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Departments of Geriatric Medicine (H.K., Y.W., K.O., R.-H.T., R.T.) and Medicine and Bioregulatory Science (T.O., T.Y., H.N.), Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan; and Department of the Second Anatomy (K.N.), Kurume University School of Medicine, Kurume, 830-0011, Japan
Address all correspondence and requests for reprints to: Ryoichi Takayanagi, Department of Geriatric Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: takayana{at}geriat.med.kyushu-u.ac.jp.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Objective: ARs with mutations in the DNA-binding domain were functionally characterized and compared with the wild-type AR.
Patients: In a complete AIS patient (subject 1), cysteine residue 579 in the first zinc finger motif of AR was substituted for phenylalanine (AR-C579F). Another mutation (AR-F582Y) was found in a partial AIS patient (subject 2).
Results: AR-F582Y retained less than 10% of the transactivation activity of the wild-type AR, whereas no ligand-dependent transactivation was detected for AR-C579F. Image analyses of the receptors fused to green fluorescent protein showed that the wild-type AR was ligand-dependently translocated into the nucleus in which it formed fine subnuclear foci. Surprisingly, after the addition of dihydrotestosterone, the two mutant ARs initially formed large cytoplasmic dots, many of which were found to be close to mitochondria by electron microscopy. Subsequently, a part of the ligand-bound mutant ARs gradually entered the nucleus to form a smaller number of larger dots, compared with the wild-type AR. Fluorescence recovery after photobleaching analysis revealed that the intranuclear mobility of the mutant ARs decreased, compared with that of the wild-type AR.
Conclusions: These results suggest that the abnormal translocation, localization, and mobility of the mutant ARs may be the cause of AIS in these subjects.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Unliganded ARs are located in the cytoplasm, in which they are sequestered with heat shock proteins. After ligand binding, a conformational change of the receptor protein results in unmasking of both the dimerization motif and the nuclear localization signal that allows translocation into the nucleus (1). Upon nuclear entry, the ligand-receptor complexes appear to move into subnuclear compartments, which are common congregation sites for steroid hormone receptors and other associated factors, such as nuclear receptor coactivators, that are required for transcriptional activation of the target genes. Complete subnuclear foci formation seems to be essential for steroid hormone receptor-mediated transactivation (4, 5, 6).
Because the human AR gene is located on the X chromosome at Xq11–12 (1, 4, 7), just a single allele mutation in the AR gene causes dysfunction of the receptor in 46, XY individuals, resulting in androgen insensitivity syndrome (AIS) (3, 8, 9, 10). Despite a high or normal level of serum testosterone, AIS patients show various phenotypic abnormalities of male sexual development. AIS is subdivided into three phenotypes: complete androgen insensitivity syndrome (CAIS), partial androgen insensitivity syndrome (PAIS), and mild androgen insensitivity syndrome. AR mutations that severely impair the function of the AR cause CAIS. The main phenotypic characteristics of individuals with CAIS are female external genitalia with a short, blind-ending vagina, absent Müllerian duct, and absence of pubic and axillary hair. The gender identity is that of a normal female, but testes are commonly located in either the abdomen or the inguinal area and the uterus is absent. Laboratory findings show the 46, XY karyotype, normal or increased synthesis of testosterone by the testes, and a normal or increased level of LH. PAIS patients also show female-like external genitalia, except for clitoromegaly and/or posterior labial fusion (3, 9). Although mutations responsible for AIS are spread throughout the AR gene, there are hot spots, especially at exon 5, which encodes part of the LBD (Androgen Receptor Gene Mutation Database: www.mcgill.ca/androgendb).
