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Endocrinology Vol. 140, No. 7 3097-3105
Copyright © 1999 by The Endocrine Society

ARTICLES

Transcription Activating and Repressing Functions of the Androgen Receptor Are Differentially Influenced by Mutations in the Deoxyribonucleic Acid-Binding Domain1

Piia Aarnisalo, Henrikki Santti, Hetti Poukka, Jorma J. Palvimo and Olli A. Jänne

Department of Physiology, Institute of Biomedicine (P.A., H.S., H.P., J.J.P, O.A.J.), and Department of Clinical Chemistry (O.A.J.), University of Helsinki, FIN-00014 Helsinki, Finland

Address all correspondence and requests for reprints to: Dr. Olli A. Jänne, M.D., Ph.D., Institute of Biomedicine, Department of Physiology, P.O. Box 9 (Siltavuorenpenger 20 J), FIN-00014 Helsinki, Finland. E-mail: olli.janne{at}helsinki.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Despite the wide spectrum of androgen receptor (AR) mutants described in androgen insensitivity syndromes (AIS), their influence on transactivating and, in particular, transrepressing functions of AR are poorly defined. Rat AR mutants with substitutions in the DNA-binding domain, corresponding to several mutations in AIS patients, were examined for these activities. AR variants (G551V and C562G) with mutations in the first zinc finger (ZF) exhibited reduced DNA-binding activity and attenuated transactivation. An R590Q substitution in the second ZF diminished transcriptional activity only from a promoter with a single androgen response element, whereas activation at multiple androgen response element sites was unaffected, despite the poor DNA-binding affinity of R590Q. Another substitution in the second ZF, A579T, yielded similar findings. In comparison to wild-type AR, G551V, and C562G variants had markedly reduced ability to repress an NF-{kappa}B/RelA-activated promoter but R590Q behaved like the native receptor. AP1 function was repressed not only by wild-type AR but also by the transactivating mutants A579T and R590Q as well as by the transcriptionally inactive mutants G551V and C562G. Furthermore, a Lys-to-Ala substitution in codon 563 of the first ZF switched AR into a ligand-dependent activator at AP1 sites but maintained the ability to repress NF-{kappa}B/RelA function. Taken together, DNA-binding domain mutations in AIS patients influence transcriptional activating and repressing functions of AR in a selective fashion, which probably contributes to the complexity in the presentation of the AIS phenotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANDROGEN RECEPTOR (AR1) belongs to the superfamily of nuclear receptors consisting of the receptors for steroid and thyroid hormones, retinoids, and vitamin D, and so-called orphan receptors whose ligands are largely unknown (1, 2). AR is a ligand-activated transcription factor capable of both activating and repressing gene expression (3, 4). It is involved in the regulation of development, differentiation, and maintenance of male reproductive functions as well as in the generation of sexually dimorphic characteristics in nongenital tissues. Upon ligand binding, the receptor acquires a new conformational state, which renders it capable of interacting with specific androgen response elements (AREs) that are usually located within or close to the promoter region of regulated genes. Liganded receptor is also able to interact with other transcription factors and transcriptional coregulators. These interactions, in turn, lead to either activation or repression of target gene expression, depending on the physiological context.

AR like other nuclear receptors displays a modular structure with three main conserved regions: an amino-terminal region harboring an autonomous activation function (AF-1), a centrally located DNA-binding domain (DBD), and a carboxyl-terminal region containing the ligand-binding domain (LBD) (1, 2). The DBD is the most conserved region among the nuclear receptors and contains two zinc finger motifs (ZF), in which four cysteines tetrahedrally coordinate a zinc ion. This domain plays an important role in both transactivation and transrepression. Three residues in the carboxyl-terminal part of the first ZF (the P box) are involved in the discrimination between response elements (5, 6, 7, 8). There is a conserved Lys residue between the P box residues, K580 in the human and K563 in the rat AR (9). Substitution of this Lys by Ala in rat glucocorticoid receptor (GR) yields a receptor (K461A) that activates transcription from both simple, composite and tethering glucocorticoid response elements (10, 11). Whereas the first ZF is primarily responsible for the response element recognition, the second ZF is required to stabilize receptor-DNA interactions. In addition to contacting the DNA phosphate backbone, a subdomain in the amino-terminus of the second ZF (the D box) is involved in receptor homodimerization and in half-site spacing requirements (8, 12, 13). The DBD is needed not only for transcriptional activation but also for transcriptional repression of genes up-regulated by other transcription factors; for example, activities of AP1, NF-{kappa}B, Ets, Oct-1, and some other factors are repressed by AR (3, 4, 14, 15, 16, 17, 18, 19, 20, 21). By and large, transcriptional repression does not involve binding of the receptor to DNA; rather, it results from protein-protein interactions and competition for common coactivators, such as the cAMP response element binding protein (CREB)-binding protein, CBP (18).

