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Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 540 (V.G., S.L., C.S., J.-C.N.); Centre de Biochimie Structurale (W.B.), Centre National de la Recherche Scientifique (CNRS); Université Montpellier 1, F-34090, Montpellier, France; Laboratoire d’Hormonologie du Développement et de la Reproduction (S.L., C.S.), Hôpital Lapeyronie, Centre hospitalier Universitaire, F34 295, Montpellier, France; and Service de Pédiatrie (S.M.), Hôpital d’Enfants de Tunis, 1000 Tunis, Tunisia
Address all correspondence and requests for reprints to: Dr. Jean-Claude Nicolas, Institut National de la Santé et de la Recherche Médicale, Unité 540, Endocrinologie Moléculaire et Cellulaire des Cancers, 60 rue de Navacelles, 34090 Montpellier, France. E-mail: nicolas{at}montp.inserm.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The unliganded AR (apo-receptor) is primarily localized in the cytoplasm. Upon ligand binding (holo-receptor), AR is translocated into the nucleus, moves to different subnuclear sites, binds to specific DNA sequences, and activates androgen-specific genes through protein-protein interactions with coregulatory proteins and general transcription factors. Like other members of the NR superfamily, AR is modular and includes an amino-terminal region containing a ligand-independent transcriptional activation domain [activation function (AF)-1], a central DNA-binding domain that specifically recognizes response elements upstream of target genes, and a carboxy-terminal ligand binding domain (LBD). The LBD is a multifunctional domain, capable of ligand binding, dimerization and interaction with transcriptional coregulators that enhance (coactivators) or decrease (corepressors) the transcriptional activity of the receptor.
The crystal structures of many NR LBDs, including AR, have now been determined, revealing a conserved fold and a common mechanism of activation (3, 4). Upon agonist binding, NR LBDs undergo conformational changes in such a way that some residues belonging to helices H3, H4, and H11 are clustered to form a predominantly hydrophobic surface onto which the apolar side of the highly mobile C-terminal LBD helix H12, encompassing the core of the activation function-2 (AF-2), binds in a stable conformation. In this so-called active- or holo-conformation, helices H3, H4, and H12 define a binding surface that specifically interacts with the LxxLL motifs of coactivators or with the closely related FXXLF motif contained in the amino-terminal (AF-1) domain of AR (5). Binding of antagonists prevents the formation of this specific surface by interfering directly or indirectly with the active conformation of H12.
Mutations in the AR gene can alter the receptor function, which leads to androgen insensitivity syndrome (AIS), a major cause of male pseudohermaphroditism. AIS encompasses a wide spectrum of undervirilization phenotypes ranging from complete AIS in subjects with female phenotype to partial AIS (PAIS) in men with isolated infertility. Mutation of a single amino acid in the AR LBD can lead to abnormalities in androgen binding (6), active conformation stability (7), or interaction with coactivators (8). For example, it has been shown that some AR mutants display altered ligand specificity, resulting in activation of gene transcription upon binding of antiandrogens or other related steroids (9, 10, 11). Recently, Hara et al. (12) described the W741L mutation that converts the antiandrogen bicalutamide into a partial agonist. This AR gene mutation is thought to contribute to the bicalutamide withdrawal syndrome observed in the treatment of prostate cancer. The characterization of such mutations significantly contributes to our understanding of the structure-function relationships of AR.
Here, we report on a functional and structural study of the ARE709K mutant that we identified in a patient referred in the neonatal period for micropenis (15 mm) and perineoscrotal hypospadias. The same mutation was previously isolated from a patient with a similar PAIS phenotype (13) as well as from a yeast genetic screening (14). We introduced several mutations at position 709 and tested the consequences of these changes on AR structure and activity in the presence of androgens and antiandrogens. These studies allowed us to characterize the functional defects of ARE709K and to provide a molecular rationale for the PAIS phenotype associated with the mutant. Moreover, we demonstrate the key role of glutamic acid 709 in the stabilization of the active conformation of AR and in androgen and antiandrogen activities.