We previously reported two unrelated AIS patients (one CAIS and one PAIS) carrying amino acid substitutions in the first zinc finger motif of the AR-DBD (11). In the CAIS patient (subject 1), cysteine residue 579, which coordinates a zinc ion, was substituted with phenylalanine, and the mutant AR showed almost no ligand-induced transcriptional activation. On the other hand, the AR-F582Y mutant found in the PAIS patient (subject 2) showed much less transactivation than the wild-type AR. Here, to visualize any dynamic changes of the intracellular localizations of these mutant ARs, the wild-type and mutant ARs were fused with green fluorescent protein (GFP) and their intracellular movements were analyzed using a laser confocal microscope. After treatment with the ligand, the wild-type AR was translocated from the cytoplasm into the nucleus in which it formed fine subnuclear foci. In contrast, the AR-DBD mutants initially formed large cytoplasmic dots, many of which were located close to mitochondria after the addition of dihydrotestosterone (DHT), and a proportion of the proteins subsequently moved into the nucleus to become located in subnuclear bodies. The subnuclear foci of the mutant ARs were characterized by their larger size, much smaller number, and lower mobility, compared with the wild-type AR. These results indicate that the pathogenesis of AIS in these two patients may be caused by the mislocation and lower mobility of their mutant ARs.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects 1 and 2 were diagnosed with CAIS and PAIS, respectively, and had missense mutations of Cys579 to Phe and Phe582 to Tyr, respectively. The clinical history and data of the AIS patients characterized in the present study were previously reported (11).
Cells
COS-7 monkey kidney cells were obtained from the Riken Cell Bank (Tokyo, Japan) and maintained in DMEM (Invitrogen, Carlsbad, CA) containing antibiotics and 10% fetal bovine serum (FBS; Cansera International Inc., Etobicoke, Canada).
Plasmids
The firefly luciferase reporter plasmids, pGL3-MMTV (5) and pGL3-PSA (12), and the expression vectors for AR (pCMV-AR) were prepared as previously described (13, 14). The plasmid vectors pAR-GFP (5) and pAR-CFP (6) for expression of the AR-GFP and AR-CFP fusion proteins were constructed as described previously. Expression plasmids of the mutant ARs for mammalian cells, designated pCMV-AR-C579F (from subject 1) and pCMV-AR-F582Y (from subject 2), were constructed as previously reported (11). To construct a GFP fusion protein of the mutant AR for subject 1, a KpnI-ScaI fragment of pCMV-AR-C579F, which contained the mutated site, was replaced with that of pAR-GFP to generate pAR-C579F-GFP. Similarly, for subject 2, a HindIII-ScaI fragment of pAR-GFP was replaced with that of pCMV-AR-F582Y to construct pAR-F582Y-GFP. The validity of the structure of each construct was confirmed by DNA sequencing using an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA).
Immunoblotting
COS-7 cells were seeded in 100-mm plates and incubated for 24 h in 5% CO2 at 37 C. Five micrograms of plasmid DNA carrying the wild-type or a mutant AR was transfected into the cells using 20 µl Superfect transfection reagent (QIAGEN GmbH, Hilden, Germany). Twenty-four hours after the transfection, the cells were washed twice with PBS and then 400 µl of Nonidet P-40 lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% Nonidet P-40] was added to each dish, followed by rocking for 30 min at 4 C. Lysates were collected by centrifugation and the protein concentrations were measured using a BCA protein assay kit (Pierce, Rockford IL). Next, 40 µg of each lysate in 1x SDS-PAGE sample buffer [2% sodium dodecyl sulfate, 100 mM dithiothreitol, 60 mM Tris-HCl (pH 6.8), and 0.01% bromophenol blue] was loaded onto a sodium dodecyl sulfate-polyacrylamide gel (10% separating gel) and electrophoresed at 20 mA for 4 h. Proteins were transferred onto nitrocellulose membrane (Hybond P; Amersham Biosciences, Piscataway, NJ) using a Trans-Blot SD Semi-Dry transfer cell (Bio-Rad Laboratories, Hercules, CA) at 250 V for 1 h at 25 C. After blocking the membrane in 1x Block-Ace (Dainippon Pharmaceutical Co., Osaka, Japan), an anti-AR polyclonal antibody (sc-816; Santa Cruz Biotechnology Inc., Santa Cruz, CA) was reacted with the membrane in 0.1x Block-Ace for 1 h at 25 C. After a brief wash with Tris-buffered saline-Tween 20 (10 mM Tris-HCl, 0.9% NaCl, and 0.05% Tween 20), horseradish peroxidase-conjugated antirabbit Ig antibodies (Amersham Biosciences) were added in 0.1x Block-Ace as the secondary antibody, and the membrane was incubated for 45 min at 25 C with gentle shaking. After washing with Tris-buffered saline-Tween 20, the membrane was reacted with Western blotting detection reagents (Amersham Biosciences) for 1 min in a dark room. The labeled protein bands were visualized and analyzed using a VersaDoc imaging system 5000 (Bio-Rad).
