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Molecular Endocrinology, doi:10.1210/me.2003-0145
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Molecular Endocrinology 18 (4): 834-850
Copyright © 2004 by The Endocrine Society

Transient, Ligand-Dependent Arrest of the Androgen Receptor in Subnuclear Foci Alters Phosphorylation and Coactivator Interactions

Ben E. Black, Michael J. Vitto, Daniel Gioeli, Adam Spencer, Nima Afshar, Mark R. Conaway, Michael J. Weber and Bryce M. Paschal

Center for Cell Signaling (B.E.B., M.J.V., A.S., N.A., B.M.P.), Department of Biochemistry and Molecular Genetics (B.E.B., N.A., B.M.P.), Cell and Molecular Biology Program (B.E.B., N.A., M.J.W., B.M.P.), and Department of Microbiology (D.G., M.J.W.), Department of Health Evaluation Sciences (M.R.C.), University of Virginia, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Bryce M. Paschal, Ph.D, Center for Cell Signaling, Box 800577 Health Systems, University of Virginia, Charlottesville, Virginia 22908. E-mail: paschal{at}virginia.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Here we report that mutations within the DNA-binding domain of AR, shown previously to inhibit nuclear export to the cytoplasm, cause an androgen-dependent defect in intranuclear trafficking of AR. Mutation of two conserved phenylalanines within the DNA recognition helix (F582, 583A) results in androgen-dependent arrest of AR in multiple subnuclear foci. A point mutation in one of the conserved phenylalanines ({Delta}F582, F582Y) is known to cause androgen insensitivity syndrome (AIS). Both AIS mutants ({Delta}F582, F582Y) and the export mutant (F582, 583A) displayed androgen-dependent arrest in foci, and all three mutants promoted androgen-dependent accumulation of the histone acetyl transferase CREB binding protein (CBP) in the foci. The foci correspond to a subnuclear compartment that is highly enriched for the steroid receptor coactivator glucocorticoid receptor-interacting protein (GRIP)-1. Agonist-bound wild-type AR induces the redistribution of GRIP-1 from foci to the nucleoplasm. This likely reflects a direct interaction between these proteins because mutation of a conserved residue within the major coactivator binding site on AR (K720A) inhibits AR-dependent dissociation of GRIP-1 from foci. GRIP-1 also remains foci-associated in the presence of agonist-bound F582, 583A, {Delta}F582, or F582Y forms of AR. Two-dimensional phospho-peptide mapping and analysis with a phospho-specific antibody revealed that mutant forms of AR that arrest in the subnuclear foci are hypophosphorylated at Ser81, a site that normally undergoes androgen-dependent phosphorylation. Our working model is that the subnuclear foci are sites where AR undergoes ligand-dependent engagement with GRIP-1 and CBP, a recruitment step that occurs before Ser81 phosphorylation and association with promoters of target genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ANDROGEN RECEPTOR (AR) is a member of the nuclear receptor (NR) superfamily that comprises the largest family of transcription factors including the steroid receptors, nonsteroid receptors, and orphan nuclear receptors (1, 2). NRs are ligand-activated transcription factors that contain a modular structure, including a structurally conserved DNA-binding domain (DBD) that contains two zinc-binding loops. Within the DBD are residues that are conserved in virtually all NRs, and other residues that are variable including those that confer sequence-specific binding to DNA response elements. Likewise, the C-terminal domain contains a ligand-binding domain (LBD) that has a conserved structure, but it is sufficiently different among NRs to permit ligand discrimination. Transcriptional activation or repression generally depends on whether the ligand is an agonist or an antagonist, and it is mediated by NR-dependent recruitment of coactivators or corepressors to promoters (3). The best characterized coactivators for NRs are the three p160 family members steroid receptor coactivator (SRC)-1/NCOA1, glucocorticoid receptor interacting protein (GRIP)-1/TIF-2, and amplified in breast cancer (AIB)-1/ACTR/RAC3 (4). The coactivators bind directly to NRs, and because they contain multiple protein interaction motifs, they are thought to function as platforms for attracting important functional partners to the promoter, most notably histone acetyl transferases. Additional multiprotein complexes are brought to the promoter to carry out ATP-dependent chromatin remodeling and histone methylation, and to facilitate recruitment of RNA polymerase II (3). Currently, it is not known whether there is temporal and spatial regulation of these recruitment events.

Ligand binding is also used to regulate the subcellular distribution of certain NRs. The GR and AR are predominantly cytoplasmic in the absence of their cognate hormone but undergo rapid import in response to corticosteroids and androgens, respectively (5, 6, 7). The interpretation of these data is that the nuclear localization signal (NLS) within GR and AR is each masked by conformation or by chaperones, when the receptor is in the cytoplasm. Ligand-binding, which is known to alter the conformation of GR and AR, switches the receptor to an import competent form by exposing the NLS to cytoplasmic receptors that mediate import of the NR within 30–60 min (8).

Translocation of NRs from the nucleus back to the cytoplasm occurs even in the continued presence of saturating concentrations of ligand (9, 10, 11, 12, 13, 14). Because ligand-bound NRs undergo nuclear export, a nucleocytoplasmic shuttling cycle of NRs is likely to be integrated with the intranuclear movements of NRs that may modulate their transactivation functions (9, 12, 14, 15). These may include the rapid association with, and dissociation from, promoter element DNA, and interactions with the nuclear matrix (16, 17, 18). Inhibiting nuclear export of NRs increases transcription in reporter assays, suggesting that nuclear export might in fact contribute to NR regulation (9, 19).

