tested, ELE1{alpha} expression is highest in DU-145 cells, and the expression is not substantially affected by androgen treatment.



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  		Figure 2. ELE1 Expression in Human Prostate Cancer-Derived Cell Lines and Rat Tissues

Northern blot analysis of total RNA from three human prostate cancer-derived cell lines, LNCaP, PC-3, and DU 145, grown in either the absence or the presence of 1 nM R1881(panel A), and of total RNA from different rat tissues (panel B). The blots were hybridized with a radiolabeled probe encompassing the first 638 bp of the ELE1{alpha} and the ELE1ß cDNA. RNA integrity and equal sample loading were verified with a 18S probe. The positions of the 28S and the 18S RNA bands are indicated as size markers. The asterisk in panel B marks a band that possibly corresponds to the ELE1ß mRNA.

 
Since the main targets for androgens are the organs and tissues of the male reproductive tract, we also analyzed the expression of ELE1 in rat prostate and testis and made a comparison with other tissues. The blot was probed with a radiolabeled cDNA fragment encompassing the first 638 bp of the ELE1 cDNA, which can hybridize to both the ELE1{alpha} and the ELE1ß mRNAs. As shown in Fig. 2BGo, ELE1{alpha} is expressed in all tissues examined at comparable and moderate levels. The lowest expression is found in the brain and the prostate, whereas the testis clearly contains much higher levels of ELE1{alpha}-mRNA. Moreover, in the testis a second and faster migrating mRNA band appears, possibly corresponding to the shorter ELE1ß variant. We could not detect it in any of the other tissues examined, even after prolonged exposure. It should be noted that a human cDNA fragment was used to probe the rat multiple tissue RNA blot that therefore had to be exposed approximately 10 times longer to obtain a clear signal.

In Vitro Interaction of ELE1 with NRs
For the characterization of the interaction between ELE1 and NRs, we performed in vitro glutathione S-transferase (GST) pull-down experiments. To ensure that the in vitro produced human AR (hAR) binds hormone correctly and that it undergoes the associated conformational changes, we measured the binding affinity of the AR for [3H]mibolerone and analyzed the tryptic digest patterns of the ligand-free receptor and its complexes with agonists and antagonists. We measured a Kd for mibolerone of 0.46 nM, which is consistent with the values reported by others (47) (Fig. 3AGo, left panel). Furthermore, in the absence of ligand the receptor is completely digested by limited amounts of trypsin. Addition of the agonists dihydrotestosterone (DHT) and R1881 or the antagonist hydroxyflutamide (HO-F), however, results in the protection of small but different ‘cores’ against trypsinization, indicating that the specific conformational changes that are associated to ligand binding do occur (Fig. 3AGo, right panel) (48).



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  		Figure 3. Analysis of the in Vitro Interaction of ELE1 with NRs

A (left panel), In vitro produced hAR was incubated with different concentrations of [3H]mibolerone, ranging from 0.1 to 10 nM, in the absence or the presence of a 100-fold excess of unlabeled hormone. Free (F) and bound (B) mibolerone were separated with dextran-coated charcoal. The Kd (0.46 nM) was calculated from the slope of the Scatchard plot. A (right panel), 35S-labeled hAR was incubated without hormone, with 10 nM of DHT or R1881 or with 10 µM of hydroxyflutamide (HO-F) and subjected to a limited proteolytic digest as described (47 ). The reaction products were separated by SDS-PAGE and visualized by fluorography. B, 35S-labeled full-length hAR was produced by in vitro coupled transcription-translation in the absence of ligand or in the presence of 10 nM R1881 or 10 µM HO-F and incubated with GST, GST-ELE1{alpha}, or GST-ELE1ß immobilized on Glutathione Sepharose resin. After stringent washings, the resin-bound proteins were eluted with SDS-loading buffer, analyzed by SDS-PAGE, and visualized by fluorography. The input lane represents 10% of the reticulocyte lysate that was used in each experiment. C, 35S-labeled hAR, mER, and rGR were produced in vitro and tested for the interaction with GST, GST-ELE1{alpha}, and GST-ELE1ß in the absence of ligand as described for panel B and in Materials and Methods. D (upper panel), Linear diagram of the hAR and the deletion mutants used in this study. The numbers refer to amino acid positions. The thin line represents the N-terminal domain, the shaded box represents the DBD, and the bold line represents the LBD. D, (lower panel), The hAR and the deletion mutants depicted in the upper panel were produced and radiolabeled by in vitro transcription translation and subsequently analyzed for interaction with GST or GST-ELE1{alpha} by the GST-pull down assay. Bound proteins were eluted with SDS-loading buffer, separated by SDS-PAGE, and visualized by fluorography.