Androgen insensitivity syndromes (AIS) are relatively rare X-linked diseases with mutations in the androgen receptor gene (22, 23, 24). Androgen insensitivity can be classified as being complete (CAIS) or partial (PAIS). AR defects described in CAIS and PAIS patients include deletions, insertions, missense, and nonsense mutations, and expansion of the polyglutamine repeat in the amino-terminal region of the receptor (22). To date, over 300 mutations have been characterized; most of them are located in the LBD (24). Point mutations in the DBD result in either CAIS or PAIS, predominantly depending on the mutant’s ability to bind DNA and dimerize. Substitution of Gly 568 by Val in the loop of the first ZF of the human AR (corresponding to G551 in the rat AR) leads to partial androgen insensitivity (25). Mutation of Cys 579 (C562 in rAR), which binds the zinc ion in the first ZF, is associated with CAIS (26). This mutant has normal androgen-binding affinity but is defective in DNA binding and, consequently, lacks transcriptional activity. Substitution of Arg residues at codons 607 and 608 (R590 and R591 in rAR) in the second ZF by Glu and Lys, respectively, causes PAIS and is associated with the development of male breast cancer (27). Transcriptional activity of the R607Q mutant has been reported to be attenuated only at low androgen concentrations (28), and androgen insensitivity of a patient with this mutation was partially overcome by high-dose androgen treatment (29).

Despite the fairly large number of AIS mutations described in the AR DBD, only a few mutants have been examined for their DNA-binding characteristics and transcriptional activities. To our knowledge, the mutants’ ability to repress transcription has not been previously addressed. In the present work, we have examined the influence of some AR DBD mutants, corresponding to those in AIS patients, on DNA binding, transactivation, and transrepression by the receptor. Our results indicate that AR DBD substitutions influence transactivating and transrepressing functions of the protein in a distinct fashion and suggest that, even in CAIS patients, some of the physiological functions of AR are maintained.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Rat AR (rAR) expression vector (pSG5-rAR) and the mutants rAR{Delta}38–296/{Delta}641–902, rAR{Delta}619–902, and rARC562G have been previously described (30, 31, 32). The mutants rARG551V, rARK563A, rARA579T, and rARR590Q were constructed by PCR. Expression vector encoding human RelA and 6 x {kappa}B-luciferase (LUC) reporter vector were gifts from Dr. Patrik Baeuerle (Genentech, Inc., South San Francisco, CA) (33). Fragments of the rat probasin promoter were generated from pBH500 (a gift from Dr. Robert J. Matusik, Vanderbilt University, Nashville, TN) (34) using PCR. pPB(-285/+32)-LUC, pPB(-150/+32)-LUC, and pPB(-115/+32)-LUC reporters were constructed by inserting the respective nucleotides of the probasin promoter into pGL3-basic vector (Promega Corp., Madison, WI). p75–1050-chloramphenical acetyl transferase (CAT) reporter was provided by Dr. Mart Saarma (Institute of Biotechnology, University of Helsinki, Helsinki, Finland), and it includes the first 1050 nucleotide (nt) upstream of the translation start site of the p75 (low-affinity neurotrophin receptor) promoter (3). p-73Col-CAT reporter containing the first 73 nt of the collagenase promoter (35) was a gift from Dr. Tom Kerppola (Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI). pIL-6(-225/+11)-LUC containing nt -225/+11 of the human IL-6 promoter (4) was a gift from Dr. Bernd Stein (Signal Pharmaceuticals, San Diego, CA). pARE4tk-LUC and pCMV-ARE2-CAT have been described (18, 36). pCMV-ß was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). Expression vector encoding amino acids 1–1099 of CBP fused to glutathione S-transferase (GST) (GST-CBP-NT) was a gift from Dr. Tony Kouzarides (Wellcome/CRC Institute and Department of Pathology, University of Cambridge, Cambridge, UK) (37).

