The Presence of Both the Amino- and Carboxyl-Terminal Domains in the AR Is Essential for the Completion of a Transcriptionally Active Form with Coactivators and Intranuclear Compartmentalization Common to the Steroid Hormone Receptors: A Three-Dimensional Imaging Study
Masayuki Saitoh,
Ryoichi Takayanagi,
Kiminobu Goto,
Akiyoshi Fukamizu,
Arihiro Tomura,
Toshihiko Yanase and
Hajime Nawata
Departments of Medicine and Bioregulatory Science (M.S., K.G., A.T., T.Y., H.N.) and Geriatric Medicine (R.T.), Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan; and Center for Tsukuba Advanced Research Alliance (A.F.), Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan; and CREST (Core Research for Evolutional Science and Technology), JST (Japan Science and Technology) (R.T., K.G., T.Y., H.N.), Kawaguchi 332-0012 Japan
Address all correspondence and requests for reprints to: Hajime Nawata, M.D., Ph.D., Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: nawata{at}intmed3.med.kyushu-u.ac.jp.
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ABSTRACT
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To clarify the physiological significance of the intranuclear speckled distribution, or foci formation, of liganded steroid receptors, the subnuclear distribution of green (GFP), yellow (YFP), and cyan (CFP) fluorescent protein-tagged receptors and coactivators was investigated. The foci formation of 5
-dihydrotestosterone (DHT)-bound AR-GFP in COS7 cells was abolished by the cotransfection of a CBP
(1182393) fragment eliciting a dominant negative effect on the transactivation capacity of the AR. The N-terminal AR fragment (AR-AF-1-YFP), which has a strong constitutive transactivation function, formed foci without DHT, whereas the C-terminal AR fragment (AR-AF-2-CFP), which has a quite low transactivation function, was distributed homogeneously even in the presence of DHT. The reporter gene assay showed a synergism between the transactivation functions of AR-AF-1 and AR-AF-2. This synergism was not reflected by the above two-dimensional imaging. In contrast, a three-dimensional imaging method clearly showed a difference in the intranuclear spatial distribution. The DHT-bound wild-type AR-GFP alone or AR-AF-1-YFP plus DHT-bound AR-AF-2-CFP was distributed as approximately 300 discrete spots in one nucleus, whereas AR-AF-1-YFP alone was distributed as one volume in a reticular pattern. Furthermore, not only AR but also the glucocorticoid receptor-YFP, ER
-GFP, and YFP-tagged SRC-1, TIF2, and CBP were found to be accumulated in identical spots in the presence of ligand. All of the above results indicate that CBP is one of the factors essential for foci formation of the AR, and may propose the hypothesis that transcriptionally activated steroid receptors, regardless of the type of receptor, are transferred to common compartments (foci) and form a complex with coactivators, and this process is essential to full transactivation.
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INTRODUCTION
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SINCE GREEN FLUORESCENT protein (GFP), which can be observed in living cells, was first introduced into the research of steroid hormone receptors (1, 2, 3), understanding of the intracellular dynamics of these receptors along with transcriptional activation has been further deepened. The GFP-GR chimera was found to shift upon ligand-binding from the cytoplasm to the nucleus (1) and to be distributed in a speckled pattern (indicating foci formation) in the nucleus (2). Similar foci formation in the nucleus was thereafter reported in the MR (4), ER
(5, 6), vitamin D receptor (7), and AR (8, 9). Furthermore, in the MR and AR the foci formation of the steroid hormone receptor was shown to be closely linked to transcriptional activation by the receptor. As evidence, the MR and AR were also translocated from the cytoplasm to the nucleus by the addition of an antagonist that inhibited transcriptional activation; however, they were diffusely distributed in the nucleus without making any foci (4, 8, 9). In the MR, ER
, and AR, the foci, distributed in a speckled pattern, were reported to be closely associated with the nuclear matrix (4, 6, 8). However, the mechanism and physiological significance of such foci formation have yet to be elucidated. The steroid hormone receptors have two major domains for transcriptional activation, activation function 1 (AF-1) in the N-terminal domain (NTD) and activation function 2 (AF-2) in the C-terminal ligand-binding domain (LBD). The transactivation function of the AF-1 region is constitutive, namely, ligand independent and autonomous, and that of the AF-2 is ligand dependent (10). It is known that the transcriptional activation function of the steroid receptor is enhanced by direct binding with coactivators (11) such as CREB-binding protein (CBP) (12), steroid receptor coactivator-1 (SRC-1) (13) and transcriptional intermediary factor 2 (TIF2) (14). The AR has been reported to be different from the other steroid hormone receptors such as the ER, GR, TR, and PR in that the AF-2 activity in the LBD is negligible or quite low in mammalian cells (15), whereas the intrinsic AF-1 activity is clearly detectable (16, 17, 18, 19). In the present study, based on analysis of a chimera of the AR and the fluorescent protein, CBP was found to be essential for the foci formation of the AR in the nucleus. Furthermore, a three-dimensional imaging method that has recently been developed (9) demonstrated that the intranuclear distribution pattern of the N-terminal fragment of the AR containing AF-1 was different than that of the full-length AR, although such differences in foci formation could not be detected by the previous two-dimensional imaging method. Three-dimensional imaging also revealed that in the presence of ligand, the AR, GR, and ER
were all accumulated in identical compartments (foci) containing coactivators. These present findings may suggest that compartmentalization (complete foci formation) in the nucleus is one of the indispensable and common steps for steroid hormone receptor-mediated transactivation.