 
To analyze the ELE1-AR interaction, 35S-labeled full-length hAR was added to a glutathione Sepharose matrix to which either GST alone or a fusion protein of GST with ELE1{alpha} or ELE1ß was coupled. The proteins were allowed to interact in the absence of ligand or in the presence of an agonist (R1881) or an antagonist (HO-F). As shown in Fig. 3BGo, the AR interacts with both ELE1{alpha} and ELE1ß, although this interaction is not ligand dependent and is not antagonized by HO-F. No differences were observed when either R1881 or DHT were used to activate the receptor (data not shown). Furthermore, the interaction is not specific for the AR, at least in vitro, since the rat GR (rGR) and the mouse ER (mER) also bind to ELE1 {alpha} and ß with affinities comparable to that of the hAR (Fig. 3CGo).

To further characterize the region of the AR that is required for this interaction, we performed GST pull-downs with full-length ELE1{alpha} and C-terminally truncated receptors. In a first series of mutants, we deleted the C-terminal half of the LBD and the complete LBD and found that the region between aa 563 and 772 is sufficient for interaction with ELE1{alpha} (data not shown). Next, we further deleted the receptor to P722 (end of exon 4, mutant {Delta}1), P670 (mutant {Delta}4), and G626 (end of exon 3, mutant {Delta}5). As shown in Fig. 3DGo, mutant {Delta}1 still interacts with ELE1{alpha}, whereas {Delta}4 does not. The N-terminal domain is not required for the interaction, since deletion of aa 1–537 had no effect on the binding (data not shown). Based on the alignment made by Wurtz et al. (49), the region between P670 and P772 corresponds to helices 1–3 at the N terminus of the LBD. We therefore generated mutants ending after helix 2 (mutant {Delta}2, aa 1–696) and after helix 1 (mutant {Delta}3, aa 1–683). These truncated receptors do not interact with ELE1{alpha} (Fig. 3DGo).

Thus, the domain that is sufficient for the in vitro interaction of the hAR with ELE1{alpha} is located between E696 and P722 and corresponds to the predicted helix 3 (H3).

Cotransfection of the hAR with ELE1 and TIF2
DU-145 prostate cancer cells were cotransfected with the mouse mammary tumor virus (MMTV)-luciferase reporter vector and expression plasmids for the hAR and either ELE1{alpha} or ELE1ß, but we did not observe strong coactivator properties (data not shown). Since we have demonstrated that DU-145 cells express ELE1{alpha}, they offer no obvious advantage over HeLa cells for use as a model system to analyze the properties of ELE1. Therefore, these cells were also cotransfected with the hAR and ELE1{alpha} or ELE1ß. As shown in Fig. 4Go (upper panel), coexpression of either ELE1 isoform results in a maximal 2-fold increase of AR activity (compare lanes 2 and 3 to lane 1). As a control, we included the p160 coactivator TIF2 (lane 4) which, under the same conditions, stimulated the hAR 4-fold. Since TIF2 has been shown to function better in COS cells than in HeLa cells (30), we performed the same experiments in COS-7 cells. As shown in Fig. 4Go (lower panel), also in these cells coexpression of ELE1{alpha} or ELE1ß has only moderate effects on the activity of the hAR (2- to 3-fold increase in receptor activity), whereas coexpression of TIF2 results in an almost 20-fold increase.



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  		Figure 4. Analysis of the Coactivator Activity of ELE1

HeLa cells (upper panel) or COS7 cells (lower panel) were transfected with 250 ng of pCMV-ßGAL, 300 ng of MMTV-luc reporter vector, 100 ng of pSG5-hAR, and 600 ng of either empty pSG5, pSG5-ELE1{alpha}, pSG5-ELE1ß, or pSG5-TIF2. The cells were treated for 24 h without (open bars) or with (solid bars) 1 nM of R1881 before the luciferase and ß-galactosidase activities were measured. The results shown are the mean ± SEM of three measurements. The luciferase activity measured in the absence of coactivator and in the presence of R1881 was arbitrarily set to 100.

 
In Vitro Interaction of ELE1 with p/CAF and TFIIB
Many transcriptional coactivators have intrinsic HAT activity or act as a platform for the recruitment of other HATs and the basal preinitiation complex. Using the liquid HAT assay, we could clearly demonstrate HAT activity for bacterially produced p/CAF but not for a GST-ELE1{alpha} fusion protein. Single hybrid experiments in HeLa cells also did not reveal an intrinsic transcription activation function for a GAL4-ELE1{alpha} fusion protein (data not shown).