Testosterone was purchased from Makor Chemicals (Jerusalem, Israel). [{alpha}-32P]dCTP and [{alpha}-32P]dATP were obtained from Amersham International (Aylesbury, UK), and [3H]acetyl-coenzyme A was from New England Nuclear Corp. (Boston, MA). Luciferase assay reagent and TNT T7-coupled rabbit reticulocyte lysate were obtained from Promega Corp. FuGene transfection reagent was purchased from Boehringer Mannheim (Mannheim, Germany).

Cell culture and transfections
CV-1, COS-1, and PC-3 cells were from American Type Culture Collection (Rockville, MD). The cells were maintained in DMEM containing penicillin (25 U/ml), streptomycin (25 U/ml), and 10% FBS. Transfections were performed by using FuGene reagent according to manufacturer’s instructions. Briefly, 60,000 or 200,000 cells were plated on a 12-well plate or a 6-well plate, respectively, 24 h before adding the DNA. Eighteen hours later, the cells received fresh medium containing 2% charcoal-stripped FBS with vehicle or testosterone at indicated concentrations. CAT and LUC activities were determined as previously described (38). Transfections were performed with triplicate dishes, and the results were repeated in two to five independent experiments. Statistical analyses were carried out with two-tailed Student’s t test.

Immunoblotting
Whole cell extracts from transfected COS-1 cells were prepared as previously described (39) and resolved by electrophoresis on a 7.5% polyacrylamide gel under denaturing conditions (40). Proteins were transferred onto an Immobilon-P membrane (Millipore Corp., Bedford, MA) and blotted with ARp3 antibody raised against a synthetic peptide corresponding to residues 14–32 of rAR (4).

Electrophoretic mobility shift assay (EMSA)
EMSAs with whole cell extracts from transfected COS-1 cells or in vitro translated proteins were carried out as described previously (38). Binding reaction consisted of a 1-h preincubation at 4 C, followed by addition of 20 fmol 32P-labeled, double-stranded oligonucleotide corresponding to a single or duplicated AREs of the rat C3 (1) gene. After a 30-min incubation at room temperature, protein-DNA complexes were resolved by electrophoresis on 4% polyacrylamide gel under nondenaturing conditions (41).

Promoter interference assay
CV-1 cells were transfected with 0.1 µg of vectors encoding different AR variants and 0.5 µg of pCMV-ARE2-CAT reporter as previously described (36), except that FuGene reagent was used for transfections.

In vitro protein binding assays
GST pull-down experiments were conducted as previously described (42), by using either purified GST alone or GST-CBP-NT adsorbed to Glutathione Sepharose and [35S]methionine-labeled rAR variants produced by translation in vitro.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of transcription by AR DNA-binding domain mutants
To study the transcriptional activity of AR DBD mutants, single amino acid substitutions were introduced into rAR (Fig. 1Go). Consequences of the substitutions, in the context of a full-length receptor protein, were then examined in CV-1 cells that do not express endogenous AR. As observed previously (32), conversion of the zinc-coordinating Cys to Gly (C562G) in the first ZF abolished the ability of AR to activate a reporter driven by four AREs in front of the minimal thymidine kinase (tk) promoter (pARE4tk-LUC) (Fig. 2AGo). Substitution of Gly 551 by Val (G551V) or Lys 563 by Ala (K563A) retained 35% and 40% of the wild-type activity, respectively. Conversion of Arg 590 to Glu (R590Q) in the second ZF, a mutation identified in PAIS patients (22, 24, 28), did not diminish transcriptional activity; rather, R590Q was more active (P < 0.05) than wild-type AR (Fig. 2AGo). In these and subsequent experiments, the dissimilar behavior of various AR mutants was not explainable by differences in their expression levels, as immunoblot analyses on extracts from transfected COS-1 cells revealed similar amounts of immunoreactive proteins (Fig. 2AGo, inset).