The nuclear export pathway of NRs has not been fully characterized. Where examined, it has been found that nuclear export of NRs is not inhibited by the Crm1 inhibitor leptomycin B (10, 20, 21, 22, 23), a clear indication that NRs use an export receptor distinct from Crm1 for translocation through the nuclear pore complex. This view is supported by the fact that NRs lack the leucine-rich nuclear export signal recognized by Crm1. In the process of analyzing the nuclear export pathway of GR, our laboratory found that the DBD of GR is necessary and sufficient for nuclear export (9). Moreover, the DBDs from nine additional NRs were determined to function as export signals. We have not ruled out the formal possibility that NRs may contain additional export signals (24). Nonetheless, the available information suggests that NRs could use a common, DBD-dependent pathway for nuclear export. One of the components of this pathway is the chaperone calreticulin, which binds the GR DBD in vitro and is necessary for GR export to the cytoplasm in vivo (23). Calreticulin overexpression antagonizes the transcriptional activity of several NRs (25, 26, 27), which is consistent with a potential role in regulating NR localization and function. Our mutational analysis of nuclear export mediated by the GR DBD revealed that two phenylalanines in the DNA recognition helix are critical for export activity. Mutation of one or both phenylalanines resulted in a dramatic reduction in nuclear export of the GR DBD fused to a green fluorescent protein (GFP) reporter protein (23). Mutation of these phenylalanines also inhibited nuclear export of full-length GR without affecting its ligand-dependent nuclear import (9). Similarly, mutation of the two phenylalanines in the DNA recognition helix of AR resulted in a nuclear export defect (9). In contrast to GR, these mutations in the context of AR cause ligand-dependent arrest in a distinct compartment within the nucleus. In the present study, we show that arrest of the AR export mutant is agonist specific, that it occurs in subnuclear foci that contain the coactivator GRIP-1, and that it results in CREB binding protein (CBP) recruitment to the foci. Peptide mapping and phospho-antibody detection were used to show the arrest of AR in subnuclear foci is correlated with hypophosphorylation at Ser81, a site of androgen-dependent phosphorylation.

The phenylalanines in the DNA recognition helix are mutated in certain patients with complete androgen insensitivity syndrome (AIS), a human disease that is generally associated with male pseudohermaphroditism and varies widely in clinical severity. We constructed the AIS mutations in AR, and found that the AIS mutants of AR ({Delta}F582, F582Y) display the same phenotype as the export mutant of AR (F582, 583A). Defects in protein trafficking are now linked to AR activity in the human disorders spinal and bulbar muscular atrophy, prostate cancer, and AIS (28, 29). Wild-type (WT) AR induces rapid, agonist- and LBD-dependent dissociation of GRIP-1 from subnuclear foci, which we speculate is an intermediate compartment that facilitates interactions between NRs and coactivators before transcription.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors including AR undergo a cycle of ligand binding, nuclear import, and nuclear export (2, 30, 31). The AR transport cycle can be observed in the heterokaryon shuttling assay. In brief, the assay involves transient transfection of AR into Cos7 cells, androgen treatment to induce nuclear import, and polyethylene glycol-induced fusion with 3T3 cells. Fusion of the plasma membranes of Cos7 and 3T3 cells results in a shared cytoplasm, allowing WT AR to undergo nuclear export from donor nuclei (Cos7) and nuclear import into acceptor nuclei (3T3) (Fig. 1AGo, top row). Mutation of conserved phenylalanine residues in the DNA recognition helix of the DBD of AR and GR inhibit export from the nucleus in vivo (9). The corresponding residues also have been shown to be critical for efficient nuclear export of full-length vitamin D and retinoid X receptors (32). In the heterokaryon shuttling assay, an export defect in a mutant form of AR (F582, 583A) resulted in a relatively high concentration of AR in donor nuclei, as compared with acceptor nuclei (Fig. 1AGo, middle row). In this assay, we noted that the mutant AR (F582, 583A) was concentrated in small foci in the donor nuclei. Because mutation of these phenylalanines reduces DNA binding, we examined whether loss of DNA-binding is sufficient to inhibit AR export from the nucleus, or sufficient to promote AR concentration in the subnuclear foci. The underlying logic was that nuclear export of AR might be temporally linked to transcription, analogous to the linkage between nuclear export of mRNA and RNA processing (33). Alternatively, mutant forms of AR that fail to bind DNA might be directed to a subnuclear compartment as part of a degradation pathway.



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  		Fig. 1. Mutations that Inhibit AR Export Cause Ligand-Dependent Arrest in the Nucleus

A, Disruption of AR trafficking is not a consequence of defective DNA binding. Nucleocytoplasmic shuttling assays were performed for WT, F582, 583A, and V581F proteins as described (9 ). Twenty-four hours before fusion the Cos7 (donor) cells are transfected with FLAG-tagged AR proteins, and the NIH3T3 (acceptor) cells are labeled with an inert dye (CMTMR; Molecular Probes, Eugene, OR) that is visible in the rhodamine channel. CMTMR, (5 (and 6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine. One hour before fusion, the Cos7 donor cells were treated with R1881 (10 nM) to induce nuclear import of AR, which is maintained throughout the experiment. Cells are fused with a brief (30 sec) incubation with PEG-1500 (Roche), washed six times, and incubated for 4 h in standard media to allow shuttling to occur. Cycloheximide (10 µg/ml) is included to inhibit protein synthesis. The cells were fixed for IF and AR detected by anti-Flag monoclonal antibody. Fusions that contain both red (CMTMR) and green (AR) fluorescence are heterokaryons. Acceptor nuclei are identified by brightly staining heterochromatin in the 4',6-diamidino-2-phenylindole (DAPI) channel and are denoted by white arrowheads. B, Ligand-specific arrest of the AR export mutant (F582, 583A) in subnuclear foci. WT and mutant AR (F582, 583A) were expressed as Flag-tagged proteins in Cos7 cells, and were treated with Veh (0.1% ethanol), R1881 (10 nM), or Cas (50 µM) for 1 h before fixation. R1881 and Cas promote nuclear import of both WT and mutant AR (F582, 583A) (middle, right panels). The mutant AR (F582, 583A) arrests in discreet subnuclear foci when treated with the agonist R1881, but localizes throughout the nucleoplasm when treated with the pure antiandrogen Cas. Insets show the distribution of WT and mutant AR in the presence of R1881 (middle panels). Bar, 10 µm.

 
We addressed this question by constructing an AR mutation that results in a complete loss of DNA binding, and a failure to activate transcription (34). A conserved P-box residue that in NRs makes direct contact with hormone response element DNA (35) was mutated to phenylalanine (V581F), and the AR mutant was tested in the heterokaryon shuttling assay. We found that the P-box mutant AR (V581F) underwent nucleocytoplasmic shuttling comparable to WT AR, and also that the V581F mutant failed to concentrate in subnuclear foci. Thus, a DNA binding defect per se is insufficient to account for the inhibition of shuttling or for the accumulation of AR in subnuclear foci. These AR transport phenotypes might, however, be related, because the arrest of mutant AR in subnuclear foci could preclude access to the export machinery.