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RESULTS |
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After verification by Western blot of expression levels in transfected cells (Fig. 1A
), the functional properties of these mutants were evaluated and compared with those of the wild-type receptor (wt-AR). The maximum binding capacities were in the same range for wt-AR and AR variants (Fig. 1B; 558, 614, 507, and 529 fmol/mg protein for wt-AR, ARE709K, ARE709A, and ARE709Y, respectively). The ligand-binding and transactivation properties of the ARE709Y mutant were almost identical to those of wt-AR, with apparent equilibrium binding affinity (Kd) values of 0.25 nM and 0.19 nM (Fig. 1B), respectively, and an EC50 of 2.10–11 M (Fig. 1D). In contrast, substitution of E709 with lysine or alanine slightly altered the Kd (0.82 and 0.42 nM, respectively) (Fig. 1B), while increasing the dissociation rates of R1881-receptor complexes (t1/2 of 25 and 27 min for ARE709K and ARE709A, respectively, vs. 60 min for wt-AR) (Fig. 1C). Similarly, the hormone-response curves for transcriptional activity of ARE709K and ARE709A were shifted in EC50 from 2.10–11 M to 2.10–10 M (Fig. 1D).
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). 10–8 M R1881 was sufficient to protect the receptor in a 29-kDa resistant fragment previously demonstrated as being characteristic of AR holo-conformation (16). Similarly, all three AR mutants were also digested in the absence of ligand. However, although 10–8 M R1881 was sufficient to stabilize ARE709Y in the 29-kDa resistant band, a maximum protection of ARE709K and ARE709A mutants was only obtained with 10–6 M R1881.
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, E709 in helix H3 is solvent-exposed and projects toward helix H12 to form hydrogen bonds with the backbone amides of M894 and M895 (17, 18). Therefore, E709 belongs to the H3/H4/H11 surface and participates in the stabilization of holo-H12. Of note, a similar stabilizing interaction was previously observed in ER
between D351 (homologous to E709 in AR) and the backbone amides of L539 and L540 from H12 (19, 20, 21, 22, 23). Our biochemical and functional analyses revealed that losing this interaction by substitution for glutamate by alanine or lysine at amino acid 709 produces AR mutants with altered holo-conformation stability and reduced transcriptional potency. Surprisingly, when E709 was substituted by a tyrosine residue, neither the stability nor the activity of AR was affected, suggesting that at least some H3-H12 stabilizing interactions are restored in this mutant. Indeed, a constructed model of the ARE709Y variant reveals that, in opposition to smaller or linear amino acids like alanine or lysine, the bulky tyrosine residue can form improved van der Waals contacts with the side chains of residues P892, E893, M894, and M895 of H12 (3.92 Å, 4.25 Å, 3.77 Å, and 4.12 Å, respectively), thereby stabilizing holo-H12 (Fig. 3B).
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Mutations at Position 709 Convert Cyproterone Acetate to a Pure Antiandrogen and Bicalutamide to a Partial Antiandrogen
We investigated the agonist and antagonist properties of antiandrogens on the various AR constructs. In this study, we used steroidal (cyproterone acetate; CPA) and nonsteroidal (hydroxyflutamide, nilutamide, and bicalutamide) antiandrogens (Fig. 4). Antagonist activities were measured by using increasing concentrations of antiandrogen in competition with a constant R1881 concentration. The R1881 concentration used in each experiment was proportional to the EC50 obtained in R1881-induced transcriptional activity (10–10 M R1881 for wt-AR and ARE709Y, and 10–9 M R1881 for ARE709K and ARE709A).