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RESULTS
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Effects of SRC-1, TIF2, and CBP on the Subnuclear Distribution of Fluorescent Protein-Fused AR
When pCMV-AR-cyan fluorescent protein (CFP), in which CFP was fused to the C terminus of AR, was transfected into COS7 cells, the AR-CFP was located in the cytosol in the absence of ligand (Fig. 1A
) but was translocated into the nucleus upon exposure to dihydrotestosterone (DHT). Moreover, the nuclear distribution of AR-CFP was not uniform, but showed a speckled pattern with the formation of foci (Fig. 1B
). When pCMV-yellow fluorescent protein (YFP)-SRC-1 and pCMV-YFP-TIF2, in which YFP was fused to the N termini of SRC-1 and TIF2, respectively, were transfected into COS7 cells, these YFP-tagged coactivators of AR were distributed diffusely in the nucleus (Fig. 1
, C and I). Cotransfection of pCMV-AR-CFP in the presence of DHT in addition to pCMV-YFP-SRC-1 or pCMV-YFP-TIF2 changed the subnuclear distribution of these coactivators from a uniform pattern to a foci-forming one (Fig. 1
, F and J). The liganded AR-CFP was colocalized with these foci of the coactivators, indicating that the transcriptionally activated AR recruited YFP-SRC-1 and YFP-TIF2 and rearranged them in the nucleus (Fig. 1
, G, H, K, and L). The cotransfection of pCMV-AR-CFP without DHT or the addition of DHT alone did not change the diffuse distribution pattern of the coactivators (Fig. 1
, E and D). YFP-CBP, a general integrator for nuclear receptors, was distributed in a mixed pattern with fine foci formation in a diffuse background in the nucleus of COS7 cells (Fig. 2A
), unlike YFP-SRC-1 and YFP-TIF2. Upon exposure to DHT, the expressed AR-CFP was translocated from the cytosol (Fig. 2A
) to the nucleus, coinciding with CBP and making clear foci (Fig. 2B
). Endogenous CBP, which was detected by immunostaining using commercially available anti-CBP antibodies, was found to be distributed in a finely speckled (microparticulate) pattern (Fig. 2C
) in the nucleus of pCMV-AR-CFP-transfected cells in the absence of DHT. However, endogenous CBP was not stained immunologically in the AR foci in DHT-exposed cells (Fig. 2D
). AR-CFP was diffusely distributed without forming any foci in the nucleus by exposure to the antiandrogenic agent hydroxyflutamide (OHF) instead of DHT as reported (8, 9) (Fig. 2E
), i.e. the transcriptionally inactive AR does not form foci in the nucleus. Endogenous CBP was detectable immunostaining when this antiandrogen was added (Fig. 2F
). These observations suggest that the AR, activated by the addition of DHT, bound to endogenous CBP and masked the anti-CBP antibody recognition site of CBP, and thus might suggest that the DHT-bound AR was immediately recruited by CBP and distributed in a foci-forming pattern. To confirm the essential role of CBP in foci formation, the effect of mutated CBP, which exerts a dominant negative effect on the transactivation function of the AR, on the foci formation of the AR was examined. A series of domain-deletion mutants of CBP were prepared. Among these CBP mutants, the CBP
(1182393) fragment, consisting of only the N-terminal and C-terminal regions, suppressed the AR-dependent transactivation in a dose-dependent manner, and this suppression was recovered by cotransfection of the expression vector for wild-type CBP (Fig. 3
). When this CBP
(1182393) expression vector was cotransfected, the foci of AR-GFP were not formed (Fig. 2G
), but the foci formation was recovered by further transfection of the wild-type CBP expression vector (Fig. 2H
). The foci formation was not affected by cotransfection of the expression vector for CBP
(7382393) (Fig. 2I
), CBP
(313468) or CBP
(15701891) (data not shown), which did not show any dominant negative effect on transactivation (Fig. 3
). The above findings show endogenous CBP to be indispensable for the foci formation of the AR. When only the CBP
(1182393) expression vector was transfected, this mutated CBP was found to be distributed diffusely in the nucleus (Fig. 2J
), but the distribution of endogenous CBP was not affected by the forced expression of CBP
(1182393) and showed a microparticulate pattern (Fig. 2K
) as seen in Fig. 2C
. Similar results were obtained when pCMV-AR-CFP was cotransfected and exposed to DHT (Fig. 2
, L and M). These observations suggest that CBP
(1182393) exerted its dominant negative effect by occupying a CBP-binding site of the AR but not by binding to a transactivation complex to which endogenous CBP binds. This explanation for the dominant negative effect of CBP
(1182393) is not inconsistent because the N-terminal 100 amino acid residues of CBP have been reported to contain a domain for nuclear receptor binding (12).

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Figure 1. Subcellular Localization of AR-CFP, YFP-SRC-1, and YFP-TIF2 in COS7 Cells
COS7 cells were transfected with the expression plasmids for AR-CFP (0.5 µg) (A and B), YFP-SRC-1 (0.5 µg) (C and D), YFP-TIF2 (0.5 µg) (I), or YFP-SRC-1 (0.3 µg) (EH) or YFP-TIF2 (0.3 µg) (JL) in addition to AR-CFP (0.25 µg), and the expressed chimeric fluorescent proteins were observed in living cells by confocal laser scanning microscopy as described in Materials and Methods. EH and JL, The transfected amounts of pCMV-YFP-SRC-1, -YFP-TIF2, and -AR-CFP were equivalent on a molar basis. The treatment or absence of treatment with 10-8 M 5 -DHT, the expression plasmid transfected, and the fluorescent signal observed are indicated in or just below each panel. The fluorescent signals from CFP and YFP are represented as red and yellow, respectively, by digital color conversion on a computer. Bar, 10 µm.
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Figure 2. Subcellular Distribution of AR-CFP and YFP-CBP, and Effect of the Truncated CBP on the Subnuclear Localization of AR-GFP in COS7 Cells
COS7 cells were transfected with the expression vectors for AR-CFP alone (0.15 µg) (CF), YFP-CBP (0.35 µg) plus AR-CFP (0.15 µg) (A and B), CBP (1182393) (1.0 µg) alone (J and K), AR-GFP (0.15 µg) in combination with CBP (1182393) (1.0 µg) (G), CBP (1182393) (1.0 µg) plus wild-type CBP (0.2 µg) (H), or CBP (7382393) (1.5 µg) (I), or AR-CFP (0.15 µg) in combination with CBP (1182393) (1.0 µg) (L and M). The molar equivalents of the transfected amount of the plasmids for AR-CFP, YFP-CBP, AR-GFP, CBP (1182393), wild-type CBP, and CBP (7382393) were 1, 1, 1, 48, 0.7, and 15, respectively. The treatment or absence of treatment with 10-8 M DHT or 10-6 M OHF, the expression plasmid transfected and the fluorescent signal observed are indicated in or just below each panel. CFP (AF) and GFP (GI) signals are represented as red and green, respectively. The YFP signal from YFP-CBP (A and B) and the Alexa Fluor 594 signal due to immunostaining of endogenous CBP (C, D, and F) are represented as yellow. The Alexa Fluor 488 signal due to immunostaining of only endogenous CBP (K and M) is represented as green. The Alexa Fluor 594 signal due to immunostaining of both CBP (1182393) and endogenous CBP (J and L) is represented as red. Primary anti-CBP antibodies used were CBP (451) in J and L and CBP (C-1) in K and M. In C, D, and F, CBP(A-22) was used in addition to CBP (451) and CBP (C-1), and essentially the same results were obtained. Panels J and L show a diffusely stained pattern, reflecting overexpressed CBP (1182393), although endogenous CBP was also stained.