Ligand-Specific Arrest of Mutant AR
We set out to characterize the subnuclear foci, reasoning that they might represent an intranuclear, intermediate compartment for AR. Because the heterokaryon shuttling assays are performed in the presence of synthetic androgen (R1881) to promote nuclear import into acceptor nuclei before cell fusion, we first examined whether AR arrest in the subnuclear foci is dependent on ligand binding. WT and mutant AR (F582, 583A) proteins were transfected into Cos7 cells, and after 1 h of treatment with vehicle (Veh, ethanol), R1881, or the anti-androgen casodex (Cas), the cells were fixed and processed for immunofluorescence (IF) microscopy. Mutant AR that localized to the nucleus in the absence of androgen, and mutant AR that was induced to enter the nucleus with Cas, both failed to accumulate in subnuclear foci (Fig. 1BGo, lower row and data not shown). Moreover, WT AR failed to accumulate in subnuclear foci under any of the conditions tested (Fig. 1BGo, upper row). These results show that the localization of AR to subnuclear foci is strictly dependent on the mutations (F582, 583A) and on the presence of androgen.

After agonist-dependent import, WT AR adopts a nonuniform, fibrous-like distribution within the nucleus (36, 37). Deconvolution microscopy and heterochromatin staining has been used by other laboratories to show that some of the agonist-bound AR accumulates in pericentromeric regions (38). In contrast, antagonist-bound AR is relatively diffuse throughout the nucleoplasm (21, 38). To better visualize the subnuclear distribution of AR, we used IF microscopy and three-dimensional reconstruction (z sections) of WT AR and mutant AR (F582, 583A) expressed in Cos7 cells, either in the presence of agonist (R1881) or antagonist (Cas). Mutant AR (F582, 583A) became highly concentrated in subnuclear foci in response to R1881, a pattern that was different from WT AR under the same condition (Fig. 2AGo; and see video data published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). In the presence of Cas, both WT AR and mutant AR (F582, 583A) were distributed diffusely throughout the nucleoplasm (Fig. 2AGo and video supplemental data). The ligand-sensitive localization of mutant AR to subnuclear foci was also observed in HeLa cells, indicating the distribution is not cell-type specific (Fig. 2BGo). We also found that the agonist-sensitive accumulation of mutant AR (F582, 583A) in subnuclear foci was inhibited by including excess Cas during the incubation with R1881 (Fig. 2CGo, +R1881, +Cas). The mutant AR (F582, 583A) redistributed from foci to the nucleoplasm if Cas was included during R1881 withdrawal (Fig. 2CGo, R1881->Cas). Redistribution of mutant AR to the nucleoplasm could be due to Cas-induced release of AR from foci. Alternatively, androgen-bound mutant AR could cycle between foci and the cytoplasm, and Cas binding could prevent reassociation with foci.



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  		Fig. 2. Characteristics of the Ligand-Dependent AR Arrest

A, The subnuclear foci where AR arrests are distributed throughout the nucleoplasm. Images of three-dimensional renderings of WT and MUT AR after 1 h of treatment with R1881 or Cas. See QuickTime video, published as supplemental data. B, AR arrest at subnuclear foci occurs in HeLa cells in response to R1881 (10 nM) but not Cas (50 µM). C, AR arrest at subnuclear foci can be competed and reversed by antagonist. Incubation with R1881 (2 nM for 1 h) leads to the accumulation of the mutant AR (F582, 583A) in subnuclear foci, but this is competed by including excess Cas (50 µM) in the reaction. AR arrest in subnuclear foci formation is reversible because treatment of for 1 h with R1881 (2 nM) followed by 4 h with Cas (50 µM) leads to redistribution of AR to the nucleoplasm. D, Export-defective GR does not arrest in subnuclear foci. Cells expressing WT or export-defective GR (F444, 445A) as GFP fusions were treated with Veh, dexamethasone (Dex) (1 µM), or R1881 (10 nM) for 1 h, and processed for microscopy. Bar, 10 µm.

 
DNA Recognition Helix Mutations Do Not Cause GR Arrest in Foci
Because the DBDs of AR and GR are 80% identical, we tested whether mutating the analogous phenylalanines in GR affected the subnuclear distribution in response to ligand. Unlike AR, we found that the analogous mutations in GR (F444,445A) did not induce accumulation in foci in response to agonist (dexamethasone), or in response to R1881 (Fig. 2DGo). The corresponding mutations in thyroid hormone receptor and retinoic acid receptor also did not result in ligand-dependent accumulation in subnuclear foci (Ref.9 ; and Black, B. E., and B. M. Paschal, unpublished observations). In contrast to these results, a point mutation within the DBD of MR was shown to cause ligand-dependent concentration in subnuclear foci (39). Thus, the arrest of NRs in subnuclear compartments is not seen in all NRs and is strongly influenced by both the mutation and the type of ligand.

Reduced Mobility of Foci-Associated AR
The accumulation of mutant AR in foci suggested that it might have a reduced mobility in the nucleus. It is possible that a reduction in AR mobility could further reduce the transcriptional activity of a DNA binding mutant (F582, 583A) by limiting the concentration of AR in the nucleoplasm. We tested for a reduction in nuclear mobility using a fluorescence loss in photobleaching (FLIP) assay with GFP fusions to WT and mutant (F582, 583A) AR proteins (Fig. 3Go). The principle of the assay is that reiterative photobleaching of a fluorescent protein in one region of the nucleus results in loss of fluorescence throughout the nucleus over time if the protein is mobile within the nucleus. The rate of the loss of fluorescence is proportional to mobility, which is represented as a decay curve. In the assay, we designated a region of interest (ROI) for photobleaching (120 x 20 pixels; white rectangle) and an ROI for FLIP measurement (30 x 30 pixels; red square). We transfected Cos7 cells with plasmids encoding GFP-AR (WT) and GFP-AR (F582, 583A), treated the cells with R1881 to induce import, and analyzed the cells by FLIP. We observed that the DNA binding domain mutations (F582, 583A) reduced the nuclear mobility of AR (Fig. 3AGo). In the examples shown, the time required for loss of one half of the nuclear fluorescence was 65 sec for GFP-AR WT and 110 sec for GFP-AR (F582, 583A). We analyzed the data from multiple cells (Fig. 3BGo), and determined that the time required for loss of one half of the nuclear fluorescence was, on average, 49 ± 15 sec for GFP-AR WT (n = 15) and 103 ± 18.5 sec for GFP-AR (F582, 583A) (n = 21) (P < 0.001).



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  		Fig. 3. Intranuclear Mobility of WT and Mutant AR

A, Images from FLIP analysis of GFP-AR (WT and F582, 583A mutant) expressed in Cos7 cells. The red box defines the ROI for fluorescence measurement, and the white rectangle defines the ROI for reiterative photobleaching. The graphs show the loss of fluorescence during the 180-sec experiment. B, FLIP analysis of data from cells expressing WT GFP-AR (n =15) and mutant GFP-AR (F582, 583A) (n =21). The data sets for both WT and mutant AR proteins were fit with single exponential curves. The decay constants for these curves averaged -0.070 ± 0.006 for cells expressing GFP-AR (WT), and -0.034 ± 0.007 for cells expressing GFP-AR (F582, 583A). P < 0.001, two-sample t test.