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, whereas CPA displayed partial agonist activity on wt-AR, it exhibited almost no activity on either mutant. Accordingly, 10–6 M CPA only partially reduced the transcriptional activity of wt-AR induced by R1881, whereas it drastically inhibited the R1881-dependent activity of the AR mutants. CPA inhibited the androgen-induced wt-AR activity at lower concentrations than those required with ARE709Y, revealing a decreased affinity of CPA for this variant. Except with hydroxyflutamide at very high concentration, none of the mutations affected the androgenic or antiandrogenic profile of hydroxyflutamide or nilutamide (Fig. 4). Interestingly, whereas the ARE709K and ARE709A mutations did not significantly affect the agonist properties of bicalutamide (Fig. 4), the ARE709Y mutant displayed an increased transcriptional activity in response to this antiandrogen, which reached 55% of maximal transactivation. Accordingly, bicalutamide inhibited the R1881-induced luciferase activity of ARE709K and ARE709A as efficiently as that of wt-AR, whereas only a partial deactivation of ARE709Y was obtained. Moreover, bicalutamide inhibited the R1881-induced activity at the lowest concentration used (10–8 M), indicating an increased affinity of bicalutamide for ARE709Y. These data clearly demonstrate that mutations E709K and E709A abolish the agonist activity of antiandrogens, thereby increasing their antagonist potential, and that substitution of E709 by a tyrosine transforms CPA from a partial antagonist into a pure antiandrogen, and bicalutamide from a pure antiandrogen into a partial agonist.
To evaluate the impact of mutations at position 709 on the antagonist-induced AR conformations, we performed limited proteolysis assays (Fig. 5). In line with its partial androgenic character, 10–7 M CPA stabilized wt-AR in a conformation providing a major 35-kDa resistant form reported to correspond to a receptor inactive conformation (16), whereas a clear 29-kDa band characteristic of the active AR conformation appeared at higher CPA concentration. Proteolytic digestion of wt-AR incubated with the antiandrogens hydroxyflutamide, bicalutamide or nilutamide resulted in a unique 35-kDa-resistant fragment. In full agreement with our functional data, we observed that all three AR mutants were protected in a major 35-kDa fragment in the presence of 10–7 M and 10–5 M CPA, hydroxyflutamide, and nilutamide. Bicalutamide stabilized ARE709K and ARE709A in an inactive conformation providing a 35-kDa-resistant fragment, whereas a clear additional 29-kDa band appeared with ARE709Y. The simultaneous presence of the two resistant fragments suggests that helix H12 of ARE709Y is in equilibrium between the inactive and active conformations in the presence of bicalutamide.
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Coactivator and Corepressor Interactions with wt-AR and ARE709Y
Paralleling our observations, it has been previously reported that the mutation homologous to ARE709Y in ER (ERD351Y) changes the pharmacology of raloxifene or tamoxifen by shifting their antiestrogenic activity to an estrogenic activity (19, 20, 21, 22, 23). Whereas tamoxifen promotes association between wt-ER and corepressors, Yamamoto et al. (24) reported that ERD351Y exhibits a reduced tamoxifen-induced interaction with nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and consequently a high tamoxifen-induced AF-1 activity. Therefore, to determine whether the partial agonistic activity of bicalutamide, in the context of ARE709Y, is due to a gain of coactivator interaction or a loss of corepressor binding, we analyzed the coregulator recruitment by wt-AR and ARE709Y in the presence of CPA and bicalutamide. Interactions of wt-AR and ARE709Y with the corepressor SMRT (25) or the coactivactor transcription intermediary factor 2 (TIF2) (a member of the p160 family of coactivators;26) were monitored using a modified mammalian two-hybrid system. In this assay, interaction between full-length AR and the VP16 (herpes simplex viral protein 16) fusions of the interacting domains of TIF2 (TIF2-ID) and SMRT (SMRT-ID) leads to an increase in luciferase gene expression. Figure 6 shows that both SMRT-ID and TIF2-ID interacted with R1881-bound wt-AR and ARE709Y with, however, a 10-fold stronger two-hybrid signal obtained with the coactivator. None of the antihormones tested promoted a significant interaction between SMRT-ID and wt-AR or ARE709Y. When wt-AR was cotransfected with TIF2-ID, a partial CPA-induced luciferase activity was obtained. In contrast, the partial agonist activity of CPA was almost completely abolished when using ARE709Y. Conversely, bicalutamide was able to induce a clear interaction of TIF2-ID with ARE709Y but not with wt-AR.
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shows that TIF2 redistribution was observed when wt-AR was coexpressed in the presence of R1881 or CPA. Wt-AR complexed to bicalutamide failed to recruit TIF2 in the nucleoplasm because there was no coactivator redistribution (Fig. 7). ARE709Y redistributed TIF2 after addition of R1881 or bicalutamide, whereas CPA treatment did not affect TIF2 localization in the nuclear foci.