We next tested the ability of ELE1{alpha} and ELE1ß to interact in vitro with p/CAF, TFIIB, the cointegrator CBP, and the p160 coactivators SRC-1a, SRC-1e, and TIF2 (Fig. 5Go). We observed in vitro binding to p/CAF and to TFIIB. The in vivo relevance of these interactions needs to be further analyzed. Coactivators of the p160 family and CBP did not bind to the GST-ELE1{alpha} fusion protein, whereas mutual interactions between the two first components were detected in control experiments.



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  		Figure 5. ELE1{alpha} and ELE1ß Interact in Vitro with TFIIB and with p/CAF

p/CAF, the general transcription factor TFIIB, CBP, and the p160 coactivators TIF2, SRC1a, and SRC1e were synthesized and radiolabeled in vitro in rabbit reticulocyte lysate. They were allowed to interact with purified GST, GST-ELE1{alpha}, or GST-ELE1ß (for TFIIB and p/CAF), GST-CBP[2058–2163] (for TIF2, SRC1a, and SRC1e), or GST-SRC1[781–988] (for CBP) prebound to a Glutathione Sepharose matrix, under the conditions described in Materials and Methods. After extensive washings the bound proteins were eluted, gel electrophoresed, and visualized by fluorography. The arrows indicate the positions of the respective 35S-labeled proteins.

 
Analysis of H3-Mutants of the hAR
We have shown that the region in the hAR required for the in vitro interaction with ELE1{alpha} corresponds to the predicted H3 of the LBD (Fig. 3DGo). To identify single residues that are involved in this interaction, we generated point mutations in this domain. Since in vitro the interaction is not AR specific, we chose to mutate the following moderately or highly conserved amino acids in the C-terminal part of H3: leucine 712 to arginine (L712R), valines 715 and 716 to alanine (V715A/V716A), tryptophan 718 to alanine (W718A), alanine 719 to lysine (A719K), and lysine 720 to alanine (K720A) (Fig. 6AGo). The ability of these mutants to bind hormone was addressed in a single point ligand-binding assay (1 nM mibolerone) of transiently transfected COS 7 cells (Fig. 6BGo). The mutants V715A/V716A and K720A can bind hormone equally well as the wild-type receptor, whereas L712R has a lower affinity. W718A and A719K, on the other hand, retained less than 10% of the amount of ligand bound by the wild-type receptor. A Western blot with extracts from transfected COS cells was performed to ensure adequate expression of all receptor mutants.



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  		Figure 6. In Vitro and in Vivo Analysis of hAR H3 Mutants

A, Sequence alignment of the LBD region of SRs corresponding to the predicted H3, according to Wurtz et al. (49 ). The numbers refer to amino acid positions. The mutations are indicated by asterisks and given below the sequence of the hAR. B, COS 7 cells were transiently transfected with an expression vector for either the wild-type AR or for one of the mutants described in panel A. For the single-point ligand-binding assay, the cells were incubated with 1 nM 3H-labeled mibolerone with or without 100 nM cold mibolerone. After a 2-h incubation at 37 C, they were washed and the radioactivity that was specifically retained within the cells was measured. The shown values are the mean of five measurements ± SEM. The activity measured for the wt AR was arbitrarily set to 100. The lower panel shows a Western blot of extracts of COS 7 cells, probed with an anti-AR antibody, to control the expression of the respective mutants. C, In vitro interaction assays of GST or GST-ELE1{alpha} and 35S-labeled wt or mutated hAR were performed as in Fig. 3BGo and as described in Materials and Methods. The mutations are described in panel A. D, HeLa cells were transiently transfected with the MMTV-luc (left panel) or the (GRE)2-oct-luc (right panel) reporter vectors (1 µg) together with expression vectors for either the wt hAR or one of the H3 mutants (100 ng) and pCMV-ßGAL (250 ng) as internal control. The cells were treated with (solid bars) or without (open bars) 10-9 M R1881 for 24 h before harvesting and measuring the luciferase and ß-galactosidase activities. The activity of the wt hAR in the presence of hormone was taken as 100, and the relative values of test measurements were calculated. At least three independent duplicate experiments were performed, and the results are shown as the mean value ± SEM. E, HeLa cells were transfected with MMTV-luc and expression vectors for the wild-type AR (triangles) or the L712R mutant (squares) as in Fig. 6DGo and treated for 24 h without hormone or with 0.001, 0.01, 0.1 or 1 nM of R1881 before the ß-galactosidase and the luciferase activity were measured. The activity of the wt hAR in the presence of 1 nM R1881 was taken as 100.