Functional reporter assays
COS-7 cells (1 x105 cells/well) were seeded in 12-well plates at 20 h before transfection. Cells were cotransfected with 0.5 µg/well of pGL3-MMTV as a reporter plasmid, 2 ng/well of pRL-CMV (Promega Corp., Madison, WI) as an internal control, and 0.1 µg/well of a wild-type or mutant AR expression plasmid with 1.7 µl/well of Superfect. In all transfection experiments, the total amount of plasmid DNA was fixed by adding empty vector to the transfection mixture. At 3 h after transfection, 0.5 ml of DMEM containing 10% charcoal-treated FBS was added with or without steroid hormone (10–8 M DHT). After incubation for 24 h, the cells were rinsed with PBS and lysed in the lysis buffer contained in a luciferase assay kit (Promega). The luciferase activity was assayed using the dual-luciferase assay system (Promega) and a Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany). Each value was determined as the average of three independent experiments. Data were presented as the means ± SD. One-way ANOVA followed by Scheffé’s test was used for multigroup comparisons. P < 0.05 was considered to be statistically significant
Confocal laser-scanning microscopy
For living cell microscopy, COS-7 cells (2 x105 cells/well) were seeded in 35-mm glass-bottom dishes (Asahi Techno Glass Corp., Tokyo, Japan) and transfected with 0.5 µg of plasmid DNA carrying the wild-type or a mutant AR fused with GFP or yellow fluorescent protein (YFP) using 5 µl/well Superfect. The cells were maintained in DMEM supplemented with 10% charcoal-treated FBS for 20 h, and then various steroid hormones were added to the medium. The cells were observed with an Axiovert 200M inverted microscope equipped with an LSM510META scan head (Carl Zeiss, Jena, Germany) using a x100, 1.4 numerical aperture oil immersion objective. Images were collected at a 12-bit depth resolution of intensities over 1024 x 1024 pixels. For excitation of GFP and YFP, a 488-nm argon laser was used and a series of images was obtained. The GFP and YFP signals were separated using the emission fingerprinting technique established by Carl Zeiss. Separation of the individual emission signals was based on recording a spectral signature and applying a linear unmixing algorithm using the reference spectra of each fluorescent protein (15).
To construct three-dimensional (3D) images, a series of 30–50 two-dimensional tomographic images were collected for each cell using the confocal microscope. These images were exported as TIF files and the TRI graphic program (Ratoc System Engineering, Tokyo, Japan) was used to reconstruct 3D images (6). Both the spatial distribution and calculation of the fluorescent proteins as distinct volumes were made possible by removing scattering background fluorescence and lens spherical aberrations and then separating each particle (6). The numbers of subnuclear foci determined for the wild-type and mutant ARs were representative of at least 20 cells.