 
AR Arrests in Subnuclear Foci with Coactivators
Several coregulators of transcription, including members of the p160 family of coactivators, display a nonuniform distribution within the nucleus and localize to discrete subnuclear domains. Among these, the steroid receptor coactivator GRIP-1 has been shown to concentrate in small, subnuclear foci coincident with GR (40, 41). The p160 family member SRC-1, which is approximately 40% identical with GRIP-1, can also localize to subnuclear foci. In this context, Weigel and co-workers (28) showed that SRC-1 colocalizes with AR that contains a point mutation C-terminal to the DBD (C619Y). Given these observations, we examined the distribution of a GFP fusion of GRIP-1 after expression in Cos7 cells. We found that GRIP-1 localized to subnuclear foci in a pattern similar to that observed with the mutant AR (582, 583A; Fig. 4AGo). In contrast, a GFP fusion of the histone acetyltransferase CBP was localized throughout the nucleoplasm in a diffuse distribution (Fig. 4AGo and Ref.42).



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  		Fig. 4. Subnuclear Foci Are Sites of Coactivator Localization

A, Nuclear localization of coactivators and AR proteins expressed individually. GFP-GRIP-1 is localized to discreet subnuclear foci (37 40 41 62 ), whereas GFP-CBP is found in a uniform distribution throughout the nucleoplasm (42 ). AR proteins were localized after a 1-h treatment with R1881 (10 nM). B, Colocalization of the AR mutant (F582, 583A) with GFP-GRIP-1 in the presence of R1881. Coexpression of WT AR results in redistribution of GFP-GRIP-1 throughout the nucleoplasm, whereas coexpression of the AR mutant (F582, 583A) results in GRIP-1 maintaining the subnuclear foci localization. C, CBP is recruited to subnuclear foci by the AR mutant (F582, 583A). CBP remains distributed throughout the nucleoplasm when coexpressed with WT AR. In contrast, CBP localizes to the same subnuclear foci as the AR mutant (F582, 583A) in R1881-treated cells. D, The AR mutant (F582, 583A) does not colocalize with PML bodies. Cells were cotransfected with plasmids encoding Flag-AR and a fusion of yellow fluorescent protein and PML (63 ), and processed for fluorescence microscopy. Bar, 10 µm.

 
To determine whether GRIP-1 and the mutant AR (F582, 583A) localize to the same subnuclear domains, we cotransfected GFP-GRIP-1 and AR and compared the distribution by IF microscopy. AR was detected in these experiments using anti-Flag antibody. We found that GRIP-1 cotransfected with mutant AR (F582, 583A) displayed a high degree of coincident localization, an indication that these proteins are concentrated in the same subnuclear foci (Fig. 4BGo). In contrast, cotransfected GRIP-1 and WT AR were distributed throughout the nucleoplasm and showed no evidence of concentration in foci. Moreover, we were unable to detect GRIP-1 in subnuclear foci when WT AR was coexpressed, suggesting GRIP-1 is dissociated from foci in the presence of AR. We also found that CBP accumulates in subnuclear foci when coexpressed with mutant AR (F582, 583A) and treated with androgen (Fig. 4CGo). Similarly, the CBP relative p300 accumulated in subnuclear foci when cotransfected with mutant AR (582, 583A) but not WT AR, and the effect on p300 localization was androgen dependent (Black, B. E., and B. M. Paschal, unpublished results). These observations could reflect either an AR-dependent recruitment of CBP/p300 to the subnuclear foci, or AR-dependent stabilization of histone acetyltransferase that, otherwise, associates only transiently with subnuclear foci.

Subnuclear Foci Are Not Promyelocytic Leukemia (PML) Bodies
The subnuclear foci containing mutant AR (F582, 583A) and GRIP-1 could reflect a storage site for transcriptional regulators, or a domain that facilitates protein-protein interactions or protein modifications that are important for transcription. We compared the distribution of mutant AR (F582, 583A) to PML bodies, which are known to be a storage compartment for multiple nuclear proteins including the p160 family member SRC-1 (43). We found that mutant AR (F582, 583A) shows virtually no overlap with PML in the absence or presence of androgen (Fig. 4DGo). Because SRC-1 has been shown to localize to PML bodies (43), it is possible that different p160 family members might occupy different subcompartments in the nucleus.

WT AR Alters GRIP-1 Distribution
GRIP-1 has been detected in subnuclear foci by several laboratories (36, 41, 43). The C-terminal domain of GRIP-1 is necessary for its accumulation in foci (41), but the mechanism of foci formation and why this occurs in only a subset of cells are presently unknown. The percentage of cells showing a subnuclear foci distribution does not appear to be an artifact of overexpression because Hager and co-workers (41) showed that increasing the amount of GRIP-1 plasmid DNA in the transfection did not increase the fraction of HeLa cells expressing GRIP-1 in subnuclear foci. Within a single microscopy field, GFP-GRIP-1 in the nuclei of Cos7 cells varies from a uniform distribution to a highly concentrated distribution in subnuclear foci (Fig. 5AGo). We addressed whether, under the conditions of our experiments, there is a correlation between the expression level of GRIP-1 in Cos7 cells and its distribution in subnuclear foci. We compared the nuclear fluorescence per unit area in cells expressing GFP-GRIP-1 and found no correlation between GRIP-1 expression level and the subnuclear localization (Fig. 5BGo).



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  		Fig. 5. Distribution and Dynamics of GRIP-1 in Subnuclear Foci

A, Heterogeneity of GRIP-1 distribution in cells. Field showing the typical range of GFP-GRIP-1 distributions in different cells. B, Presence of GRIP-1 in subnuclear foci is not correlated with expression level. Total nuclear GFP fluorescence was measured in Cos7 cells with GRIP-1 distributed in subnuclear foci (Foci+), distributed throughout the nucleoplasm (Foci-), and plotted as the fluorescence per unit area. C, Real-time analysis of WT AR-dependent GRIP-1 dissociation from subnuclear foci. WT AR and GFP-GRIP-1 were coexpressed in Cos7 cells, and changes in the distribution of GFP-GRIP-1 were recorded by laser scanning microscopy after the addition of androgen (10 nM R1881). Images were taken at 10-min time points.