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D351Y, the interaction between ARE709Y and SMRT-ID in the presence of CPA or bicalutamide remains unchanged relative to wt-AR. |
DISCUSSION |
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Our biochemical and functional experiments demonstrate that the ARE709K and ARE709A mutations destabilize the AR-androgen complex with an increased dissociation rate and a lower protection from trypsic digestion. This structural instability explains the partial transactivation capacities exhibited by these mutants bound to the agonist R1881. Yeh et al. (13) previously described the ARE709K mutation in a patient with PAIS. Their in vitro studies suggested that this AR mutant displays only a slightly reduced DHT-mediated AR activity, with, however, a single concentration of DHT tested (13, 30). The authors explored the response of AR to estrogens and proposed that the consequence of losing the estradiol-ARE709K-ARA70 pathway while maintaining the DHT-ARE709K-ARA70 pathway might likely be one of the explanations for the observed PAIS (13). The deficiency of androgen action that we describe in the present report appears more relevant to explain this PAIS phenotype with respect to what is known of androgen action for sex differentiation. Surprisingly, our data show that mutation of E709 by a tyrosine has no significant effect on the transcriptional activity and coactivator recruitment of androgen-bound AR. Molecular modeling and conformational studies suggest that van der Waals contacts between Y709 and some side-chain residues of H12 might help to stabilize the active conformation of the ARE709Y/agonist complex, whereas the stabilizing interactions between holo-H12 and E709 are completely abolished in the ARE709K and ARE709A mutants.
We next examined the contribution of glutamic acid 709 to the antagonist activity of several partial and full antiandrogens. We first substantiated the partial agonist/antagonist character of CPA, which appears unable to fully stabilize the proper H3/H4/H11 surface required for holo-H12 positioning. Indeed, the weak androgenic activity displayed by CPA and the proteolytic digestion pattern showing the simultaneous presence of the 29- and 35-kDa-resistant bands suggest that AR can adopt both active and inactive conformations when bound to CPA. Therefore, in contrast to pure antagonists like hydroxyflutamide, bicalutamide, or nilutamide that almost completely abrogate the active conformation of AR LBD, CPA allows the active conformation of H12 to be reached transiently, thereby accounting for its partial androgenic activity.
All the 709 AR mutations tested abrogated the residual androgenic activity displayed by CPA. The drastic effect of the mutations on the activation profile of CPA can be simply explained by the fact that the binding of a full agonist like DHT or R1881 induces the optimal H3/H4/H11 surface, offering maximum productive interactions between the LBD core and holo-H12, whereas only a subset of these interactions is generated in the presence of CPA. In consequence, whereas in the AR/R1881 complex the presence of E709 is not absolutely required for at least partial AR activation, the substitution of this residue completely abolishes the residual androgenic activity of CPA because the few remaining van der Waals contacts between H12 and the LBD core cannot compensate for the loss of interaction between E709 and holo-H12.
None of the mutations tested had a significant effect on the antiandrogenic profile of hydroxyflutamide or nilutamide. In contrast, the E709Y mutation transformed the antiandrogen bicalutamide into a partial agonist. To support this finding, we provided experimental evidence that the replacement of E709 by a tyrosine allows bicalutamide to induce AR active conformation and its interaction with coactivators. Due to the poor structural homology between steroidal and nonsteroidal ligands, no attempt was made to build a model for the antagonist-bound AR LBD. However, because of the modest molecular size of hydroxyflutamide and nilutamide and because they lack a bulky side chain, it is very likely that these ligands antagonize AR through the molecular mechanism previously described as passive antagonism (31). Conversely, due to its larger size, it is unlikely that bicalutamide can be contained within the agonist-binding cavity of AR. The x-ray structure of the ARW741L LBD mutant bound to bicalutamide was recently reported (32). This mutation confers agonist activity to bicalutamide (33) and, indeed, an active (holo) conformation of the ARW741L/bicalutamide complex was observed in the crystal. Although the structure does not provide direct experimental information on the structural antagonist mechanism of bicalutamide, it demonstrates that the sulfonyl-linked phenyl ring (B-ring) of the antiandrogen is accommodated at the location of the indole ring of W741 in wt-AR. Therefore, in wt-AR, the B-ring of bicalutamide probably protrudes out of the opening to the binding pocket between helices H3 and H11, thereby preventing H12 from adopting the active conformation. The gain in androgenic activity of bicalutamide in the context of ARE709Y is difficult to reconcile convincingly on the basis of our current structural knowledge. Further structural work will be required to understand how bicalutamide can stabilize the holo-conformation of ARE709Y and how the mutation increases the affinity of bicalutamide for the receptor.