Fluorescence recovery after photobleaching (FRAP) analysis
Cells were transfected with the wild-type AR and a mutant AR and incubated for 24 h at 37 C. After a further 5-h incubation with DHT, FRAP analysis was performed. After collection of the initial image using a Carl Zeiss LSM510META microscope, a selected area of a fixed size in the nucleus was photobleached by setting the laser wavelength to 488 nm and using the maximum power for 50 iterations. After the bleaching, images within the bleached region were taken every second at a resolution of 512 x 512 pixels to follow the recovery of the fluorescence intensity. The intensity of the fluorescence was calculated using the LSM510 software and the half-recovery time (t1/2) was determined as the time when the fluorescence intensity reached half the maximal recovery using the Microsoft Excel software. Each t1/2 was the average of six to 10 FRAP experiments.
Organelle detection
COS-7 cells were transfected with pAR-C579F-GFP. Twenty-four hours after the transfection, the cells were treated with DHT and then LysoTracker Red or MitoTracker Orange CMTMRos (Molecular Probes Inc., Eugene, OR) was added in the medium at a final concentration of 100 nM to visualize the lysosomes or mitochondria, respectively. The cells were incubated for 30 min at 37 C and then rinsed with PBS. Images were collected using the fluorescence microscope.
Immunoelectron microscopy
Twenty-four hours before transfection, COS-7 cells were seeded in a 60-mm dish. The cells were transfected with 2 µg of pAR-C579F-GFP using 3 µl of Superfect. After incubation for 20 h, the cells were treated with 10–8 M DHT for 1 h. After a brief wash with PBS, the cells were fixed in 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min at 25 C. The fixed cells were scraped off the dish, centrifuged at 5000 x g for 10 min at 4 C and resuspended in 0.1 M phosphate buffer. After dehydration in a graded series of ethanol, the cell pellet was embedded in LR White resin and ultrathin sections were cut (16). After blocking in 0.1 M phosphate buffer containing 3% BSA, the sections were incubated with rabbit polyclonal antibodies against GFP (BD Biosciences Clontech, Mountain View, CA) at 1:1000 dilution in 0.1 M phosphate buffer at 4 C overnight, followed by incubation with colloidal gold (Ø = 15 nm)-conjugated goat antirabbit IgG (Amersham Biosciences) at 1:100 dilution. After washing with distilled water, the sections was stained with 2% uranyl acetate and lead citrate, and observed with a Hitachi H-7000 electron microscope (Hitachinaka, Japan).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). This residue also forms a P-box that is important for the recognition of an androgen-responsive element in target genes. The other mutation from a PAIS patient (subject 2) was found at Phe582 next to the P-box (Fig. 1A). To compare the functions of these two mutant ARs with the wild-type AR, recombinant AR proteins were expressed in COS-7 cells. Immunoblot analysis showed that the expression levels of the wild-type and mutant ARs in transfected cells were almost the same (Fig. 1B). To examine AR-mediated transcriptional activation, luciferase assays were performed using the MMTV promoter containing several hormone-responsive elements (17). For the wild-type AR, remarkable transcriptional activation was observed after addition of the ligand DHT. On the other hand, AR-F582Y showed less than 10% of the transcriptional activation of the wild-type AR, whereas AR-C579F showed no ligand-dependent transactivation at all (Fig. 2).
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) (5). Upon treatment with the ligand, the wild-type AR was almost completely translocated from the cytoplasm into the nucleus in which it formed subnuclear fine foci (Fig. 3D). The AR-DBD mutants were also diffusely present in the cytoplasm before addition of the ligand (Fig. 3, B and C). However, in contrast to the wild-type AR, the two mutant ARs formed cytoplasmic dots after the addition of DHT (Fig. 3, E and F). Time-course experiments revealed that most of the fluorescent signals for the wild-type AR were observed in the nucleus within 30 min after the addition of DHT (Fig. 4B), whereas cells expressing the AR-C579F mutant showed two patterns (Fig. 4, E–H or I–L). Approximately 80% of cells formed the cytoplasmic dots and a limited number of nuclear foci 30 min after the addition of DHT (Fig. 4F), and the nuclear dots became clear at 3 h after the ligand addition (Fig. 4H). In the other type of AR-C579F-expressing cells (20%), cytoplasmic dots were not so striking (Fig. 4J), but delayed nuclear translocation with dot formation of mutant AR was observed (Fig. 4L). In both types of the mutant-expressing cells, a smaller number of larger-sized intranuclear dots for AR-C579F were observed, compared with those for the wild-type AR (Fig. 4, D, H, and L). Even after a 3-h treatment with the ligand, a significant amount of the mutant AR still remained in the cytoplasm (Fig. 4, H and L), whereas almost all the signals for the wild-type AR were detected in the nucleus (Fig. 4D). These data indicate that the nuclear translocation and subnuclear localization are impaired in AR-DBD mutants.