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  		Figure 1. The rat androgen receptor DNA-binding domain substitutions and deletion mutants used in this study. A, DNA-binding domain substitutions. The numbering of amino acid residues in rAR is according to Tan et al. (61 ); the numbers inparentheses refer to the corresponding residues in the human sequence. The amino acids mutated in this study are boldface. The residues of the P box are shown in gray circles and those of the D box in open boxes. B, Schematic illustration of the structures of rAR deletion mutants used. The numbering of amino acid residues is as above (61 ). TAD, Transactivation domain; DBD, DNA-binding domain; and LBD, ligand-binding domain.

 


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  		Figure 2. Transcriptional activation of pARE4tk-LUC reporter by the wild-type and mutated ARs. A, CV-1 cells (2 x 105) were cotransfected with the expression vectors for the wild-type and mutant ARs (0.1 µg) and pARE4tk-LUC reporter (0.5 µg) (in Materials and Methods). Eighteen hours after transfection, the cells received fresh medium with vehicle (cross-hatched bars) or 100 nM testosterone (solid bars) for the subsequent 30 h. LUC activities are expressed relative to that of the wild-type receptor in the presence of androgen (pSG5-rAR + testosterone = 100%). Inset, Expression levels of the wild-type and mutant ARs. Each lane contained 30 µg protein from cells transfected with vectors expressing the following AR forms: lane 1, no rAR (empty pSG5 vector); lane 2, wild-type rAR; lane 3, G551V; lane 4, K563A; lane 5, R590Q; and lane 6, C562G. Soluble cell extracts were subjected to electrophoresis under denaturing conditions followed by immunoblotting using ARp3 antibody (4 ). B, The cells were transfected as in panel A and cultured in the presence of indicated concentrations of testosterone (in nM) for 30 h. The LUC activities are expressed relative to that achieved with wild-type AR in the presence of 100 nM testosterone (= 100%). Mean ± SEM values of three independent experiments with triplicate dishes are given as percentages.

 
The report that transcriptional activity of R590Q is attenuated only at low androgen levels (28) prompted us to examine the function of the wild-type and DBD-mutated receptors at different testosterone concentrations (Fig. 2BGo). Relative induction of ARE4tk-LUC by G551V and K563A varied from 35–50% of wild-type activity, and it was not markedly influenced by the androgen concentration. The R590Q mutant activated the reporter gene more efficiently than the wild-type receptor (P < 0.05) at all hormone concentrations tested (1–100 nM). Similar results were obtained in CV-1, COS-1, and PC-3 cells when the probasin promoter containing region -285/+32 with two AREs, or a minimal promoter containing two AREs in front of a TATA-box element, was used (Fig. 3Go, A and B, and data not shown). However, R590Q and another substitution in the second ZF, A579T, were less active than native rAR with the probasin reporter containing a single ARE [(-150/+32)-LUC, Fig. 3CGo]; they exhibited 15–30% of the wild-type activity at 0.3–1 nM testosterone, even though the mutant and wild-type activities were indistinguishable at a higher steroid concentration (10 nM). R590Q also activated to some extent a reporter construct driven by region -115/+32 of the probasin promoter that is devoid of AREs (our unpublished observations). This latter activity of R590Q was, however, relatively minor in comparison to that it had on ARE-containing promoters (2- to 3-fold vs. >=100-fold).



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  		Figure 3. Activation of transcription from rat probasin promoter constructs by the wild-type and mutant ARs. CV-1 cells were transfected and treated as described in Materials and Methods and the legend to Fig. 2Go. pPB(-285/+32)-LUC was used as the reporter in panels A and B, and pPB(-150/+32)-LUC was the reporter in panel C. In A, the cells were treated with 100 nM testosterone, whereas in B and C, the indicated testosterone concentrations (in nM) were used. Values are mean ± SEM of three independent experiments. The LUC activities are expressed relative to that achieved with wild-type AR (= 100%) in the presence of 100 nM testosterone in A and B, and 10 nM testosterone in C.