 
The ability of these mutants to interact with ELE1{alpha} was tested in a GST pull-down assay. As shown in Fig. 6CGo, the mutant L712R has a reduced affinity for ELE1{alpha} compared with the wild-type receptor, since less protein is retained on the GST-ELE1{alpha} beads. The mutants V715A/V716A, W718A, and A719K have affinities comparable to that of the unmodified receptor, whereas that of K720A is slightly higher. We examined the transcription-activating properties of these mutant receptors in transient transfection experiments. HeLa cells were cotransfected with MMTV-luc or (GRE)2-oct-luc reporter constructs and expression vectors for either the wild-type receptor or one of the mutants. As shown in Fig. 6DGo, all mutants retain some ability to activate the transcription of the MMTV-driven reporter in response to 1 nM R1881. Comparable results are obtained with the (GRE)2-oct-luc reporter. Mutation of leucine 712 to arginine caused only a slight (~35%) decrease in the ligand-dependent transcriptional activity of the AR, despite the clear effects of this mutation on the AR-ELE1{alpha} interaction. Surprisingly, mutation of the highly conserved lysine at position 720 to alanine, which severely impaired the transcription activation function of the mER (50), had no effect on the hAR. The double mutant V715A/V716A showed a slightly higher activity on the (GRE)2-oct-luc reporter, but a slightly reduced activity on the MMTV-luc reporter.

Next, we compared the dose-response curve for the wild-type receptor and the L712R mutant in HeLa cells, using MMTV-luc as reporter and increasing concentrations of R1881 (0.001 to 1 nM). As shown in Fig. 6EGo, the response of the L712R mutant to a wide range of R1881 concentrations is similar to that of the unmutated receptor, except that the maximal response to androgens is allways ~35% lower, which is consistent with its reduced affinity for the ligand.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The goal of our study was to characterize the interaction of ELE1 with the hAR and to evaluate its role as an AR-specific transcriptional coactivator. Therefore we cloned the ELE1 cDNA by RT-PCR. In addition to the expected full-length cDNA (ELE1{alpha}), we obtained an internally deleted variant (ELE1ß). This deletion is likely caused by the removal of one or more exons, since the last residue before the deletion (Q238) is the last amino acid encoded by exon 5 of the ele1 gene (46). Moreover, the NH2-terminal region of 238 aa from ELE1 ({alpha} and ß) corresponds to the ELE1-derived fragment in the RET/PTC 3 fusion-protein (43). Specific base changes in the ELE1{alpha} but not in the ELE1ß cDNA indicate, furthermore, that both proteins are encoded by two nonallelic genes with different expression patterns and RNA processing characteristics.

In view of its proposed role as an AR-specific coactivator, we analyzed the expression of ELE1 RNA in different rat tissues and in three human prostate cancer-derived cell lines. All cells, including DU-145 cells, were shown to express ELE1{alpha}. A possible explanation for the lack of ELE1{alpha} expression in these cells reported by Yeh and Chang (42) could be that these authors used a high-passage cell line (personal communication) with altered characteristics compared with our cells, or that they used a different DU-145 subclone. The high level of ELE1{alpha} expression in the rat testis could be indicative of its role in AR functioning. The prostate, however, another important androgen target tissue, contains much less ELE1{alpha}.

We tested the coactivator properties of ELE1{alpha} and ELE1ß in cotransfection experiments, and for both isoforms we found a very weak (~2-fold) increase in AR transcripional activity, irrespective of the cell line used (DU-145, HeLa, or COS7). The very poor effects of cotransfection of the AR with ELE1{alpha} or ELE1ß could be due to sufficient levels of endogenous protein in all cells. It should be noted, however, that under the same conditions the p160 coactivator TIF2 effeciently enhances the AR activity, most notably in COS cells, despite the fact that the p160 family members are also ubiquitously expressed.