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, both the C579F and F582Y AR mutants had a much lower number of foci in the nucleus than the wild-type AR. The average number of nuclear foci for the wild-type AR was 300 ± 8, whereas the numbers for F582Y and C579F were 106 ± 18 and 127 ± 23, respectively.
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). Opposite experiments using AR-C579F-GFP and AR-WT-CMV led to similar results, revealing that both nuclear and cytoplasmic dots appeared after incubation with DHT (Fig. 6Ad). To confirm the colocalization of the wild-type and mutant ARs, AR-WT-CFP and AR-C579F-GFP were coexpressed, and each fluorescent signal was collected under the laser confocal microscope. As shown in Fig. 6B, wild-type and mutant ARs were colocalized in both the nuclear and cytoplasmic dots in the presence of 10 nM DHT (Fig. 6B, d–f). The magnitude of the transcriptional activation in the cells transfected with both AR-WT and AR-C579F was less than half that induced by AR-WT (Fig. 6C). In addition to this experiment using 10 nM DHT, we measured the transcriptional activation of the wild-type AR in the presence of 5 nM DHT. The reporter luciferase activity of the wild-type at 5 nM DHT was significantly higher than that by combination of the wild-type and AR-C579F at 10 nM. Even in a high dose of DHT (100 nM), which can fill all the wild-type LBD, coexpression of AR-C579F inhibited the transcriptional activation mediated by the wild-type AR (Fig. 6C). These results indicated that simple stealing of ligand by AR-C579F is not able to explain the suppression effect of AR-C579F and strongly suggested that AR-C579F is able to form heterodimers with AR-WT in the cells and inhibit the translocation of AR-WT, thereby reducing the transactivation.
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and 5, the numbers of nuclear foci for the mutant ARs were much lower than that for the wild-type AR. To evaluate whether there are any differences in dynamics between the nuclear foci of the wild-type and mutant ARs, the mobilities of the wild-type and mutant ARs in the nucleus were measured quantitatively by FRAP analysis. After a short period of photobleaching at the maximal power of the laser, continuous images were taken at 1-sec intervals. As shown in Fig. 7A, the recovery of the fluorescence intensity for AR-C579F foci was slower than that for AR-WT. The fluorescence recovery was evaluated by plotting the intensity of the bleached area against time (Fig. 7B). Compared with the wild-type AR, the slope of the fluorescence recovery of AR-C579F was relatively gentle. The t1/2 for AR-C579F was 10.5 ± 1.6 sec (n = 12), which was significantly slower than that for the wild-type AR [8.0 ± 1.1 sec (n = 10)]. In another experiment, AR-F582Y also showed reduced mobility, compared with the wild type [t1/2 = 8.4 ± 1.1 sec (n = 6)], and the t1/2 for AR-F582Y [9.9 ± 1.2 sec (n = 6)] was almost the same as that for AR-C579F [10.2 ± 1.1 sec (n = 6)]. These results indicate that the DBD amino acid substitution in the mutant AR caused decreased mobility of the AR in the nucleus.