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Figure 3. Repression of AR-Dependent Transactivation by CBP (1182393) and Its Reversal by Coexpressed Wild-Type CBP
COS7 cells were transfected with the indicated amounts (µg) of the expression vectors for AR-GFP and the truncated or wild-type CBP, along with the reporter genes, as described in Materials and Methods. The luciferase activities are represented as values relative to the activity induced by AR-GFP alone in the presence of 10-8 M DHT, which was arbitrarily set as 100. Each bar represents the mean ± SD of three independent experiments.
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Subnuclear Distribution and Transcriptional Activation Function of the N-Terminal and C-Terminal AR Fragments Fused to Fluorescent Protein
AR-AF-1-YFP, in which YFP was fused to the C terminus of the AR-AF-1 fragment, consisting of the AF-1-containing NTD and the DNA-binding domain (DBD) of the AR, was distributed in a foci-forming pattern in the nucleus of COS7 cells regardless of the presence of DHT (Fig. 4
, A and B). In contrast, AR-AF-2-CFP, in which CFP was fused to the C terminus of the AR-AF-2 fragment, consisting of the DBD and the AF-2-containing ligand-binding domain (LBD) of the AR, was distributed in a uniform pattern regardless of the presence of DHT (Fig. 4
, G and H). The subnuclear distribution pattern was similar in the AR fragments in which fluorescent protein was fused to the N termini of the AR fragments (data not shown). When, in addition to pCMV-AR-AF-1-YFP, pCMV-AR-AF-2-CFP was cotransfected at an amount lower than that of pCMV-AR-AF-1-YFP on a molar basis, the foci formation of AR-AF-1-YFP collapsed (Fig. 4C
). Cotransfection of pCMV-AR-AF-2-CFP at an amount higher than that of pCMV-AR-AF-1-YFP perfectly excluded AR-AF-1-YFP from the nucleus to the cytosol (Fig. 4D
). However, AR-AF-1-YFP was translocated again into the nucleus with foci formation by the addition of DHT (Fig. 4E
), and AR-AF-2-CFP was also distributed with foci formation coinciding with AR-AF-1-YFP (Fig. 4F
). Both the AR-AF-1-YFP and AR-AF-2-CFP fragments contained the DBD. Therefore, there was a possibility that the overlapped DBDs affected the interaction between AR-AF-1 and AR-AF-2. The DBD-deleted NTD,
(DBD)AR-AF-1-YFP, was distributed dominantly in the cytosol (Fig. 4I
), probably because this fragment lacked the nuclear localization signal (NLS).
(DBD)AR-AF-1-YFP was translocated into the nucleus with foci formation in the presence of DHT-bound AR-AF-2-CFP (Fig. 4J
) as seen in cells transfected with both pCMV-AR-AF-1-YFP and pCMV-AR-AF-2-CFP (Fig. 4
, E and F), indicating that the overlapped DBD did not hinder the function of each AR fragment. When pCMV-YFP-SRC-1 or pCMV-YFP-TIF2 was cotransfected with pCMV-AR-AF-1-CFP, these coactivators formed foci coinciding with AR-AF-1-CFP (Fig. 4
, K and L). AR-AF-1-YFP activated the transcription of the luciferase reporter gene regardless of the presence of DHT, and the level of the transactivation function was about 70% of that of the AR-AF-1 not fused to YFP. Although AR-AF-2-CFP showed ligand-dependent transcription activation, it was less than 6% of that of AR-AF-1-YFP (Fig. 5
). Ligand-independent transactivation by AR-AF-1-YFP was suppressed to about 17% of the original activity by cotransfection of equivalent amounts or twice as much of pCMV-AR-AF-2-CFP in the absence of DHT, but transactivation increased 3 to 4 times in the presence of DHT (Fig. 5
). Essentially similar results were obtained with the AR fragments, which were not fused to fluorescent protein (Fig. 5
) and in the experiments using CV-1 and LNCaP cells (data not shown).

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Figure 4. Subcellular Distribution of the N-Terminal (AR-AF-1) and C-Terminal (AR-AF-2) Fragments Tagged with Fluorescent Proteins
COS7 cells were transfected with the expression plasmids indicated in each panel, and then the living cells were scanned. The transfected amount of the plasmid was 0.5 µg in panels A, B, G, and H. The molar ratios of the transfected amounts of pCMV-AR-AF-1-YFP and pCMV-AR-AF-2-CFP were 2:1 (0.35 and 0.15 µg, respectively) in C and 1:2 (0.18 and 0.32 µg, respectively) in DF. IL, Transfected amounts of the expression plasmids for (DBD)AR-AF-1-YFP (0.2 µg), AR-AF-2-CFP (0.19 µg), YFP-SRC-1 (0.28 µg), YFP-TIF2 (0.28 µg), and AR-AF-1-CFP (0.22 µg) were equivalent on a molar basis. The addition of 10-8 M DHT and the fluorescent signal observed are indicated in or just below each panel. The CFP and YFP signals are represented as red and yellow, respectively.
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Figure 5. Transcriptional Activation by Full-Length AR, the Truncated ARs, and Their Chimeras with Fluorescent Proteins in COS7 Cells
COS7 cells were transfected with the expression vectors for full-length AR (0.2 µg), the N-terminal fragment (AR-AF-1) (0.18 µg), the C-terminal fragment (AR-AF-2) (0.16 µg), and their chimeras with fluorescent proteins as indicated, along with the reporter genes, as described in Materials and Methods. The numbers in parentheses indicate the molar equivalents of the vectors transfected by setting that of the full-length AR as 1.0. The luciferase activities are represented as values relative to the activity induced by full-length AR alone in the presence of 10-8 M DHT, which was arbitrarily set as 100. Each bar represents the mean ± SD of three independent experiments.