 
DNA-binding activity of the DBD mutants
DNA-binding properties of the wild-type and mutated ARs were analyzed by EMSA by using proteins extracted from transfected COS-1 cells (Fig. 4AGo) or produced by translation in vitro (data not shown). In comparison to wild-type AR, the affinity of all mutants for a double-stranded ARE oligonucleotide was decreased: no specific interaction was seen with C562G; G551V and R590Q formed weak complexes; and the affinity of K563A for ARE was approximately one-half of that of the wild-type protein. A579T behaved like R590Q, and similar results were seen when an oligonucleotide with two AREs was used in EMSA experiments (data not shown). The specificity of the complexes was ascertained by the use of AR antibody, which led to the formation of supershifted complexes in other cases but the mutant C562G (Fig. 4AGo, lanes 8, 12, 16, and 20; identified with • symbols). The differences in the DNA-binding activity of the AR DBD forms examined was not due to their dissimilar expression levels in transfected cells (Fig. 4BGo). The sudden increments in ARE-bound band intensities observed with the mutants G551V, K563A, and R590Q were not seen in Sp1-DNA complexes when an Sp1 oligonucleotide was used as the probe in EMSA experiments (data not shown), possibly due to cooperative binding of AR to ARE (see Ref. 41). In any event, the interaction of AR DBD mutants in vitro with ARE sequences correlated only modestly with the transactivation ability of the same receptor forms, in that the mutants R590Q and A579T bound poorly to ARE but elicited a robust activation of AR-dependent reporters with multiple AREs, whereas the mutant K563A had higher affinity for ARE than anticipated on the basis of its blunted transactivation potency (Figs. 2Go, 3AGo, and 4AGo).



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  		Figure 4. DNA-binding activity of the wild-type and mutant ARs as determined by EMSA. Extracts from COS-1 cells transfected by electroporation with the expression vectors for wild-type and mutant rARs were prepared as described in Materials and Methods. A, Three aliquots of each sample (7.5, 15, and 30 µg protein) were incubated with 32P-labeled C3 (1 )-ARE before electrophoresis on a 4% polyacrylamide gel. Before the incubation with 32P-labeled ARE, an additional aliquot of each sample (30 µg protein, lanes 4, 8, 12, 16, 20, and 24) was treated with ARp4 antibody (29 ) for 60 min. The migration of the specific AR-ARE and antibody (Ab)-AR-ARE complexes is depicted by arrowheads, and the samples treated with AR antibody are identified by the • symbol. Identical results were obtained in repeated experiments. B, The amount of immunoreactive AR proteins in the COS-1 cell extracts used for EMSA. Samples (30 µg protein) from each cell extract were subjected to electrophoresis under denaturing conditions followed by immunoblotting as described in Materials and Methods and the legend to Fig. 2AGo. wt, Wild-type.

 
A promoter interference assay was used to examine AR-ARE interaction in intact cells. In agreement with our previous observations (36), wild-type AR exhibited significant promoter-interfering activity in the presence of 10–100 nM testosterone. All AR mutants interfered with pCMV-ARE2-CAT expression less well than the wild-type protein (data not shown). These observations were in general agreement with the EMSA results and, collectively, they speak for an impaired ability of the AR mutants to recognize specific DNA sequences.

Repression of NF-{kappa}B (RelA) function by the DBD mutants
In addition to transcriptional activation, AR is also capable of transrepressing through cross-modulation of other transcription factors, such as NF-{kappa}B (RelA) (4). That AR DBD is indeed mandatory for the transrepression to occur was established by the use of an AR variant with a large deletion in DBD ({Delta}557–610, see Fig. 1Go): this AR form was inactive with respect to the AR-dependent repression of RelA-activated transcription (Fig. 5Go). A comparable DBD mutant with both zinc fingers destroyed has not been seen in AIS patients; however, deletions of one of the two fingers have been described in CAIS patients (22, 24). In concert with our previous findings (4), deletion of the LBD or most of the amino-terminal transactivation domain did not influence the receptor’s ability to repress RelA function, as studied by the use of a RelA expression vector and reporters driven by six {kappa}B-sites in front of the tk promoter (6 x {kappa}B-LUC) or the proximal human IL-6 promoter (Fig. 5Go, A and B). In the presence of androgen, wild-type AR decreased expression of both reporter genes to 40% (P < 0.01). The R590Q substitution had minimal or no influence on the transrepressive activity of AR, and the K563A mutation attenuated it only marginally. In the case of the 6 x {kappa}B-LUC reporter, G551V was almost completely devoid of the transrepressing activity, and the mutant C562G repressed RelA function only weakly; both differed significantly (P < 0.05) from the activity of wild-type AR (Fig. 5AGo).