The interaction between the hAR and ELE1 was reported to be ligand dependent in yeast and specific for the AR (42). In vitro, we observed a strong interaction of both ELE1{alpha} and ELE1ß with the hAR, although this interaction does not require the addition of ligand. The affinity of the AR is comparable for both ELE1 isoforms, indicating that the 328 amino acid region deleted in ELE1ß is not involved in this interaction. This apparent discrepancy between the in vitro ligand-independent interaction described here and the ligand dependency observed in yeast (42, 51) might be explained by the fact that in yeast cells, additional proteins may interact with the AR-LBD in the absence of ligand, thereby preventing interaction with ELE1. Addition of hormone could then release this block and allow the LBD to bind to ELE1. Apart from the hAR, other steroid receptors (mER and rGR) also interact in vitro with ELE1. Thus, the interaction is not receptor specific. Furthermore, Treuter et al. (51) recently reported the cloning of the ELE1 cDNA by a yeast double-hybrid screen using the LBD of the peroxisome proliferator-activated receptor as bait. Thus, ELE1 also interacts with NRs that do not belong to the subclass of the SRs. This is not exceptional, however, since none of the coactivators that have been described so far display receptor specificity. The ER, as well as the PR, TR, RAR, and RXR, interacts with all of the p160 family members (25, 28, 29, 30, 31, 33, 35). Likewise, Trip1 interacts with both the TR and RXR (28), and TIF1 interacts with RXR{alpha}, RAR{alpha}, vitamin D receptor, PR, and ER (23, 24).

We mapped the domain of the AR that is required for the interaction with ELE1{alpha} to the region between aa 696 and 722, which corresponds to the predicted H3 of the LBD (49). This region has been shown to be important for transcription activation by the ER (50). Moreover, crystallographic studies on the LBDs of the ER, TR, PR, RXR, and RAR have indicated that binding of the appropriate ligand repositions helix 12, comprising the core AF2, which subsequently makes direct contacts with amino acids in H3 and H4, thereby providing a new surface for interaction with coactivators (13). Feng et al. (52) systematically mutated specific residues in the LBD of the TR and found that the AF2 is formed by residues from the helices 3, 5, 6, and 12. The AF2 core is dispensable for the hAR-ELE1{alpha} interaction, which again demonstrates that the interaction between the AR and ELE1 is clearly different from the interaction between the p160 coactivators and NRs. Furthermore, both ELE1{alpha} and ELE1ß contain a LXXLL motif (LYSLL) between aa 92 and 96. It has been shown that the integrity of the AF2 core domain is essential for the interaction of NRs with this motif in p160 coactivators (37). Since a truncated receptor lacking H12 can still interact with ELE1{alpha}, it is very unlikely that this binding occurs through the LYSLL motif.

ELE1 has no intrinsic transactivating properties and does not interact with CBP, but it can bind to TFIIB and to p/CAF. Although the in vivo relevance of these protein-protein interactions needs to be examined in more detail, it is tempting to speculate that ELE1 might function as a linker protein between the NR and the basal transcription machinery, thereby recruiting or stabilizing the PIC on the promoter. Furthermore, through the interaction with p/CAF it might contribute to the recruitment of HATs and the subsequent chromatin remodeling. Such an activity, however, would not become apparent under the conditions of transient transfection as used in this work.

We tried to identify single residues that are critical for the interaction of ELE1 with the hAR. Sequence comparison of the H3 regions of steroid receptors reveals that this domain is best conserved at the C terminus of the helix. Mutation of a series of moderately or highly conserved residues in H3 revealed that the leucine at position 712 is essential for ELE1 binding. In the hGR the corresponding residue is a valine, and in the human mineralocorticoid receptor it is a methionine. All these residues have hydrophobic side chains, indicating that the binding between ELE1{alpha} and the AR could occur through hydrophobic interactions. Despite the dramatic effects of this mutation on the ELE1-hAR interaction, the mutated receptor is still transcriptionally active on both the MMTV-luc and the (GRE)2-oct-luc reporters, although the maximal activity is ~35% less than that of the wild-type receptor. This correlates well with the reduced ligand-binding affinity observed in COS 7 cells. Thus, the slight decrease in transcriptional activity most probably reflects a decrease in ligand binding, rather than resulting from an impaired interaction with ELE1. A striking observation in the analysis of the H3 mutants is the fact that mutation of the highly conserved lysine residue at position 720 to an alanine (K720A), which severely impairs the function of the ER (50), had no effect on the transcriptional activity of the AR. This indicates that the mechanism of activation by the AR may be different from the ER. Similar conclusions can de drawn from other observations: whereas the ER-LBD harbors an autonomous transcription activation function (AF2) when fused to a heterologous GAL4-DBD (7, 50), the AR-LBD requires the NH2-terminal AF1 function to gain ligand-dependent transcription activation properties (53).