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, AR-DBD mutants showed ligand-dependent formation of large cytoplasmic dots. We investigated the localizations of the mutant ARs in the cytoplasm after the ligand treatment. The mutant ARs were not localized in the lysosomes, as shown by an experiment using LysoTracker, a specific marker for lysosomes (data not shown). Next, the mitochondrial localization was examined using MitoTracker, a highly specific probe for mitochondria. Many, but not all, of the cytoplasmic dots of the mutant ARs were located close to mitochondria (Fig. 8A). Because signals for the mutant ARs were not observed in the mitochondria, the mutant ARs do not seem to cross the mitochondrial membrane. To further examine the relationship between the mutant ARs and the mitochondria, immunoelectron microscopy was performed using an anti-GFP antibody. Many gold labels were present around the mitochondria, but none were found inside the mitochondria (Fig. 8Ba). Some clusters were present in the cytoplasm, and these would correspond to the cytoplasmic dots observed by the laser-scanning microscopy (Fig. 8Bb).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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There is a significant bias in the distribution of mutations of the AR gene in AIS patients, although the mutations are spread throughout the whole gene (9). Although exon 1 of the AR gene encodes more than half of the AR protein, including the transcription activation domain of the amino terminus, the number of mutations found in this exon is only about 10% of the total number of mutations, and most of the exon 1 mutations are nonsense or frameshift mutations. The mutation hot spots in AIS patients are part of the LBD constituting the pocket for androgen binding. In the DBD, 32 different mutations have been reported and 15 of these were detected in the first zinc finger motif. Most of the mutations in the DBD or LBD are single-base substitutions (3, 9). Some AIS patients do not carry any AR mutations. In such cases, there must be some abnormality in the signal transduction between the AR and the basic transcription machinery, and an impediment in a cofactor interacting with the N-terminal transcription activation domain of the AR has been suggested (14).
The amino acid substitution in the CAIS patient (subject 1) occurred at the cysteine residue contained in the first zinc finger motif of the AR. This AR-C579F mutant would not be able to coordinate the zinc ion, resulting in complete loss of the ligand-dependent DNA-binding ability. In the PAIS patient, the amino acid substitution (F582Y) occurred next to the P-box and may cause a conformational change of the zinc finger motif. The impaired DNA-binding of these mutant ARs was confirmed by gel mobility shift assays, as we previously reported (11). Before the present imaging analysis, it was anticipated that these mutants would be able to enter the nucleus after ligand treatment but be unable to form foci like the wild-type AR due to the loss of the DNA-binding capacity. However, these mutant ARs initially formed cytoplasmic dots instead of the nuclear foci and nuclear dots subsequently appeared. Because there were no differences among the localizations of the wild-type and mutant ARs in the absence of the ligand, the conformations of the ligand-bound mutant ARs are considered to differ from that of the wild-type AR.
In the present study, ligand-induced formation of cytoplasmic dots of the mutant ARs was observed close to the mitochondria. However, the mutant AR signals were not detected inside the mitochondria by immunoelectron microscopy. Similar findings of an association between cytoplasmic protein aggregates and mitochondria have been observed for other mutant proteins, including the polyQ AR mutants (21, 22, 23, 24). The mechanism for such aggregation close to mitochondria has been speculated to be that the ubiquitin-proteasome system tries to degrade a large amount of aggregated proteins and therefore requires an increased amount of ATP around the protein inclusions. The mechanism of localization close to mitochondria for the polyQ AR mutant might also occur for our AR-DBD mutants, although pathophysiology is quite different between our AIS and polyQ diseases. However, further study is necessary for elucidation of abnormal dot formation of our AR mutants.