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  		Figure 5. Repression of RelA function by wild-type and mutated ARs. A, COS-1 cells (6 x 104) were cotransfected with p{kappa}B6tk-LUC reporter (0.15 µg), pCMV-RelA (0.03 µg), and the expression vectors for the wild-type and mutated ARs (0.15 µg). B, COS-1 cells were transfected as in panel A, except that pIL-6(-225/+11)-LUC (0.15 µg) was used as the reporter. The relative LUC activity in the absence of cotransfected rAR was set as 100% in each case. The cells were exposed to 100 nM testosterone for 30 h. Values are mean ± SEM of three independent experiments.

 
Repression of AP1 by the DBD mutants
In addition to RelA-dependent transcription, AR is also able to down-regulate AP1-activated gene expression (3). To examine how mutations in the DBD influence repression of AP1-dependent reporters by AR, CV-1 or COS-1 cells were transfected with p75–1050CAT or a reporter construct (p-73Col-CAT) driven by the collagenase promoter (Fig. 6Go, A and B). In the presence of testosterone, wild-type AR repressed the expression of these reporters to one-third or less (P < 0.01). G551V, C562G, A579T, and R590Q variants did not modify this repressive function of wild-type AR to any major extent. However, substitution of Lys 563 by Ala (the mutant K563A) either abolished the activity of wild-type AR (p75–1050CAT, Fig. 6AGo) or converted the receptor to an androgen-dependent activator of an AP1-responsive promoter (p-73Col-CAT, Fig. 6BGo). It was also remarkable that a large DBD deletion ({Delta}557–610, Fig. 1Go) totally abolished the repressive activity of AR and resulted into a strong ligand-dependent activation (P < 0.01) of the two AP1-dependent reporters (Fig. 6Go, A and B). As was the case with RelA repression, deletion of the amino-terminal transactivation domain ({Delta}46–408) or the LBD plus most of the hinge region ({Delta}619–902) did not perturb the ability of AR to repress AP1 function.



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  		Figure 6. Repression of AP1 activity by wild-type and mutated ARs. A, CV-1 cells (6 x 104) were cotransfected with p75–1050CAT reporter (0.15 µg) and expression vectors for the wild-type and mutant rARs (0.15 µg), after which they were cultured in the presence of 100 nM testosterone for 30 h. B, p-73Col-CAT was used as the reporter in COS-1 cells, otherwise the experimental design was the same as in panel A. Mean ± SEM values of three independent experiments are shown as percentages (the reporter activity in the absence of rAR = 100%). The numbers 480 (A) and 1490 (B) refer to the mean relative CAT activities measured for the {Delta}557–610 mutant.

 
Interaction of wild-type and mutated AR proteins with CBP
We have previously shown that AR interacts in intact cells with CBP and that competition for CBP is part of the mechanism underlying the repression elicited by AR on AP1- and NF-{kappa}B-activated transcription (18). These studies also revealed that AR LBD is not the only region participating in the formation of the interaction interface between AR and CBP (18). Because our preliminary in vitro experiments indicated that AR DBD is recognized by CBP (data not shown), it was pertinent to determine whether the wild-type receptor and its DBD mutants (in the context of a full-length protein) interacted differently with CBP. This was, however, not the case: similar amounts of the AR variants bound to the GST-CBP(1–1099) fusion protein, whereas a control protein (luciferase) exhibited no binding to CBP (Fig. 7Go). Thus, the dissimilar and selective properties of wild-type AR and AR DBD mutants to activate or repress transcription are not due to their grossly differing ability to interact with the coactivator CBP, at least as judged by this in vitro protein-protein interaction assay.