In conclusion, our results reveal the existence of two nonallelic variants of ELE1. They confirm that ELE1 interacts with the LBD of the hAR but also demonstrate that, at least in vitro, this interaction is not ligand dependent and that ELE1 can also interact with other members of the NR superfamily. Although we demonstrate that ELE1 may act as a bridging factor between steroid receptors and the basal transcription machinery or chromatin-remodeling enzymes, it does not act as a classic coactivator in mammalian cells in our experimental conditions. In fact, mutation of specific residues in the LBD of the hAR that severely impair the AR-ELE1 interaction have only moderate effects on the AR transcriptional activity. Thus, the in vivo function of ELE1 should be further investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
All restriction and modifying enzymes were obtained from either Pharmacia (Uppsala, Sweden), Life Technologies (Grand Island, NY), or Boehringer (Mannheim, Germany). Cell culture reagents were obtained from Life Technologies. The TNT rabbit reticulocyte lysate in vitro coupled transcription translation kit was purchased from Promega (Madison, WI). L-[35S]methionine (>1000 Ci/mmol) and [3H]mibolerone (82.3 Ci/mmol) were purchased from Amersham (Buckinghamshire, UK). The hormones 5{alpha}-dihydrotestosterone, R1881 (methyltrienolone), and mibolerone were from Dupont-New England Nuclear (Boston, MA). Hydroxyflutamide was a kind gift from Schering (Kenilworth, NJ).

Plasmids
For the construction of pSG5-hAR, the hAR cDNA was PCR amplified from pSV-ARO (54) with Expand High Fidelity polymerase (Boehringer) in the presence of 4% dimethylsulfoxide and using the primers 5'-GGTGGATCCATGGAAGTGCAG-TTAGGGCTGGGAAGGTCTAC-3' and 5'-TACGTGGATCCTCACTGGGTGTGGAAATAGATGGGCTTGACT, and the re-sulting PCR product was cloned in the BamHI restriction site of pSG5. Deletion mutants were made by PCR using the Pfu thermostable polymerase (Stratagene, La Jolla, CA) and pSG5-hAR as template and the following primers: 5'-TAGGATCCATGTTGGAGACTGCCAGGGAC-3' (forward primer; FP), 5'-TAAGATCTGGATCCTCAAGGCAAGGCCTTGGCCC-3' (reverse primer for {Delta}1), 5'-TAAGATCTGGATCCTCAAAAGGAGTCGGGCTGGTT-3' (reverse primer for {Delta}2), 5'-TAA-GATCTGGATCCTCATACACCTGGCTCAATGGC-3' (reverse primer for {Delta}3), 5'-TAAGATCTGGATCCTCAGGGCTGACA-TTCATAGCC-3' (reverse primer for {Delta}4), 5'-TAAGATCTGGATCCTCATCCCAGAGTCATCCCTGC-3' (reverse primer for {Delta}5).The resulting PCR products were digested with HindIII and BglII and exchanged for the corresponding fragment in pSG5-hAR.

Site-directed mutagenesis was performed using a standard PCR-based method. The plasmid pCR(538–918;wt) was constructed by inserting a fragment encoding the entire DBD and LBD, PCR amplified from pSG5-hAR with the primers FP and 5'-GCTGCAATAAACAAGTTCTGC-3' (reverse primer; RP), and subsequently cloned in the pCR-SCRIPT vector (Stratagene). Next, two sets of PCR reactions were performed. In the first set, with pSG5-hAR as template, oligonucleotide FP was used as forward primer and the following oligonucleotides as reverse primers: 5'-CACGTGTACACG-CTGTCTCTC-3' (PCR a), 5'-CTTGTACACGCCGCCAAGTGGGCCAAG-3' (PCR c), 5'-GTGGTCAAGGCGGCCAAGGCC-3' (PCR e), 5'-GTCAAGTGGAAGAAGGCCTTG-3' (PCR g), and 5'-CAAGTGGGCCGCGGCCTTGCC-3' (PCR i). Similarly, the oligonucleotide RP was used as reverse primer and the following oligonucleotides as forward primers: CACGTGTACACGCTGTCTCTC-3' (PCR b), 5'-CTTGGCCCACTTGGCGGCGTGTACAAG-3' (PCR d), 5'-GGCCTTGGCCGCCTTGACCAC-3' (PCR f), 5'-CAAGGCCTTCTTCCACTTGAC-3' (PCR h), and 5'-GGCAACGCCGCGGCCCACTTG-3' (PCR j). The templates for the second set of reactions, using the oligonucleotides FP and RP as primers, were a combination of the reaction products from PCRs a and b, c and d, e and f, g and h, and finally i and j. The PCR products from this second set of reactions were digested with HindIII and BglII and exchanged for the corresponding fragment in pCR-(538–918;wt), giving rise to pCR-(538–918;ab), pCR-(538–918;cd), pCR-(538–918;ef), pCR-(538–918;gh), and pCR-(538–918;ij). Finally, Tth111I-NcoI fragments from these constructs were exchanged for the corresponding fragment in pSG5-hAR, resulting in AR expression plasmids carrying the following mutations: L712R, V715A/V716A, W718A, A719K, and K720A. For the construction of pSG5-TFIIB, the TFIIB cDNA was isolated from pGEX-TFIIB (a gift from Dr. B. O’Malley) and subcloned in pSG5. Expression vectors for TIF2 and SRC-1 were kindly provided by Dr. H. Gronemeyer and Dr. M. G. Parker. The reporter gene construct (GRE)2-oct-luc, which contains two GREs in front of the minimal TATA box derived from the oct6 gene promoter and the luciferase gene, was the kind gift of Dr. A. O. Brinkmann.