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Construction of the Three-Dimensional Image of the Subnuclear Distribution of the N-Terminal and C-Terminal AR Fragments Fused to Fluorescent Protein
Although foci formation in the nucleus was closely correlated with the presence of transactivation function in not only the full-length AR but also the AR fragments (Figs. 15



), the existence of foci formation did not reflect differences in the levels of the transcriptional activation function. The formation of foci was evenly observed by two-dimensional imaging among cells transfected with the expression vectors for the full-length AR-CFP, the AR-AF-1-YFP alone and the AR-AF-1-YFP in combination with the AR-AF-2-CFP (Figs. 1B
and 4
, B and E), although the levels of transactivation were different (Fig. 5
). Therefore, to clarify whether any differences in the subnuclear localization exist among these AR fragments and the full-length AR, we analyzed the intranuclear distribution by a computer-assisted three-dimensional imaging method. Nuclear DNA in living cells was stained with Hoechst 33342, which has been used to discriminate the heterochromatin region from the euchromatin region. To obtain the heterochromatin image, the less stained areas (namely euchromatin region) were cut off and shown as blank images (Fig. 6E
). Most of the liganded full-length AR-GFP was detected as discrete multiple spots (Fig. 6A
, surface view), and the green spots were located in the euchromatin region (Fig. 6F
, surface view). In contrast, AR-AF-2-CFP was homogenously distributed as a single large spot, except for the nucleoli, regardless of the presence of DHT (Fig. 6C
, surface view), over the whole chromatin (Fig. 6
, I, surface view, and J, tomographic view). AR-AF-1-YFP was distributed in an intermediate pattern between the patterns of the full-length AR and the AR-AF-2-CFP, i.e. a reticular pattern (Fig. 6B
, surface), in which the spatial distribution of the YFP signal was not discontinuous and was calculated as one volume, mostly in the euchromatin region (Fig. 6
, G, surface, and H, tomogram). However, in combination with DHT-bound AR-AF-2-CFP, the AR-AF-1-YFP formed isolated spots and showed distribution which was essentially identical with that of the full-length AR-GFP (Fig. 6
, D, surface, and K, tomogram). The DHT-bound AR-AF-2-CFP in combination with the DBD-deleted NTD,
(DBD)AR-AF-1, also formed isolated spots (Fig. 6L
, surface), indicating that overlapped DBDs of the AR-AF-1 and AR-AF-2 fragments did not affect the interaction between the two fragments. Essentially the same results were obtained using CV-1 and LNCaP cells.

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Figure 6. The Three-Dimensional Image Analysis of the Intranuclear Localization of the N-Terminal (AR-AF-1) and C-Terminal (AR-AF-2) AR Fragments Tagged with Fluorescent Proteins in COS7 Cells
COS7 cells transfected with the expression plasmids were treated with 10-8 M DHT and stained with Hoechst 33342. Next, the confocal images of the nuclei in living cells were collected to reconstruct the three-dimensional images. The expression plasmids transfected are indicated in each panel. In panel A, 0.25 µg of pCMV-AR-GFP was transfected, and the amounts of each plasmid transfected in other panels were equivalent on a molar basis. The images were displayed as a surface view (AG, I and L) or a tomographic sectional view on the z-axis (H, J, and K). The fluorescent signals from GFP, YFP, CFP, and Hoechst 33342 are represented as green, yellow, red, and blue, respectively. A, AR-GFP foci formation in the nucleus; B, reticular distribution of AR-AF-1-YFP; C, diffuse and homogenous distribution of AR-AF-2-CFP; D, orange-colored foci formation by AR-AF-1-YFP and AR-AF-2-CFP; E, chromatin structure stained with Hoechst 33342; F, spatially superimposed three-dimensional image of A and E; G and H, superimposed image of B and the chromatin structure; I and J, superimposed image of C and the chromatin structure; K, superimposed image of D and the chromatin structure; L, foci formation by (DBD)AR-AF-1 and AR-AF-2-CFP. Bar, 5 µm.
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Quantitative Analysis of Intranuclear Foci of the AR, GR, and ER
by Three-Dimensional Imaging
The present three-dimensional construction method for the confocal images, in which a rejection of scattering fluorescence with a low degree of brightness and median filter processing were carried out, allowed us to observe intranuclear fluorescent proteins at a high resolution and to quantify the number of fluorescent spots in the nucleus. Approximately 300 AR-CFP spots (see legend for Fig. 7
) were found to exist as a distinct volume in one nucleus (Fig. 7A
). The nuclear volume occupied by these 300 spots was estimated to be approximately 10% of the total nuclear volume in the present experimental conditions. When the three-dimensional imaging method was applied to liganded GR-YFP and ER
-GFP, in which YFP and GFP were fused to the C termini of GR and ER
, respectively, a distribution of discrete spots in the nucleus was also observed (Fig. 7
, B and C), and the numbers of spots (308 ± 23, n = 8) were similar to that of AR-CFP. The number of spots was calculated from the three-dimensional images processed under the same conditions. When pCMV-AR-CFP was cotransfected with pCMV-GR-YFP, they formed identical spots in the presence of their ligands (Fig. 7D
), and naturally the number of spots was similar to that of AR-CFP or GR-YFP alone. Similar results were obtained by the cotransfection of AR-CFP and ER
-GFP (Fig. 7E
). The number of AR-CFP spots was not affected by cotransfection, i.e. overexpression, of coactivators such as YFP-SRC-1, YFP-TIF2 and YFP-CBP (see legend for Fig. 7
). Furthermore, YFP-SRC-1 (Fig. 7F
), YFP-TIF2 (Fig. 7G
) and YFP-CBP (Fig. 7H
) were all colocalized with AR-CFP, forming identical spots.

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Figure 7. The Three-Dimensional Image Analysis of the Intranuclear Localization of Liganded AR-CFP, GR-YFP and ER -GFP, and YFP-Tagged Coactivators in COS7 Cells
COS7 cells were transfected with the expression plasmids indicated in each panel. In panel A, 0.25 µg of pCMV-AR-CFP was transfected, and the amounts of each plasmid transfected in other panels were equivalent on a molar basis. The cells were then scanned after treatment with 10-8 M ligand for each receptor as indicated. The images are displayed as surface views. The GFP, YFP, and CFP signals are represented as green, yellow and red, respectively. A, Red spots due to AR-CFP foci formation; B, yellow spots due to GR-YFP foci formation; C, green spots due to ER -GFP foci formation; D, orange-colored foci formation by AR-CFP and GR-YFP; E, yellow-colored foci formation by AR-CFP and ER -GFP; F, G, and H, orange-colored foci formation by AR-CFP and YFP-tagged SRC-1, TIF2 or CBP, respectively. The numbers of spots identified as a distinct volume were quantified as 299 ± 63 (A), 301 ± 24 (B), 319 ± 22 (C), 303 ± 33 (D), 312 ± 30 (E), 262 ± 20 (F), 305 ± 27 (G), and 297 ± 23 (H) (mean ± SD) from four independent experiments.