The C579F mutation in the AR-DBD showed lower mobility than the wild-type AR. FRAP analysis has recently been used to examine the intranuclear dynamics of nuclear receptors (25, 26, 27). In the presence of ligands, the mobility of the nuclear receptors was reduced. It has been reported that ligand-induced intranuclear foci formation of steroid hormone receptors is associated with the nuclear matrix in which coactivators are also recruited (28). This nuclear matrix binding induced by ligand treatment is suggested to cause the decreased mobility of the receptors. A glucocorticoid receptor (GR) mutant carrying a deletion of the N-terminal region showed a much lower mobility (26). This mutant was deprived of all the putative phosphorylation sites of the GR. In ATP-deprived cells, GRs are dephosphorylated and tightly bound to the nuclear matrix (29, 30). Therefore, it has been speculated that appropriate reduction of the mobility of steroid hormone receptors, namely nuclear matrix binding with coactivators (6, 31, 32, 33), is an essential process for the normal transactivation functions of steroid hormone receptors. Lower or much increased mobility of ligand-bound steroid hormone receptors, as shown for the present AR C579F mutant, may indicate an impaired transactivation process.
The present reanalysis of our mutant ARs unexpectedly revealed two kinds of functional defects, i.e. lack of DNA-binding ability reported previously (11) and impairment of translocation from the cytoplasm to the nucleus. The finding that mutations in DNA-binding domain of AR impair nuclear translocation is novel and suggests the existence of an important intramolecular domain for nuclear translocation except for the hinge region. This finding is expected to contribute to the study of translocation mechanism. A mutant AR, K632A/K633A, which has mutations in the hinge region, has an intact DNA-binding domain, but its nuclear translocation is impaired (34). The pattern of the translocation impairment of this AR-K632A/K633A mutant was quite similar to that of our AR mutants, namely the AR-K632A/K633A mutant formed cytoplasmic aggregates (large dots) and its transactivation function examined by a reporter luciferase assay was markedly low (34, 35).
These reported results clearly indicate that impairment of nuclear translocation such as cytoplasmic dot formation can be responsible for the suppression of transactivation function of AR. As is well known, for transactivation function of AR, AR first must be translocated into the nucleus, secondly form a complex with coactivators, and then bind to target genes. Therefore, it would be reasonable that impairment of nuclear translocation in AR-C579F and AR-F582Y is largely responsible for AIS. Abnormal dot formation and decreased mobility of liganded AR-DBD mutants in the nucleus might be due to lack of DNA-binding ability, and thus, a defect in DNA-binding also would be responsible for AIS to some extent. Recent studies have revealed dynamic movement of nuclear receptors during a transactivation process within the nucleus (25, 36, 37). Liganded steroid hormone receptors including AR are transferred to subnuclear compartments (foci) and form a complex with coactivators. These receptor-coactivator complexes are mobilized to the target genes. The steroid hormone receptors and coactivators show multiphasic on and off for binding to promoter elements of genes. The receptor-coactivator complexes also undergo a rapid exchange between target genes and the compartments. The present AR-DBD mutants are not able to access target genes. This may disturb the dynamic movement (mobility), resulting in prolonged stay at nuclear matrix and abnormal dot formation. This speculation may be supported by the reported observation that the AR-K632A/K633A mutant did not show abnormal intranuclear dot formation, although the authors did not touch on it.
In conclusion, the AR-DBD mutations, C579F and F582Y, found in our AIS patients showed abnormalities in ligand-dependent nuclear translocation, nuclear matrix targeting, and intranuclear mobility of the receptor, which may cause AIS in these patients.
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Acknowledgments |
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Footnotes |
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First Published Online August 23, 2005
Abbreviations: AIS, Androgen insensitivity syndrome; AR, androgen receptor; CAIS, complete androgen insensitivity syndrome; 3D, three-dimensional; DBD, DNA-binding domain; DHT, dihydrotestosterone; FBS, fetal bovine serum; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; GR, glucocorticoid receptor; LBD, ligand-binding domain; PAIS, partial androgen insensitivity syndrome; t1/2, half-recovery time; YFP, yellow fluorescent protein.
Received January 27, 2005.
Accepted August 15, 2005.
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References |
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