In conclusion, we analyzed the functional role of E709 in helix H3 of AR LBD. We provide evidence that the H3/H12 interaction mediated by E709 is required for optimal androgen-dependent coactivator recruitment and transcriptional activation. Furthermore, the relative agonist vs. antagonist activity of antiandrogens is highly dependent on the side-chain residue at position 709. Our data show that the mutation of E709 to tyrosine suffices to abolish the androgenic activity of CPA and to transform bicalutamide into a partial androgen. Both our structural and functional data reveal the key role of glutamic acid 709 for androgenic and antiandrogenic activities.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Direct Sequencing of Patient DNA
DNA was extracted from peripheral blood leukocytes. The eight exons and flanking intron regions of the human AR gene were screened for mutations by direct sequencing of the amplified PCR products, using sets of primers previously described (34) and an ABI Prism Big Dye Terminator Sequencing kit (Applied Biosystems, Courtaboeuf, France). The sequencing reactions were analyzed on a ABI 310 genetic analyzer.
Construction of the Variant AR cDNA Expression Vectors
To obtain the natural mutant vector (ARE709K), a KpnI-BamHI fragment (containing both DNA- and androgen-binding domains) was cleaved from pSG5-human AR (hAR) and subcloned into the corresponding site of pUC19 (pUC19-hAR). The amplified fragment of the patient’s exon 4 was digested by Tth 111-I and StuI, and the purified fragment was ligated to pUC19-hAR, whose normal Tth 111-I and StuI fragment was previously cleaved. Finally, the mutated exon 4 cloned into the KpnI-BamHI fragment was inserted into the corresponding sites of pSG5-hAR to yield pSG5-hAR-E709K, the expression vector of mutated AR.
Artificial E709A and E709Y mutants were obtained in AR cDNA in pSG5-hAR by site-directed mutagenesis (Stratagene, La Jolla, CA) using PCR amplification with specific primer containing mutation, and digestion of parental DNA with DpnI according to the manufacturer’s procedures. The primers used were:
5'-GCCTCAATGAACTGGGAGCGAGACAGCTTGTACACGTGG-3' for E709A, 5'-GCCTCAATGAACTGGGATATCGACAGCTTGTACACGTGG-3' for E709Y, and the complementary sequences for antisense primers. Amplified and DpnI digested products were directly transformed into Escherichia coli DH5 (Life Technologies, Cergy, France). Different clones were amplified and the extracted DNA was sequenced to verify the mutation insertion.
The plasmid pECFP-AR was obtained by insertion of AR excised by the NheI and BglII restriction sites from pGFP-AR, as previously described (35), into pECFP-C1 (CLONTECH, Palo Alto, CA). pECFP-ARE709Y was constructed by exchange of the KpnI-PvuI fragment from pSG5-hAR-E709Y containing the mutation with the identical fragment into pECFP-wt-AR. pEYFP-TIF2 was produced by insertion of TIF2 cDNA amplified by PCR from psg5-TIF2 into pcDNA3-EYFP by KpnI and EcoRI cloning sites. All amplifications were performed in E. coli DH5, and all steps were verified by sequencing.
Coupled in Vitro Transcription and Translation
Expression plasmids (pSG5-hAR wild-type or mutants) were transcribed and translated with the TNT T7 Quick Coupled Transcription/Translation System (Promega, Charbonnieres, France) in the presence of [35S]methionine (1000 Ci/mmol; Amersham, Orsay, France), according to the manufacturer’s instruction, for 2 h at 30 C.