 
Because the heterogeneity of GRIP-1 distribution could limit analysis in populations of fixed cells, we used fluorescence microscopy to score the number of cells that display GRIP-1 association with foci, and the potential effect on agonist-bound AR on the GRIP-1 distribution (Table 1Go). We found that, in the absence of AR, GFP-GRIP-1 is found in foci in 36.6% of Cos7 cells expressing the protein. WT AR has little effect on this distribution, unless agonist is added, whereby the percentage of cells with GFP-GRIP-1 in foci is reduced to 2.9%. Mutant AR (F582, 583A) in the presence of agonist not only fails to promote GRIP-1 dissociation from foci, but it actually increases the percentage of cells expressing GRIP-1 in foci to 67.1%. These data further establish a link between AR and the intranuclear distribution of the coactivator GRIP-1.


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  		Table 1. AR Regulates the Association of GRIP-1 with Subnuclear Foci

 
We used live cell imaging to examine the time course of AR and androgen-induced GRIP-1 dissociation from subnuclear foci. GFP-GRIP-1 and AR were coexpressed in Cos7 cells, and 24 h post transfection, androgen was added and the cells were imaged by confocal fluorescence microscopy at 10-min time points. The redistribution of GFP fluorescence from a small number of subnuclear foci was detected as early as 10 min after R1881 addition; by 20 min there was a substantial redistribution from subnuclear foci to the nucleoplasm (Fig. 5CGo). The GRIP-1 redistribution in this assay requires coexpression of AR and addition of R1881 (data not shown). These live-cell imaging results support the concept of an AR-dependent mechanism for regulating GRIP-1 distribution within the nucleus. This may involve ligand-bound AR that triggers GRIP-1 dissociation from foci, or ligand-bound AR that captures GRIP-1 that is undergoing rapid exchange between subnuclear foci and the nucleoplasm.

The LBD Is Involved in GRIP-1 Dissociation from Foci
The ligand specificity of AR-induced GRIP-1 dissociation reported here and by Palvimo and colleagues (36) suggested the LBD could play a critical role in this process. We tested this by mutating a lysine (K720) within the AR LBD that, based on studies with estrogen receptor LBD (44), is predicted to modulate contact between the LBD and nuclear receptor boxes in p160 family members including GRIP-1 (3). We found that the K720A mutant form of AR is expressed at levels comparable to WT AR, is predominantly cytoplasmic in the absence of androgen, and undergoes nuclear import in response to R1881 (Fig. 6Go). Unlike WT AR, the K720A mutant form of AR is defective in promoting GRIP-1 dissociation from foci (Fig. 6Go and Table 1Go). These data implicate the coactivator binding surface on the AR LBD in GRIP-1 dissociation from foci.



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  		Fig. 6. Mutation of the AR LBD Inhibits GRIP-1 Dissociation from Foci

GFP-GRIP-1 was coexpressed with WT or mutant AR (K720A) in Cos7 cells, and the proteins were localized by fluorescence microscopy after treatment with androgen for 1 h (+R1881).

 
AIS Mutants Localize to Subnuclear Foci
The highly conserved phenylalanines characterized in this study are mutated in patients diagnosed with AIS (45, 46). Although the majority of AIS mutations map to the LBD of AR, complete AIS can also be caused by mutations within the DBD (45, 46). We tested whether the AIS mutants (F582Y and {Delta}F582) localize to subnuclear foci that contain GRIP-1 by coexpression in Cos7 cells. The AR mutants F582Y and {Delta}F582 were expressed at levels comparable to WT AR and localized predominantly to the cytoplasm in the absence of androgen (Fig. 7AGo). The distribution of GFP-GRIP-1 to subnuclear foci appeared to be unaffected by expression of WT and mutant AR proteins in cells that were not treated with androgen. Androgen treatment (+R1881) resulted in complete nuclear translocation of WT, F582Y, and {Delta}F582 AR proteins, but the WT and mutant proteins had strikingly different effects on GRIP-1 distribution within the nucleus (Fig. 7BGo). GRIP-1 was found throughout the nucleoplasm in the presence of WT AR, however, GRIP-1 remained localized to subnuclear foci in the presence of the AIS mutants F582Y and {Delta}F582 (Fig. 7BGo). Colocalization of the AIS mutants of AR in subnuclear foci that contain GRIP-1, and the failure of GRIP-1 to dissociate from foci under these conditions, suggests that these AIS mutations predispose AR to ligand-dependent arrest within an intermediate compartment in the nucleus.



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  		Fig. 7. AIS Mutations Cause Ligand-Dependent Localization of AR and CBP to Subnuclear Foci

A and B, WT AR and AIS mutants of AR were coexpressed with GFP-GRIP-1, and the proteins were localized after no treatment (-R1881) or treatment with synthetic androgen for 1 h (+R1881). The AIS mutants are predominantly cytoplasmic in the absence of androgen, undergo androgen-dependent nuclear import, and concentrate in subnuclear foci that contain GRIP-1. C, AIS mutants of AR recruit CBP into subnuclear foci. WT AR and AIS mutants of AR were coexpressed with GFP-CBP, and the proteins were localized after treatment with synthetic androgen for 1 h (+R1881).

 
Given the similarities between the AR mutants F582, 583A, F582Y, and {Delta}F582 in subnuclear targeting and arrest in GRIP-1 subnuclear foci, we tested whether expression of AIS mutants could induce accumulation of CBP in subnuclear foci. GFP-CBP, when coexpressed with the AIS mutant F582Y or {Delta}F582, colocalized to subnuclear foci with the mutant AR in the presence of androgen, but not in the absence of androgen (Fig. 7CGo, and data not shown). In certain instances, we found that a considerable amount of CBP did not arrest in foci and instead remained nucleoplasmic. We speculate that this could reflect a limited number of binding sites for CBP in the foci, or a dynamic association of CBP with foci that favors dissociation. Our results show that AIS mutations can cause the arrest of AR along with critical coactivators of transcription in a subnuclear compartment.