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DISCUSSION
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The present study demonstrates that the coactivator CBP is involved in the intranuclear foci formation of AR. YFP-SRC-1 and YFP-TIF2 were homogenously distributed in the nucleus but converged to the foci of the DHT-bound AR-CFP upon cotransfection, whereas YFP-CBP was distributed in the nucleus in a fine speckled pattern but became colocalized with the liganded AR-CFP with an increased YFP intensity upon cotransfection (Fig. 2
). The endogenous CBP was also distributed in a speckled pattern, and it was found, although indirectly, that the liganded AR-CFP bound to the endogenous CBP (Fig. 2
, DF). The introduction of a CBP mutant with a dominant negative effect on the AR-dependent transactivation destroyed the foci formation of AR-GFP, and AR-GFP was distributed in a uniform pattern. The involvement of CBP in the AR foci formation is also supported by reports that the addition of 12-O-tetradecanoylphorbol-13-acetate (20) or transfection of the expression plasmid for NF-
B (21) repressed the AR-dependent transactivation, and competition for endogenous CBP was speculated as the mechanism of this repression (20, 21). A diffuse intranuclear distribution of GFP-SRC-1 in cells transfected with the GFP-SRC-1 expression vector alone and a distribution of endogenous CBP in a microparticulate (finely speckled) pattern in nontransfected cells have also been reported by other groups (6, 22). A physiological significance of the difference in the intranuclear distribution between CBP and p160 coactivators such as SRC-1 and TIF2 remains unknown, but one of the speculations is as follows. CBP is an integrator of multiple signal transductions and interacts with various transcription regulators (12). Therefore, endogenous or exogenous CBP may make discrete foci by forming complexes with transcription factors other than the steroid receptors as well.
The AR is unusual among steroid hormone receptors in its AF-1 and AF-2 functions. Most of the transactivation functions of the AR exist in AF-1 in the NTD (16, 17, 18, 19), and the ligand-dependent transactivation function of AF-2 in the LBD is extremely low in mammalian cells (15). In the absence of ligand, the LBD of the AR is thought to keep the AR retained in the cytosol (17) by masking the NLS, which was reported to exist within the DBD and the hinge region corresponding to amino acid residues 557653 (23), 617633 (17, 24), or 627658 (25). The unliganded LBD has also been reported to suppress the constitutive transactivation function of the NTD (17, 23). The NTD of the AR has recently been shown to interact directly with TIF2 and SRC-1 (26, 27). The present study succeeded in perfectly visualizing these reports on AF-1 and AF-2 function in the AR (Fig. 4
), as follows. Both the AR-AF-1-YFP and the AR-AF-2-CFP fragments prepared in the present study contained the exposed NLS, and therefore, each of them was translocated into the nucleus without ligand binding. The cotransfection of AR-AF-2-CFP and AR-AF-1-YFP suppressed the foci formation of AR-AF-1-YFP (Fig. 4C
) and excluded the AR-AF-1-YFP to the cytosol (Fig. 4D
). The transfection of AR-AF-1-YFP alone formed foci in the nucleus, reflecting its strong constitutive transactivation function, whereas the AR-AF-2-CFP alone did not produce foci even in the presence of ligand. YFP-SRC-1 and YFP-TIF2 were shown to be localized within the AR-AF-1-CFP foci (Fig. 4
, K and L). The rapid recruitment of SRC-1 to the ER
foci upon exposure to E2 was studied by Stenoien et al. (6, 28), who reported that helix 12 and the AF-2 domain in the LBD are essential for the agonist-induced recruitment of coactivators, but the AF-1 domain and DBD are dispensable. In contrast to the ER
, AR-AF-1 can recruit SRC-1 and TIF2. Thus, the recruitment of coactivators is suggested to be an essential and common process for transactivation by the various steroid receptors, but the molecular mechanism is different among the receptors.
AR-AF-1 (NTD) and AR-AF-2 (LBD) showed a synergistic action. If the degree of transactivation of MMTV (mouse mammarian tumor virus) promoter induced by both AR-AF-1 (NTD) and liganded AR-AF-2 (LBD) is set at 100%, the transactivation induced by AR-AF-1 alone was 28% in COS7 cells, and that by liganded AR-AF-2 alone was less than 2% (Fig. 5
). Similarly, the degree of transactivation by the AR NTD alone and by the liganded AR LBD alone have been reported to be 1075% (18, 24, 29) and 03% (18, 19, 24, 25, 29), respectively, of that by the liganded wild-type AR in various types of cells. For an explanation of this synergistic mechanism, the following has been speculated. AR-AF-2, although its intrinsic activity is negligibly low, can associate with AR-AF-1 in the presence of ligand, and this intramolecular interaction results in the formation of a complete platform to recruit coactivators. This conformational change then elicits the full activity of the AR (19, 26, 27, 30, 31, 32). The above speculation has been deduced from overall consideration of the results obtained by a reporter gene assay, a GST pull-down assay and a two-hybrid assay. If intramolecular interplay between the AF-1 and AF-2 domains really proceeds in living cells, there must be a difference between the structures of the receptor/coactivator complex in the case with AR-AF-1 alone vs. the case with full-length AR or the coexistence of AR-AF-1 and AR-AF-2. However, that difference was not detectable by two-dimensional image analysis. Therefore, a three-dimensional imaging method, which has recently been developed by us to detect accurately any antiandrogenic effect (9), was applied to the analyses of the subnuclear distribution of the fluorescent protein-tagged receptors. Using this method, to our knowledge, we for the first time succeeded in obtaining direct evidence of structural differences between the receptor/coactivator complex of AR-AF-1 alone and that of the full-length AR (Fig. 6
, A and B). Three-dimensional imaging also revealed that the native AR/coactivator complexes, i.e. the AR foci, are distributed inside euchromatin, where transcription is thought to be active. This three-dimensional imaging method allowed analysis of the accurate subnuclear spatial localization and quantitation of the foci formed by the steroid hormone receptor labeled with fluorescent protein. The following was thus revealed. The AR, GR, and ER
were accumulated in identical subnuclear compartments in the presence of ligand. The maximum number of subnuclear compartments, which was considered to be observed with the overexpression of the steroid hormone receptors, i.e. with the transfection of their expression vectors, was approximately 300 in one COS7 cell nucleus. Coactivators such as SRC-1, TIF2, and CBP were also accumulated in the same subnuclear compartments with those of the steroid hormone receptors. An overexpression of these coactivators did not affect the maximum number of compartments. These findings might propose the hypothesis that transcriptionally activated steroid hormone receptors, regardless of the type of the receptor, are transferred to common compartments located in the euchromatin region and form a complex with coactivators. These receptor/coactivator complexes are then mobilized to the target genes of each receptor and are then rapidly returned to the compartment after use. This hypothesis would be supported by recent reports that liganded GFP-GR undergoes a rapid exchange between chromatin and the nucleoplasmic compartment (33) and that liganded GR and ER
are retained in the nuclear matrix even after DNase-digestion of the nucleus (6, 34). Furthermore, the dynamics of the ER
in the nucleus have recently been revealed by fluorescence recovery after photobleaching. It was shown that the E2-bound CFP-ER
/YFP-SRC-1 complex associates with the nuclear matrix (i.e. formation of foci) but undergoes rapid exchange within seconds (35).