Molecular Cloning of the ELE1 cDNA
The ELE1 cDNA was cloned by RT-PCR from RNA derived from HeLa cells, LNCaP cells, and T-47D cells. Briefly, 3 µg of total RNA were reverse transcribed with the primer 5'-CTAGGAATTCTCACATCTGTAGAGGAGTTC-3' (30 min at 42 C), which generates an EcoRI restriction site immediately downstream of the ELE1 stop codon. The remaining RNA was digested with RNase H for 10 min at 55 C, and the first-strand cDNA was PCR amplified using oligonucleotide 5'-GATCGAATTCATATGAATACCTTCCAAGAC-3' as forward primer and the above mentioned oligonucleotide as reverse primer. PCR was performed with either Taq or Pfu (Stratagene) thermostable polymerases. The reaction products were gel purified and subsequently cloned in the pGEM-T (Promega) or pCR-SCRIPT (Stratagene) vectors, respectively, and analyzed by sequencing. For expression as a GST-fusion protein, the cDNA was cloned in the EcoRI site of the pGEX-2TK vector (Pharmacia); for expression in mammalian cells or in vitro transcription translation, it was cloned in the EcoRI site of pSG5.

Northern Blotting
RNA from LNCaP, PC-3, and DU-145 cells, grown in either the absence or the presence of 1 nM R1881, was prepared, electrophoresed, and blotted as described (55, 56). The probe for Northern hybridization was prepared by PCR with the primers 5'-GATCGAATTCATATGAATACCTTCCAAGACC-3' and 5'-C-CACTGGCAGGTTTGCTTCC-3', which generates a fragment comprising the first 638 bp of the ELE1 cDNA. One microliter of this reaction was used in a 100-µl labeling reaction as previously described (55). The 18S RNA probe was labeled by random priming. The membrane was prehybridized for 2 h at 42 C in 5 x SSC (20 x SSC: 3 M NaCl and 3 M sodium citrate), 2 x Denhardt’s (100 x Denhardt’s: 2% Ficoll 400, 2% polyvinylpyrolidone) containing 50% (vol/vol) formamide, 0.1% SDS, and 100 µg/ml sonicated salmon sperm DNA and subsequently hybridized with probe (106 cpm/ml) for 18 h at 42 C in the same buffer. The blot was washed once at room temperature with 2 x SSC containing 0.5% SDS and twice for 20 min at 65 C with 0.2 x SSC containing 0.1% SDS and finally autoradiographed at -80 C using intensifying screens.

In Vitro Ligand Binding and Limited Trypsinization Assay
The in vitro ligand-binding and limited trypsinization assays were performed as described previously (47, 48).

GST Pull-Down Assay
Expression and purification of GST or the GST fusion proteins using the pGEX-system (Pharmacia) and coupled in vitro transcription-translation with the TNT system (Promega) were performed according to the manufacturer’s instructions. 35S-labeled proteins were added to the glutathione Sepharose containing either GST or the GST fusion protein in NENT/B [NENT buffer (100 mM NaCl, 1 mM EDTA, 0.02% NP-40, 20 mM Tris, pH 8) containing 1 mg/ml BSA] in a total volume of 200 µl and incubated for 30 min at room temperature and for 30 min at 4 C. The glutathione Sepharose was washed five times with 1 ml of NENT/B. Bound proteins were eluted by boiling the resin in 30 µl of 2 x SDS loading buffer (20% glycerol, 2% SDS, 0.0025% bromophenol blue, 10% ß-mercaptoethanol), separated by SDS-PAGE and visualized by fluorography. For the interactions of ELE1 with TFIIB, SRC-1, TIF2, p/CAF, and CBP, NENT buffer without BSA but containing 0.1% NP-40 was used.