Subnuclear Foci Localization and Phosphorylation
AR is phosphorylated on defined serines in response to stimuli, which include androgen, growth factors, and pharmacological reagents that activate kinase pathways. To determine whether the arrest of AR in GRIP-1-containing subnuclear foci affects its phosphorylation state we analyzed WT and mutant (F582, 583A) forms of AR by metabolic labeling and two-dimensional phospho-peptide mapping (Fig. 8AGo). The migration positions of phospho-peptides derived from AR and the identities of individual phosphorylation sites were established in a previous study by thin layer electrophoresis and ascending chromatography, direct sequencing, mass spectrometry, and mutagenesis (47). One of the major sites of phosphorylation induced in response to treatment with R1881 is Ser81 (Refs.47, 48, 49 ; Fig. 8AGo). We compared the phospho-peptide maps of WT and mutant (F582, 583A) forms of AR and found that in the absence of androgen, the phospho-peptide maps were virtually the same. After treatment with androgen (+R1881), we observed that the peptide from WT AR spanning the Ser81 phosphorylation site contained a significantly higher level of radioactive phosphate, as reported (47). In contrast, the mutant AR (F582, 583A) failed to display the robust the R1881-induced phosphorylation at Ser81 that was detected with WT AR (Fig. 8AGo).



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  		Fig. 8. Hypophosphorylation of Ser81 in AR Mutants Localized to Subnuclear Foci

A, Phospho-peptide maps of WT and mutant AR (F582, 593A). Without androgen treatment, similar levels of 32P are detected in the peptide that contains Ser81, derived from both the WT and mutant AR (F582, 583A) proteins (left panels). After androgen treatment, the marked increase in 32P detected in the WT AR-derived peptide is not observed in the mutant AR-derived peptide (right panels). Phosphorylation of Ser650 (S650) is similar in the WT and mutant AR proteins, both in the absence and presence of androgen. B, Immunoblots of AR proteins showing the specificity of the anti-phospho-Ser81 antibody. Cos-1 cells were transfected with WT AR or the mutant AR (S81A), treated with R1881 for 2 h, and processed for immunoprecipitation and immunoblotting. The robust, androgen-dependent reactivity of the antibody is observed with WT AR, but not with AR where the phospho-acceptor serine is mutated to alanine. Total AR in the samples (anti-AR) was detected with PG-21, which recognizes the N terminus of AR and is not phospho specific. C, Immunoblots of AR proteins showing the level of phospho-Ser81 reactivity in response to androgen addition. WT and mutant AR proteins were expressed and processed as described above. Mutants that localize to subnuclear foci (F582, 583A, {Delta}F582, and F582Y) are hypophosphorylated at Ser81, whereas the P-box DNA binding mutant of AR (V581F) displays a WT level of phospho-Ser81 reactivity.

 
To further investigate the phosphorylation state of mutant AR proteins, we used affinity-purified antibodies against the phospho-peptide corresponding to the Ser81 phosphorylation site. The specificity of the anti-phospho-Ser81 antibody is demonstrated by the significant enhancement of immunoreactivity after treatment with R1881, and by the absence of immunoreactivity with AR that contains a mutation in the epitope (S81A; Fig. 8BGo). We used the anti-phospho-Ser81 antibody to probe immunoblots of Cos7 cells expressing WT and mutant AR proteins. Consistent with the phospho-peptide mapping, the antibody-based detection revealed R1881-dependent Ser81 phosphorylation in WT AR (Fig. 8CGo). The R1881-dependent phosphorylation was significantly reduced in the AR mutant (F582, 583A) that undergoes R1881-dependent arrest in subnuclear foci (Fig. 8CGo). Moreover, a reduced level of Ser81 phosphorylation was observed with the AIS mutants F582Y and {Delta}F582. The hypophosphorylation at Ser81 in these mutants is apparently correlated with AR arrest in subnuclear foci and not with a failure to bind to DNA because the P-box mutant AR (V581F) displays a level of R1881-induced phosphorylation that is comparable to WT AR (Fig. 8CGo). Thus, the AIS mutations that cause ligand-dependent arrest of AR in subnuclear foci also prevent subsequent modification by a Ser81 kinase. We interpret these results as evidence that AR undergoes a sequence of intranuclear steps before engagement with androgen response element DNA (Fig. 9Go).



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  		Fig. 9. Model of AR Trafficking

See text for details.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
AR functions as a molecular sensor that detects subnanomolar concentrations of cellular androgens and converts the androgen signal into changes in gene expression (50). This pathway is critical for growth and development of the normal prostate and in prostate cancer, as well (51). In the LNCaP human prostate cancer cell line, which expresses AR, the expression profiles of more than 100 genes are increased in the presence of androgen (52). The exact number of genes that are regulated directly by AR has not been determined, but sequences matching or showing high similarity to androgen response element DNA sequences are present in the majority of these genes. AR function is likely to extend beyond the male reproductive system, given that AR mRNA and protein is detected in a variety of tissues, and AR knockout mice show histological defects in bone and adipose tissue (53).

The pathway of AR-dependent gene expression is initiated when circulating androgens cross the plasma membrane and bind to cytoplasmic AR. Androgen binding induces a conformational change in AR in vitro, and this change in structure is believed to provide the basis for AR translocation into the nucleus in vivo. Physical changes in receptor structure and possibly chaperone composition induced by steroid binding were originally observed in GR and referred to as receptor transformation (54). Whereas it is clear that the androgen-dependent transformation switches AR to a form that is competent for import, the molecular mechanism underlying this switch has not been defined. The transformation is certain to involve the bipartite NLS within the hinge region (6, 7), but there is also evidence for an NLS activity associated with the LBD of AR (55). In the cytoplasm, these transport signals within AR could be masked by chaperone proteins, or they have limited solvent exposure because of AR tertiary structure. In both scenarios, androgen binding should result in exposure of at least one NLS to a cytoplasmic import receptor. By analogy with other import receptor-mediated transport pathways, the AR-import receptor complex binds to, and translocates through, the nuclear pore complex. RanGTP binding to the import receptor on the nuclear side of the nuclear pore complex should release AR into the nucleoplasm.

In the present study, we have analyzed post-import trafficking of AR and presented evidence, suggesting that AR travels through a coactivator compartment in the nucleus (Fig. 9Go). We observed that AR mutations (F582, 583A), originally characterized as loss-of-function with regard to nuclear export (9), result in androgen-dependent arrest in subnuclear foci. Importantly, we determined that mutation of only one of these phenylalanine residues, as occurs in complete AIS, is sufficient to arrest AR in subnuclear foci. The molecular basis of AIS is generally interpreted in the context of LBD mutations that reduce or eliminate androgen binding, or DBD mutations that reduce or eliminate DNA binding. We speculate that defects in post-import nuclear trafficking might contribute to the AIS disease phenotype either directly or through secondary effects. The arrest of mutant AR in subnuclear foci could limit the effective concentration of AR, or as discussed below, limit the effective concentration of transcriptional coactivators. In addition, defects in post-import nuclear trafficking of AR are not limited to AIS. An analysis of AR mutations that occur in prostate cancer revealed that mutation of a cysteine proximal to the second zinc finger in the DBD (C619Y) is sufficient to cause AR arrest in subnuclear foci that contain SRC-1 (28). Presently, it is not known whether different p160 family members occupy the same subnuclear foci.