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  		Figure 7. Interaction of wild-type and mutated AR proteins with CBP in vitro. 35S-Labeled rAR proteins were produced by translation in vitro and incubated with GST alone or GST-CBP-NT adsorbed onto Glutathione Sepharose as indicated. The matrix was subsequently washed, and the bound proteins resolved by PAGE under denaturing conditions and visualized by fluorography (Materials and Methods). A portion (5%) of each AR sample was also subjected to electrophoresis without a preincubation with GST or GST-CBP-NT. Abbreviations: wt, wild-type; luc, luciferase.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Nuclear receptors regulate gene transcription by at least three mechanisms: 1) activation of transcription via binding to cis-acting positive DNA elements, 2) repression of transcription via binding to cis-acting negative DNA motifs, and 3) transcriptional interference through protein-protein interaction with other trans-acting factors (1, 2, 43). The DBDs of nuclear receptors are required for most, if not all, of these mechanisms, even though transcriptional interference, also referred to as cross-modulation, does not have to involve direct DBD-DNA element interactions. Mechanisms used for transactivation and transrepression by steroid receptors are distinct, in that some receptor forms that are unable to transactivate nevertheless repress transcription activated by specific proteins, such as AP1, NF-{kappa}B, Ets, and several other factors (3, 4, 14, 15, 16, 17, 18, 19, 20, 21, 44, 45, 46, 47).

In the present study, we sought to determine how several single amino acid substitutions in the AR DBD influence transactivating and transrepressing properties of the receptor. The results are summarized in Table 1Go and indicate that each mutant has a distinct functional profile. For example, mutants in the first ZF (G551V and C562G), which are not able to transactivate, repress AP1-inducible reporters almost as efficiently as wild-type AR. Their competence to interfere with NF-{kappa}B function is, however, significantly compromised. On the other hand, the K563A substitution abolishes transactivation almost entirely, it does not alter the receptor’s ability to repress NF-{kappa}B function and, finally, instead of down-regulating AP1-dependent transcription, it switches the receptor into an androgen-dependent activator at an AP1 site. The mutants A579T and R590Q, which exhibit reduced DNA-binding activity both in vitro and in vivo, show attenuated transcriptional activity only at a single ARE site and possess close to wild-type repressive actions on both AP1- and NF-{kappa}B-dependent transcription (Table 1Go). In view of these data, it is conceivable that patients suffering from PAIS or CAIS due to mutations in the AR DBD are able to maintain some of the normal functions of the wild-type receptor, even though the actions that require binding of AR to cis-acting DNA motifs (AREs) are lost or severely attenuated.


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  		Table 1. Comparison of relative levels of transcriptional activation [ARE4-tk-LUC and PB(-285/+32)-LUC], RelA repression (6 x {kappa}B-LUC and IL-6-LUC), and AP-1 repression (1050-CAT and -73Col-CAT) of the AR variants

 
Mice with a GR mutation that prevents receptor dimerization and thus glucocorticoid response element (GRE) binding are viable, as opposed to GR null mutant mice that do not survive (48). The dimerization-defective GR does not activate transcription from glucocorticoid-responsive promoters but interferes with AP1-activated transcription (48). These data suggest that glucocorticoid actions dependent on GR-GRE interaction are not essential for survival, whereas the cross-talk with other transcription factors plays a major role. Very little is known about the consequences of androgen insensitivity in the extragenital tissues, such as the immune system, the neural tissues, and the skeleton, the development and function of which are influenced by androgens (22). The effects of androgens in these tissues are most likely mediated by AR, but whether they are dependent on the receptor’s ability to interact with AREs or rely predominantly on cross-talk with other transcription factors has not been addressed. In view of our data, it is easy to envision that AIS patients present varying phenotypes in extragenital tissues, depending on the location of the mutation and its influence on different aspects of AR function.