Limited Proteolysis Assays
Five microliters of [35S]-receptor synthesized in vitro were preincubated at 37 C for 30 min with 0.5 µl of vehicle or ligand. Limited proteolysis was performed by the addition of 5 µl of various trypsin amounts (0, 25, and 50 µg/ml; final concentration). Incubations with protease were conducted at 27 C for 10 min and stopped by the addition of 10 µl of sodium dodecyl sulfate (SDS) sample buffer and chilling on ice. The samples were boiled for 5 min. The products of proteolysis were separated on a 0.75-mm-thick, 12% SDS polyacrylamide gel. After electrophoresis, the gels were washed in distilled water and vacuum-dried for 20 min. Gels were exposed to a Fujix (Tokyo, Japan) film imaging plate for 1 h and to autoradiography overnight. Band intensity was semiquantified with Fujix software.
Cell Culture
COS-7 cells and CV-1 cells were cultured in DMEM (Life Technologies) supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 µg/ml) in a humidified atmosphere containing 5% CO2. Cells were transiently transfected using the calcium phosphate DNA precipitation method. After overnight incubation, precipitates were removed and replaced by fresh DMEM without fetal calf serum.
Immunoblot
In 100-mm dishes, COS-7 cells (109 cells) were transfected with 10 µg of pSG5-hAR wt or mutants. Forty-four hours later, cells were lysed directly in the dish with 50 µl of lysis buffer [40 mM Tris (pH 7.4), 1 mM EDTA, 10% glycerol, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.08% SDS] supplemented with a cocktail of protease inhibitors (Sigma, Saint Quentin Fallavier, France). The cellular debris was pelleted at 13,000 x g for 10 min and the protein concentration was determined on a supernatant aliquot by the Lowry quantification method. The lysate was mixed vol/vol with SDS sample buffer and boiled for 10 min. The total proteins (50 µg) of each extract were subjected to 10% SDS-PAGE and Western transfer by electroblotting. Nitrocellulose filters were saturated in TBS [20 mM Tris-HCl (pH 7.4), 500 mM NaCl] plus 10% milk and 0.05% Tween 20 at room temperature. Filters were then incubated with polyclonal anti-AR antibody (SpO61) diluted 1/2000 (36) and antiactin (Sigma) diluted 1/2000, followed by hybridation of peroxidase-conjugated antirabbit IgG diluted 1/5000 (Amersham). Blots were developed using the ECL chemiluminescent detection system (Pierce, Rockford, IL).
Androgen-Binding and Dissociation Assays
To determine the binding characteristics of the hAR mutants compared with the wild-type receptor, COS-7 cells were transiently transfected in 12-well dishes with 50 ng of pSG5-hAR wild-type or mutant and 250 ng of pCMV-ß-galactosidase. After 48 h at 37 C, the transfected cells were incubated at 37 C for 2 h with increasing concentrations (0.05–4 nM) of [3H]R1881 for total binding. Nonspecific binding was measured in parallel incubations containing an additional 1000-fold molar excess of radioinert ligand. In parallel, the dissociation rate of the R1881-receptor complex in COS-7 cells was measured in transfected cells by incubation with 4 nM of [3H]R1881 at 37 C for 2 h, followed by the addition of a 1000-fold excess of unlabeled ligand for various times. Nonspecific binding was determined from cells that were treated with 4 nM [3H]R1881 in the presence of a 1000-fold excess of unlabeled R1881.
The cells were harvested in lysis buffer: 25 mM Tris-H3PO4 (pH 7.8), 2 mM dithiothreitol, 2 mM EDTA, 1% Triton X-100, and 10% glycerol. Aliquots were used for radioactivity measurement, ß-galactosidase activity, and protein assay. After subtraction of nonspecific from total binding, the dissociation constants (Kd) and the maximum androgen-binding sites (Bmax) were derived from Scatchard plots, and the percentage of remaining basal R1881-binding was plotted semi-logarithmically against time.