The subnuclear foci characterized in this study and observed in previous studies (36, 40, 41) contain the p160 family member GRIP-1, a factor originally described as a coactivator for GR (56). Using several approaches including live cell imaging, we showed that WT AR changes the distribution of GRIP-1 from subnuclear foci to a more uniform nucleoplasmic distribution. We interpret this as evidence that AR promotes the dissociation of GRIP-1 from a nuclear structure that, in the absence of AR and androgen, can facilitate a substantial accumulation of GRIP-1. Aside from GRIP-1 and potentially other p160 family members (28), the composition of the subnuclear foci is undefined. Because GRIP-1 release from subnuclear foci in situ is resistant to salt extraction and DNase I treatment, it is likely that the foci are part of a nonchromatin compartment (41).

An important feature of the AR-induced dissociation of GRIP-1 from subnuclear foci is the ligand specificity of the reaction. With both the WT and mutant AR proteins, androgen or Cas binding to the LBD of AR is sufficient to induce the transformation of AR necessary for nuclear translocation. However, Cas-bound WT AR fails to dissociate GRIP-1 from foci, and Cas-bound mutant AR fails to arrest at foci. Palvimo and colleagues (36) have shown that the antiandrogens Cas and flutamide can block AR- and androgen-dependent redistribution of GRIP-1 from subnuclear foci to the nucleoplasm. Taken together, the available data indicate that specific molecular features of agonist vs. antagonist-bound AR determine its repertoire of interactions, which, in turn, dictate its intranuclear distribution and function. In chromatin immunoprecipitation experiments, androgen-bound AR recruits the coactivators GRIP-1 and CBP to the prostate-specific antigen promoter and enhancer to activate transcription, whereas Cas-bound AR recruits the corepressors, nuclear receptor corepressor and silencing mediator of retinoic acid and thyroid hormone receptor, to negatively regulate transcription (57, 58).

Potential insight into the biochemical basis of AR recruitment of GRIP-1 derives from the structural basis of agonist-dependent binding of GRIP-1 to ER, which may be prototypical of coactivator-nuclear receptor interactions (44). GRIP-1 and other p160 family members contain LXXLL motifs, and direct contact of a single LXXLL motif with a hydrophobic depression on the LBD surface provides the coactivator-receptor interface. The interaction is agonist-specific because, at least in the case of ER, the flexible helix 12 in the LBD obscures the binding site for the LXXLL motif when antagonist is bound. Thus, AR-dependent and androgen-specific dissociation of GRIP-1 from subnuclear foci could rely on the same AR-GRIP-1 interface used to coordinate the assembly of these proteins at promoters and enhancers of target genes. Our finding that mutation of K720 in the LBD abrogates AR-dependent GRIP-1 dissociation from foci is consistent with such a scenario. It is also possible that AR-dependent dissociation of GRIP-1 from foci relies on contact between the N-terminal activation function-1 domain of AR and the activation domain 2 of GRIP-1 because the interaction between these domains is also ligand dependent (59). In this scenario, the dissociation of GRIP1 from subnuclear foci and the recruitment of GRIP-1 to promoters would depend on distinct coactivator-nuclear receptor interfaces, and the K720A mutation indirectly affects GRIP-1 recruitment by altering activation function-1 structure. It should be noted that the K720A mutation reduces, but does not abolish, GRIP-1-stimulated AR transcriptional activity (60). Because the AR K720A mutant is strongly defective for GRIP-1 dissociation, we infer that the transcriptional activity of AR K720A measured in transient transfection assays (60) occurs in cells where GRIP-1 is localized throughout the nucleoplasm. Under this condition, the AR LBD-dependent GRIP-1 dissociation reaction could be dispensable for transcription because GRIP-1 is available in the nucleoplasm.

We propose that AR undergoes several post-import, intranuclear reactions that precede transcription (Fig. 9Go). These include agonist-dependent interactions with subnuclear foci that facilitate the recruitment of GRIP-1 and probably CBP, and modification by a nuclear kinase at Ser81. The identity of the kinase and the functional significance of this phosphorylation remain to be established, but there are clues regarding when phosphorylation occurs on this pathway. First, because the kinetics of 32P-labeling at Ser81 in vivo are significantly slower than the kinetics of nuclear import (47), we infer that phosphorylation at Ser81 occurs post import. Second, phosphorylation is likely to occur after AR interacts with subnuclear foci because the AR mutants that are arrested in subnuclear foci are hypophosphorylated at Ser81. Third, phosphorylation at Ser81 lags behind AR-induced dissociation of GRIP-1 from subnuclear foci, which is observed within 20 min of androgen addition. Although hypophosphorylation of Ser81 provides a molecular marker for AR localization in subnuclear foci, Ser81 phosphorylation is not a prerequisite for AR movement to or from foci because mutation (S81A) does not cause androgen-dependent arrest at subnuclear foci. It is also possible that a foci-associated phosphatase is responsible for the hypophosphorylated state of Ser81 in AR.

GRIP-1 and the other p160 family members are thought to provide a platform function by bridging the interactions between nuclear receptors and coregulators such as CBP/p300 (3). We suggest that the subnuclear foci represent a recruitment site where androgen-bound AR engages GRIP-1 and CBP. After this engagement, these proteins could relocate to the promoters and enhancers of target genes where GRIP-1 and CBP facilitate the recruitment of the additional components necessary for transcription. After transcription, the coactivators are recycled and AR is exported from the nucleus.

The importance of spatial organization for the coactivator function of GRIP-1 appears to extend beyond the nuclear receptor superfamily. GRIP-1 functions as a coactivator for the muscle differentiation mediated by the enhancer binding factor MEF2-C (61). The distribution of GRIP-1 and MEF2-C changes from diffuse nuclear to subnuclear foci during the differentiation program. The localization of GRIP-1 and MEF2-C in subnuclear foci, the physical interaction between these proteins, and the differentiation program are antagonized by ectopic expression of cyclin D and cyclin-dependent kinase 4 (61). The apparent correlation between the localization and biological activity of GRIP-1 and MEF2-C lends support for the idea that the subnuclear foci could be a nuclear structure that facilitates protein-protein interactions important for transcription.