Patients with the R607Q mutation (corresponding to R590Q in the rAR) display features of PAIS with male phenotype (androgen insensitivity grade 3) (22, 28). Development of breast cancer has been described in connection with this substitution, a condition very rare in men and not known to be associated with other AIS mutations (27, 49). Surprisingly, reduced transcriptional activation was observed for R590Q only with a reporter construct driven by a single ARE and, even then, only at low ligand concentrations. Similar data have been described for another AR DBD mutation in the second ZF, an Ala-to-Thr substitution at position 596 of hAR (A579T in rAR) in a Reifenstein syndrome patient (50). In this case, impaired DNA binding was associated with diminished transcriptional activity from promoters with a single ARE but not with multiple AREs. The results pertaining to DNA binding and transactivation by A579T were confirmed in this work that showed furthermore that transrepressive actions of the A579T mutant on AP1 and NF-{kappa}B function were very similar to those of R590Q. The disparity between DNA binding and transcriptional activation of A579T and R590Q mutants is potentially explainable by the findings of Liu et al. (51), who showed that destabilization of the GR DBD dimer interface through mutations in the second ZF disrupts binding and activity of GR at a single response element but markedly increases the receptor’s synergistic activity on a reporter gene containing multiple DNA elements.

The second ZF of rGR has been reported to be responsible for the interference with NF-{kappa}B/RelA (45). Arg 488 (corresponding to R590 in rAR) and Lys 490 (K592 in rAR) have been identified as critical residues in this cross-modulation. In our current study, substitution of R590 in rAR did not abolish the ability of the receptor to repress RelA-activated transcription. The function of the corresponding Arg in GR differs in other respects as well; in contrast to rAR R590Q, GR with an R488Q substitution binds normally to DNA but fails to transactivate (52, 53). Thus, despite this Arg being conserved among steroid receptors, its role varies depending on the general amino acid composition of the particular receptor’s DNA-binding domain and other functional regions.

Interference with transcription activated by AP1 and NF-{kappa}B/RelA family members is a feature typical of all steroid receptors (14, 15, 16, 17, 18, 54). In the case of the estrogen receptor, modulation of these functions is dependent on the type of ligand and the form of the receptor, whether {alpha} or ß (55, 56, 57). ER{alpha} and ERß signal from an AP1 site in opposite ways when complexed with estradiol-17ß, in that ER{alpha}-estrogen complex activates, whereas ERß-estrogen complex inhibits transcription (56). The action of estrogen, probably mediated by ER{alpha}, at an NF-{kappa}B/RelA site is inhibitory (54). As the transcriptional activities of AR and ERs are expected to be functionally integrated at AP1 and NF-{kappa}B sites, it is an intriguing possibility that AR mutants, such as those affecting AR DBD, would modulate the actions of ER{alpha} or ERß at these sites in fashions that are either identical with or opposite to those by the native AR. It remains to be established whether this has any bearing on the association of the R607Q and R608K substitutions with male breast cancer (27, 49). Collectively, AR DBD mutants in AIS patients are not functionally inert and can potentially modulate transcriptional activity of other proteins, including other nuclear receptors, which may be an important contributing factor to the wide spectrum of presentations in androgen insensitivity.

Steroid receptors, including AR, convey their activating or repressing actions on the transcription machinery via interaction with a growing number of bridging proteins, usually referred to as coactivators and corepressors (58). Altered interaction of AR mutants with these proteins is likely to participate in the development of AIS phenotype, even though such an alteration was not observed in this work between CBP and AR DBD mutants in vitro. Most of the coactivators and corepressors interact with the LBDs of nuclear receptors (58) and, therefore, a mutation in AR LBD could potentially maintain androgen-binding ability but lead to the development of AIS owing to impaired interaction with coactivators. In a similar fashion, AR DBD mutants may exhibit attenuated ability to recognize auxiliary proteins participating in transcriptional activation, such as those recently shown to interact with the zinc finger region of AR (42, 59, 60).


   Acknowledgments
 
The authors would like to thank Drs. Patrik Baeuerle, Benita S. Katzenellenbogen, Tom Kerppola, Tony Kouzarides, Robert J. Matusik, Mart Saarma, and Bernd Stein for plasmids.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council (Academy of Finland), the Emil Aaltonen Foundation, the Sigrid Jusélius Foundation, the Research and Science Foundation of Farmos, the Paulo Foundation, and Biocentrum Helsinki. Back

Received October 14, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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