Transfection and Luciferase Activity Assay
CV-1 cells or COS-7 cells were transiently transfected in 12-well dishes with 50 ng of pSG5-hAR, 250 ng of pCMV-ß-galactosidase to correct for transfection efficiency, and 0.5 µg of p-mouse-mammary-tumor-virus-luciferase (MMTV-luc). For the modified mammalian two-hybrid experiments, 0.5 µg of psg5-, -VP16-TIF2 (interaction domain) or -VP16-SMRT (interaction domain) was added. Precipitate was removed after 16 h and cells were maintained in DMEM with vehicle alone or various ligand concentrations. After 30 h, the cells were lysed by 300 µl of the lysis buffer described above. Luciferase activity was measured by the reaction of lysate with luciferin solution: 270 µM coenzyme A, 470 µM luciferin, 530 µM ATP, 20 mM Tricine (pH 7.8), 1.07 mM (MgCO3)4 Mg(OH)2 5H2O, 2.67 mM MgSO4 and 1 mM EDTA. Luciferase activity was measured on a centro LB960 luminometer (Berthold, Thoiry, France). Each incubation was performed in duplicate. ß-Galactosidase activity was determined to control the efficiency of each transfection. The presented results are the averages of three independent experiments.
Microscopy and Imaging Analysis
COS-7 cells were cultured on coverslips and then transfected with 1 µg of plasmids using 3 µl/dish of Fugene reagent (Roche, Meylan, France). Twenty-four hours after transfection, the culture medium was replaced with serum-free DMEM for overnight starvation. Cells were incubated with R1881 (10–8 M) or antihormones (10–6 M) for 8 h, fixed with 4% paraformaldehyde for 15 min, washed three times with PBS and mounted on slides with Dako (Carpinteria, CA) mounting medium. The cells were imaged using confocal laser scanning microscopy (Leica SP2 UV system; Leica Microsystems, Heidelberg, Germany). The cells were imaged for cyan fluorescence by excitation with the 457-nm line from an argon laser, and emission was viewed through a 460- to 490-nm band pass filter. The yellow fluorescence was scanned using the 514-nm excitation line and the 520- to 550-nm band pass filter as emission filter.
Receptor Modeling
The ARE709Y LBD model was generated from the AR/DHT crystal structure (18). The E709Y substitution was incorporated using the program O (37) and a conjugate gradient energy minimization was performed with CNS (38). To avoid deviations of the LBD fold during minimization, a harmonic restraint of 10 kcal/Å2 was applied to all main-chain atoms and crystallographic symmetry-related molecules were incorporated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present address for S.L.: Laboratoire de Biochimie, Hôpital Carémeau, Centre Hospitalier Universitaire, Nîmes 30000, France.
Potential conflicts of interest: Authors have nothing to declare.
First Published Online December 22, 2005
Abbreviations: AF, Activation function; AIS, androgen insensitivity syndrome; AR, androgen receptor; Bmax, maximum androgen binding; CFP, cyan fluorescent protein; CPA, cyproterone acetate; DHT, dihydrotestosterone; ECFP, enhanced CFP; EYFP, enhanced YFP; hAR, human AR; Kd, dissociation constant; LBD, ligand binding domain; MMTV-luc, mouse-mammary-tumor-virus-luciferase; NR, nuclear receptor; PAIS, partial AIS; SDS, sodium dodecyl sulfate; SMRT, silencing mediator of retinoic acid and thyroid hormone receptor; T, testosterone; TIF, transcription intermediary factor; VP16, herpes simplex viral protein 16; wt-AR, wild-type AR; YFP, yellow fluorescent protein.
Received for publication October 21, 2005. Accepted for publication December 13, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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is not crucial for the antagonist activity of antiestrogens. J Biol Chem 275:20867–20872 mutant (D351Y) shows weak AF-2 activity in the presence of tamoxifen. J Biol Chem 275:37552–37558 mutant D351Y shows reduced tamoxifen-dependent interaction with corepressor complexes. J Biol Chem 276:42684–42691NURSA Molecule Pages Link:
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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, wt-AR; 
, ARE709K; 
, ARE709A; 
, ARE709Y). B/F, Bound/free; E, glutamic acid; K, lysine; A, alanine; Y, tyrosine.