In conclusion, the present study on the AR and foci formation in the nucleus provides novel findings regarding the physiological significance of the subnuclear foci formation of the steroid hormone receptors. CBP is at least one of the factors essential to foci formation of the AR. Foci formation indicates the formation of a complete steric conformation of the AR essential for transactivation, for which both the NTD and LBD are necessary, and is also evidence of the intranuclear compartmentalization that may be common to other steroid receptors and essential for full transcriptional activation. In addition, the three-dimensional imaging method performed in the present study seems to be an effective modality for further investigating the mechanism of nuclear receptor-mediated transcription.
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MATERIALS AND METHODS
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Plasmid Constructs
The firefly luciferase reporter vector (pGL3-MMTV) and the expression vectors for the human AR (pCMX-AR) (36), the C-terminal-truncated AR (AR-AF-1) corresponding to AR 1703 (pCMX-AR-AF-1) and the N-terminal-truncated AR (AR-AF-2) corresponding to AR 502919 (pCMX-AR-AF-2) were prepared as previously described (29). The expression plasmid for the DBD-deleted AR-AF-1 (
(DBD)AR-AF-1), corresponding to AR 1501, was prepared by excising the DBD between HindIII and KpnI sites of pCMX-AR-AF-1. The expression plasmids for the full-length mouse CBP 12443 (pcDNA/mCBP) and for the truncated mutants, pcDNA/mCBP
(1182393), pcDNA/mCBP
(313468), pcDNA/mCBP
(7382393), and pcDNA/mCBP
(15701891), were prepared as previously described (37). Human cDNAs for the GR, SRC-1, and TIF2 were prepared as reported previously (29), and the human ER
cDNA was provided by Dr. Shigeaki Kato (Tokyo University, Japan). The expression plasmids for AR-GFP (pCMV-AR-GFP) and AR-CFP (pCMV-AR-CFP) chimeras were constructed by inserting the full-length AR cDNA into the NheI-SmaI sites of pEGFP-N1 and pECFP-N1 (CLONTECH Laboratories, Inc., Palo Alto, CA), respectively. The expression plasmids for AR-AF-1-YFP (pCMV-AR-AF-1-YFP) and AR-AF-2-CFP (pCMV-AR-AF-2-CFP) chimeras, in which fluorescent proteins were fused to the C termini of the AR fragments, were prepared by inserting the AR-AF-1 and AR-AF-2 cDNAs into the NheI-XhoI sites of pEYFP-N1 (CLONTECH Laboratories, Inc.) and the NheI-KpnI sites of pECFP-N1, respectively, using PCR techniques. The expression plasmid for AR-AF-1-CFP (pCMV-AR-AF-1-CFP) was prepared by inserting the AR-AF-1 cDNA into the NheI-XhoI sites of pECFP-N1. Similarly, the expression plasmids for GR-YFP (pCMV-GR-YFP), ER
-GFP (pCMV-ER
-GFP), YFP-SRC-1 (pCMV-YFP-SRC-1), YFP-TIF2 (pCMV-YFP-TIF2) and
(DBD)AR-AF-1-YFP were constructed. The plasmids for YFP-AR-AF-1 (pCMV-YFP-AR-AF-1) and CFP-AR-AF-2 (pCMV-CFP-AR-AF-2), in which fluorescent proteins were fused to the N termini of the AR fragments, were prepared by inserting the AR-AF-1 and AR-AF-2 cDNAs into the NheI-XhoI sites of pEYFP-C1 and the BglII-XhoI sites of pECFP-C1 (CLONTECH Laboratories, Inc.), respectively. The expression vector for YFP-CBP chimera (pCMV-YFP-CBP) was constructed by inserting the YFP cDNA into the HindIII site of pcDNA/mCBP, upstream of the CBP sequence.
Reporter Assay
Kidney-derived cell lines, COS7 and CV-1, and a human prostatic cancer cell-line, LNCaP, were obtained from American Type Culture Collection (Manassas, VA). COS7 and CV-1 cells were maintained in DMEM (Life Technologies, Inc.) supplemented with 10% FBS, 2 mM L-glutamine and 100 U/ml of penicillin-streptomycin. LNCaP cells were similarly maintained except for the use of Roswell Park Memorial Institute 1640 medium instead of DMEM. The cells, cultured in 6-well plates (3 x 105 cells per well), were transfected with 1 µg/well of pGL3-MMTV as the reporter, 2 ng/well of pRL-CMV (a Renilla luciferase vector, Promega Corp., Madison, WI) as the internal control, and 0.1 to 0.2 µg/well of the expression vector for the AR, the AR fragment, the chimeric receptor or the mutated CBP, using 7 µl/well of SuperFect reagent (QIAGEN, Hilden, Germany). For coexpression studies, the total amount of vector added to each well was equalized by the addition of empty vector, unless otherwise indicated. Starting 3 h after transfection, the cells were incubated for 48 h in DMEM with 10% charcoal-treated FBS in the presence or absence of 10-8 M 5
-dihydrotestosterone, 10-8 M dexamethasone, 10-8 M E2, or 10-6 M OHF. The cells were then solubilized with 500 µl of lysis buffer (Promega Corp.) and the activities of the reporter gene were determined by the Dual-Luciferase Reporter Assay System (Promega Corp.). One-way analysis of variance followed by Scheffés test was used for multi-group comparisons.