Cell Culture
HeLa, T-47D, LNCaP, DU-145, and COS 7 cells were obtained from the Amercian Tissue Type Collection (ATTC, Manassas, VA) and were routinely maintained in DMEM (Life Technologies) containing 1 g/liter glucose and supplemented with antibiotics (penicillin, streptomycin; Life Technologies) and 10% heat-inactivated FBS. For DU-145 cells, L-glutamine was added to a concentration of 2 mM.

Transfections
Cells were transfected using the standard calcium phosphate coprecipitation procedure (57). Twenty four hours before transfection, HeLa cells, COS 7 cells, or DU-145 cells were trypsinized and seeded in 24-well culture plates at a density of 7.5 x 104 cells per well in medium containing 5% dextran-coated charcoal-stripped FBS (DCC-FBS). For cotransfection studies, the following amounts of plasmids were used (per well): 300 ng MMTV-luc, 100 ng pSG5-hAR, and 600 ng of either empty pSG5, pSG5-ELE1{alpha}, pSG5-ELE1ß, or pSG5-TIF2. For the analysis of the different receptor mutants, HeLa cells were transfected with 1 µg of reporter vector (MMTV-luc or (GRE)2-oct-luc) and 100 ng of expression vector for the wild-type or mutated AR per well. p-CMV-ß-GAL (250 ng) was always included as internal control. The cells were incubated for 24 h in the absence or the presence of 1 nM R1881 before the luciferase, ß-galactosidase, and protein concentrations were determined.

For the single-point ligand-binding assay and for Western blotting, COS 7 cells were transfected with pSG5-hAR or expression vectors for the respective receptor mutants. Forty eight hours after transfection, the medium was replaced with DCC-FBS containing 1 nM [3H]mibolerone, either with or without a 100-fold excess of cold mibolerone. After 2 h incubation at 37 C, the cells were placed on melting ice, washed twice with ice-cold PBS, and lysed in 1% Triton X-100, 0.1 N NaOH, and their radioactivity was counted. For Western blot analysis, the cells were scraped in 1 ml PBS and pelleted by centrifugation. The pellet was dissolved in 50 µl of high-salt extraction buffer (20 mM Tris, pH 7.8, 420 mM NaCl, 1 mM EDTA), and the cells were lysed by two cycles of freezing in liquid nitrogen and thawing on ice. Insoluble material was pelleted by centrifugation, and 10 µl of the soluble extract were separated by SDS-PAGE in a 9% gel and electroblotted onto a polyvinylidene fluoride membrane. The blot was probed with a polyclonal antiserum against the N terminus of the hAR, and the proteins were visualized with the enhanced chemiluminescence system (Amersham).


   ACKNOWLEDGMENTS
 
We thank Dr. A. O. Brinkmann (Erasmus University, Rotterdam, The Netherlands), Dr. B. W. O’Malley (Baylor University, Houston, TX), Dr. M. G. Parker (Imperial Cancer Research Fund, London, UK) and Dr. H. Gronemeyer (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch-Cedex, France) for the kind gift of plasmids; Dr. Neri (Schering Plough, Kenilworth, NJ) for the gift of hydroxyflutamide; R. Bollen and H. De Bruyn for excellent technical assistance; and V. Feytons for the expert synthesis of many oligo-nucleotides.


   FOOTNOTES
 
Address requests for reprints to: Dr. B. Peeters, Department of Biochemistry, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. e-mail: ben.peeters{at}med.kuleuven.ac.be

This work was supported in part by a grant ‘Geconcerteerde Onderzoeksactie van de Vlaamse Gemeenschap,’ by grants from the Belgian ‘Fonds voor Geneeskundig Wetenschappelijk Onderzoek’ and by a grant ‘Interuniversity Poles of Attraction Programme, Belgian State, Prime Minister’s Office, Federal Office for Scientific, Technical and Cultural Affairs.’ P.A. was holder of a scholarship ‘Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie.’ F.C. and J.V.S. are Senior Research Assistants of the Fund for Scientific Research Flanders (Belgium).

Received for publication June 4, 1998. Revision received August 31, 1998. Accepted for publication September 21, 1998.


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 DISCUSSION
 MATERIALS AND METHODS
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