Further analysis of the composition and properties of subnuclear foci should help clarify the role of these structures in transcription, including whether AIS-induced accumulation of shared coactivators in foci impacts on the expression of genes not directly regulated by AR.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Transfection and Fixed Cell Microscopy
Cos7 cells were transfected using Fugene6 and HeLa cells were transfected using Extremegene, both according to the manufacturer’s instructions (Roche, Indianapolis, IN). Twenty-four hours post transfection, cells were treated with R1881 or Cas (1000x stocks) or ethanol (Veh) as indicated in the figure legends, fixed in 3.7% formaldehyde, and permeabilized with 0.2% Triton X-100. GFP and GFP-variant proteins were detected by direct fluorescence, and Flag-tagged AR was detected with the M2 anti-Flag monoclonal antibody, or with the AR441 anti-AR antibody. Primary antibodies were detected using Cy3-coupled antimouse goat antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) and fluorescein isothiocyanate-coupled antimouse goat antibody (Pierce Chemical Co., Inc., Rockford, IL). Heterokaryon analysis of Cos7–3T3 fusions was performed as described (9). Fluorescence microscopy was performed on either a Nikon (Melville, NY) Microphot-SA or Nikon E800 microscope, both equipped with a charge-coupled device camera, and images captured and assembled using Improvision Openlab (Lexington, MA) (version 3.0.8), Adobe (San Jose, CA) Photoshop (version 6.0), and Macromedia (San Francisco, CA) Freehand (version 9.0) software. Three-dimensional renderings were generated using Openlab software after compiling z-sections. Three-dimensional rotations of the cells shown in Fig. 2AGo (videos 1–4) are provided as Apple QuickTime (Cupertino, CA) movies.

Live Cell Microscopy
AR and androgen-dependent dissociation of GRIP-1 from subnuclear foci was recorded in live Cos7 cells by confocal imaging on a Zeiss LSM 510 microscope. The Cos7 cells were plated into Bioptech Delta T dishes (Fisher Scientific, Pittsburgh, PA) and transfected with GFP-GRIP-1 plasmid using Fugene6. Before imaging, the media were changed to phenol red-free DMEM containing 10% newborn calf serum, and supplemented with 15 mM HEPES (pH 7.4). During the experiment the cells were maintained at 37 C on a Bioptech heated stage (Delta T system). GFP fluorescence was measured at 10-min intervals using the argon laser for excitation (488 nm, 20% power), a Plan-Apochromat x100/1.4 numerical aperture oil objective, a 505-nm long-pass filter, a 215-nm pinhole, and a scan time of 0.9 sec. For FLIP analysis, Cos7 cells expressing GFP-AR were cultured and transfected as above for GFP-GRIP-1. An ROI (120 x 20 pixels) within the nucleus of each cell was photobleached with the argon laser (458, 477, and 488 nm, 100% power each) for five iterations, and images were taken every 5 sec over the 180 sec time course. The fluorescence intensity at each 5-sec time point was measured in a separate ROI (30 x 30 pixels), which was normalized to the prebleach value and plotted as a function of time.

Quantitation of GFP-GRIP-1 Expression
Images of GFP-GRIP-1 expressing cells were captured in OpenLab and TIFF images exported to ImageQuant. The summed intensities and areas of nuclei of foci-positive and foci-negative cells were recorded and fluorescence per pixel area was calculated, and the averages plotted using Microsoft Excel (Redmond, WA).

Two-Dimensional Phospho-Peptide Mapping
Two-dimensional phospho-peptide mapping was performed as described (47). In brief, Cos-1 cells were transfected with WT and mutant AR plasmids as indicated in the legend. Twenty-four to 36-h post transfection, the cells were metabolically labeled in media containing 3 mCi of carrier-free 32Pi per ml for 6 h in the absence or presence of R1881 (5 nM). The cells were washed and lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. The metabolically labeled AR was purified by immunoprecipitation (PG-21; Upstate Biotechnology, Lake Placid, NY), digested with trypsin N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin (Worthington Biochemical Corp., Lakewood, NJ) and endoproteinase Glu-C (Calbiochem, San Diego, CA). The resulting phospho-peptides were resolved by two-dimensional thin-layer electrophoresis and ascending chromatography. Equivalent counts (10,000 cpm) were loaded on each thin-layer chromatography plate.

Phospho-Specific Antibody to AR
The anti-phospho-Ser81 antibody was raised against a peptide spanning the Ser81 phosphorylation site in human AR by standard methods. The peptide was synthesized with phospho-serine and an N-terminal cysteine, it was coupled to keyhole limpet hemocyanin, and it was used for antibody production in rabbits (Upstate Biotechnology, Lake Placid, NY). Antibody titers were monitored by immunblotting, and the terminal bleeds were affinity purified using Sepharose-immobilized peptide. The specificity of the antibody was verified by immunoblot analysis against WT and S81A mutant AR. The increase in immunoreactivity with the phospho-Ser81 antibody during a timecourse of R1881 treatment is parallel to the increase in phospho-peptide labeling observed on two-dimensional peptide maps (Gioeli, D., and M. J. Weber, unpublished observations).


   ACKNOWLEDGMENTS
 
We thank Vicki Gordon for expert technical assistance, R. Day, D. Wotton, and D. Edwards for reagents, and members of the Paschal laboratory for helpful discussions.


   FOOTNOTES
 
These studies were supported by the NIH and by the Paul Mellon Prostate Cancer Research Institute at the University of Virginia.

Present address for B.E.B.: Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California 92093.

B.E.B. and M.J.V. are co-first authors.

For convenience, we refer to the AIS phenylalanine deletion in the DBD as {Delta}F582. Because the codons for amino acids 582 and 583 in human AR are identical, the codon deleted in the patient with complete AIS cannot be determined (45 ).

Abbreviations: AIS, Androgen insensitivity syndrome; AR, androgen receptor; Cas, casodex; CBP, CREB binding protein; DBD, DNA-binding domain; FLIP, fluorescence loss in photobleaching; GFP, green fluorescent protein; GR, glucocorticoid receptor; GRIP, GR-interacting protein; IF, immunofluorescence; LBD, ligand-binding domain; NLS, nuclear localization signal; NR, nuclear receptor; PML, promyelocytic leukemia; ROI, region of interest; SRC, steroid receptor coactivator; Veh, vehicle; WT, wild-type.

Received for publication April 17, 2003. Accepted for publication November 26, 2003.


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NURSA Molecule Pages Link:

Nuclear Receptors:   COUP-TFII  |  GR  |  AR
Coregulators:   CBP  |  GRIP1  |  SMRT
Ligands:   Dexamethasone  |  R1881



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