Fluorescence Microscopy and Three-Dimensional Image Analysis
The cells were cultured in 35-mm glass-bottom dishes (MatTek) (3 x 105 cells/dish) and then transfected with various plasmids in a total amount of 0.5 µg/dish using 2.5 µl of SuperFect. For coexpression studies, the total amount of vector added to each dish was equalized by the addition of empty vector, unless otherwise indicated. Sixteen to 24 h after incubation in DMEM containing 10% charcoal-treated FBS, the culture media were replaced with fresh DMEM in the presence or absence of the hormones or chemicals, and then the cells were observed with a Leica Corp. TSP-SP invert confocal laser scanning microscope (Leica Corp. Microsystems, Heidelberg, Germany), using a 100x, 1.4 numerical aperture PL APO oil immersion objective. Imaging for GFP, YFP, and CFP was performed by excitation with the 488-nm, 514-nm, and 450-nm lines, respectively, from an argon laser, and the emissions were viewed through band passes ranging from 500 to 550 nm, from 530 to 590 nm, and from 470 to 500 nm, respectively, by band pass regulation with a Prism System (Leica Corp. Microsystems). For simultaneous imaging of multiple fluorescent proteins, the laser line was changed, and the band pass was further finely controlled so as not to overlap emissions. For example, for the simultaneous observation of AR-CFP and ER
-GFP, the chimeras were imaged by excitation with 450-nm and 514-nm lines, respectively, and the emissions through band passes ranging from 460 to 480 nm and from 580 to 620 nm, respectively, were observed. The nuclei were stained with Hoechst 33342 (Molecular Probes, Inc., Eugene, OR) (2 µg/ml) and excited with the 350-nm line from a UV laser, and the emission was viewed through a band pass ranging from 400 to 450 nm. For immunostaining, the cells were washed using PBS and fixed in 4% (vol/vol) paraformaldehyde in PBS for 30 min at 25 C. After being blocked in 10% goat serum for 1 h, the cells were incubated with anti-CBP mouse monoclonal antibody [CBP (C-1) against the C terminus of CBP] or anti-CBP rabbit polyclonal antibodies [CBP (A-22) against the N terminus and CBP (451) against the CREB-binding domain] (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:100-fold dilution for 1 h, followed by incubation with 0.33 µg/ml of Alexa Fluor 594-labeled antimouse or antirabbit IgG or Alexa Fluor 488-labeled antirabbit IgG (Molecular Probes, Inc.) for 1 h at 25 C. After being washed by PBS, the stained cells were observed with a confocal microscope by excitation with the 568-nm line from a krypton laser and emission from 600640 nm for Alexa Fluor 594-labeled IgG or by excitation with the 514-nm line from an argon laser and emission from 520 to 560 nm for Alexa Fluor 488-labeled IgG. A three-dimensional imaging study was performed essentially in the same manner as previously reported (9). In brief, a series of 3050 scanning images were collected for each single nucleus, and these two-dimensional tomograms were reconstructed using the three-dimensional analysis software of TRI Graphics Program (Ratoc System Engineering, Tokyo, Japan). Both the spatial distribution and calculations of the fluorescent proteins as a distinct volume were made possible by removing scattering background fluorescence and lens spherical aberrations and then by separating each particle.
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ACKNOWLEDGMENTS
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This work was performed in part at the Kyushu University Station for Collaborative Research. The authors gratefully thank Mitoshi Toki for his valuable technical assistance in performing the three-dimensional imaging analyses, and Pamela J. Tamura for assistance in preparing the manuscript.
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FOOTNOTES
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This work was supported in part by a Grant-in-Aid for Scientific Research from The Ministry of Education, Science, Sports and Culture, Japan.
Abbreviations: AF, Activation function; CBP, CREB-binding protein; CFP, cyan fluorescent protein; DBD, DNA-binding domain; DHT, dihydrotestosterone; GFP, green fluorescent protein; LBD, ligand-binding domain; MMTV, mouse mammarian tumor virus; NLS, nuclear localization signal; NTD, N-terminal domain; OHF, hydroxyflutamide; SRC-1, steroid receptor coactivator-1; TIF2, transcriptional intermediary factor 2; YFP, yellow fluorescent protein.
Received for publication July 17, 2001.
Accepted for publication December 14, 2001.
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REFERENCES
|
---|
-
Ogawa H, Inouye S, Tsuji FI, Yasuda K, Umesono K 1995 Localization, trafficking, and temperature-dependence of the Aequorea green fluorescent protein in cultured vertebrate cells. Proc Natl Acad Sci USA 92:1189911903[Abstract]
-
Htun H, Barsony J, Renyi I, Gould DL, Hager GL 1996 Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera. Proc Natl Acad Sci USA 93:48454850[Abstract/Free Full Text]
-
Georget V, Lobaccaro JM, Terouanne B, Mangeat P, Nicolas J-C, Sultan C 1997 Trafficking of the androgen receptor in living cells with fused green fluorescent protein-androgen receptor. Mol Cell Endocrinol 129:1726[CrossRef][Medline]
-
Fejes-Tóth G, Pearce D, Náray-Fejes-Tóth A 1998 Subcellular localization of mineralocorticoid receptors in living cells: effects of receptor agonists and antagonists. Proc Natl Acad Sci USA 95:29732978[Abstract/Free Full Text]
-
Htun H, Holth LT, Walker D, Davie JR, Hager GL 1999 Direct visualization of the human estrogen receptor reveals a role for ligand in the nuclear distribution of the receptor. Mol Biol Cell 10:471486[Abstract/Free Full Text]
-
Stenoien DL, Mancini MG, Patel K, Allegretto EA, Smith CL, Mancini MA 2000 Subnuclear trafficking of estrogen receptor-
and steroid receptor coactivator-1. Mol Endocrinol 14:518534[Abstract/Free Full Text]
-
Racz A, Barsony J 1999 Hormone-dependent translocation of vitamin D receptors is linked to transactivation. J Biol Chem 274:1935219360[Abstract/Free Full Text]
-
Tyagi RK, Lavrovsky Y, Ahn SC, Song CS, Chatterjee B, Roy AK 2000 Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells. Mol Endocrinol 14:11621174[Abstract/Free Full Text]
-
Tomura A, Goto K, Morinaga H, Nomura M, Okabe T, Yanase T, Takayanagi R, Nawata H 2001 The subnuclear three dimensional image analysis of androgen receptor fused to green fluorescence protein. J Biol Chem 276:2839528401[Abstract/Free Full Text]
-
Beato M, Herrlich P, Schutz G 1995 Steroid hormone receptors: many actors in search of a plot. Cell 83:851857[Medline]
-
Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:11671177[Abstract]
-
Kamei K, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin S-C, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85: 403414
-
Onate SA, Tsai SY, Tsai M-J, OMalley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:13541357[Abstract]
-
Voegel JJ, Heine MJS, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDa transcriptional mediator for the ligand-dependent activation function AF-2 of nuclear receptors. EMBO J 15:36673675[Abstract]
-
Moilanen A, Rouleau N, Ikonen T, Palvimo JJ, Jänne OA 1997 The presence of a transcription activation function in the hormone-binding domain of androgen receptor is revealed by studies in yeast cells. FEBS Lett 412:355358[CrossRef][Medline]
-
Rundlett SE, Wu XP, Miesfeld RL 1990 Functional characterizations of the androgen receptor confirm that the molecular basis of androgen action is transcriptional regulation. Mol Endocrinol 4:708714[Abstract]
-
Zhou ZX, Sar M, Simental JA, Lane MV, Wilson EM 1994 A ligand-dependent bipartite nuclear targeting signal in the human androgen receptor. Requirement for the DNA-binding domain and modulation by NH2-terminal and carboxyl-terminal sequences. J Biol Chem 269:1311513123[Abstract/Free Full Text]
-
Jenster G, van der Korput H AGM, Trapman J 1995 Identification of two transcription activation units in the N-terminal domain of the human androgen receptor. J Biol Chem 270:73417346[Abstract/Free Full Text]
-
Ikonen T, Palvimo JJ, Jänne OA 1997 Interaction between the amino- and carboxyl-terminal regions of the rat androgen receptor modulates transcriptional activity and is influenced by nuclear receptor coactivators. J Biol Chem 272:2982129828[Abstract/Free Full Text]
-
Frønsdal K, Engedal N, Slagsvold T, Saatcioglu F 1998 CREB binding protein is a coactivator for the androgen receptor and mediates cross-talk with AP-1. J Biol Chem 273:3185331859[Abstract/Free Full Text]
-
Aarnisalo P, Palvimo JJ, Jänne OA 1998 CREB-binding protein in androgen receptor-mediated signaling. Proc Natl Acad Sci USA 95:21222127[Abstract/Free Full Text]
-
LaMorte VJ, Dyck JA, Ochs RL, and Evans RM 1998 Localization of nascent RNA and CREB binding protein with the PML-containing nuclear body. Proc Natl Acad Sci USA 95:49914996[Abstract/Free Full Text]
-
Jenster G, van der Korput HA, van Vroonhoven C, van der Kwast TH, Trapman J, Brinkmann AO 1991 Domains of the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization. Mol Endocrinol 5:13961404[Abstract]
-
Poukka H, Karvonen U, Yoshikawa N, Tanaka H, Palvimo JJ, Jänne OA 2000 The RING finger protein SNURF modulates nuclear trafficking of the androgen receptor. J Cell Sci 113:29913001[Abstract/Free Full Text]
-
Simental JA, Sar M, Lane MV, French FS, Wilson EM 1991 Transcriptional activation and nuclear targeting signals of the human androgen receptor. J Biol Chem 266:510518[Abstract/Free Full Text]
-
Alen P, Claessens F, Verhoeven G, Rombauts W, Peeters B 1999 The androgen receptor amino-terminal domain plays a key role in p160 coactivator-stimulated gene transcription. Mol Cell Biol 19:60856097[Abstract/Free Full Text]
-
Bevan CL, Hoare S, Claessens F, Heery DM, Parker MG 1999 The AF1 and AF2 domains of the androgen receptor interact with distinct regions of SRC1. Mol Cell Biol 19:83838392[Abstract/Free Full Text]
-
Stenoien DL, Nye AC, Mancini MG, Patel K, Dutertre M, OMalley BW, Smith CL, Belmont AS, and Mancini MA 2001 Ligand-mediated assembly and real-time cellular dynamics of estrogen receptor-coactivator complexes in living cells. Mol Cell Biol 21:44044412[Abstract/Free Full Text]
-
Adachi M, Takayanagi R, Tomura A, Imasaki K, Kato S, Goto K, Yanase T, Ikuyama S, Nawata H 2000 Androgen-insensitivity syndrome as a possible coactivator disease. N Engl J Med 343:856862[Free Full Text]
-
Doesburg P, Kuil CW, Berrevoets CA, Steketee K, Faber PW, Mulder E, Brinkmann AO, Trapman J 1996 Functional in vivo interaction between the amino-terminal, transactivation domain and the ligand binding domain of the androgen receptor. Biochemistry 36:10521064[CrossRef]
-
Berrevoets CA, Doesburg P, Steketee K, Trapman J, Brinkmann AO 2012 1998 Functional interactions of the AF-2 activation domain core region of the human androgen receptor with the amino-terminal domain and with the transcriptional coactivator TIF2 (transcriptional intermediary factor 2). Mol Endocrinol 12:11721183[Abstract/Free Full Text]
-
He B, Kemppainen JA, Wilson EM 2000 FXXLF and WXXLF sequences mediate the NH2-terminal interaction with the ligand binding domain of the androgen receptor. J Biol Chem 275:2298622994[Abstract/Free Full Text]
-
McNally JG, Müller WG, Walker D, Wolford R, Hager GL 2000 The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science 287:12621265[Abstract/Free Full Text]
-
van Steensel B, Brink M, van der Meulen K, van Binnendijk EP, Wansink DG, de Jong L, de Kloet ER, van Driel R 1995 Localization of the glucocorticoid receptor in discrete clusters in the cell nucleus. J Cell Sci 108:30033011[Abstract/Free Full Text]
-
Stenoien DL, Patel K, Mancini MG, Dutertre M, Smith CL, OMalley BW, Mancini MA 2001 FRAP reveals that mobility of oestrogen receptor-
is ligand- and proteasome-dependent. Nat Cell Biol 3:1523[CrossRef][Medline]
-
Nakao R, Haji M, Yanase T, Ogo A, Takayanagi R, Katsube T, Fukumaki Y, Nawata H 1992 A single amino acid substitution (Met786
Val) in the steroid-binding domain of human androgen receptor leads to complete androgen insensitivity syndrome. J Clin Endocrinol Metab 74:11521157[Abstract]
-
Miyagishi M, Fujii R, Hatta M, Yoshida E, Araya N, Nagafuchi A, Ishihara S, Nakajima T, Fukamizu A 2000 Regulation of Lef-mediated transcription and p53-dependent pathway by associating ß-catenin with CBP/p300. J Biol Chem 275:3517035175[Abstract/Free Full Text]