Androgen Receptor Nuclear Translocation Is Facilitated by the f-Actin Cross-Linking Protein Filamin

Daniel M. Ozanne, Mark E. Brady, Susan Cook, Luke Gaughan, David E. Neal and Craig N. Robson

Prostate Research Group School of Surgical and Reproductive Sciences Medical School, University of Newcastle upon Tyne Newcastle upon Tyne, England NE2 4HH


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human androgen receptor (hAR) is a ligand-dependent transcription factor responsible for the development of the male phenotype. The mechanism whereby nuclear translocation of the hAR is induced by its natural ligand 5{alpha}-dihydrotestosterone is a phenomenon not fully understood. The two-hybrid interaction trap assay has been used to isolate proteins that interact with the hAR in an attempt to identify molecules involved in hAR transactivation and movement. We have identified the actin-binding protein filamin, a 280-kDa component of the cytoskeleton, as an hAR interacting protein. This interaction is ligand independent but is enhanced in its presence. The functional significance of this interaction was analyzed using a cell line deficient in filamin via transient expression of a green fluorescent protein-hAR chimera. In filamin-deficient cells this revealed that hAR remained cytoplasmic even after prolonged exposure to synthetic ligand. Nuclear shuttling was restored when this cell line regained wild-type expression of filamin. These data suggest a novel role for filamin, implicating it as an important molecule in AR movement from the cytoplasm to the nucleus.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid hormone receptors (SHRs) exact their effects after activation by their cognate ligands and binding to specific responsive elements on their target genes within the nucleus (reviewed in Refs. 1, 2). The cellular localization of different SHRs is varied and dependent upon the specific receptor. Both the unliganded progesterone receptor and estrogen receptor are nuclear in localization, whereas unliganded glucocorticoid receptor (GR) exists within the cytoplasm and is translocated to the nucleus upon activation (3, 4, 5). The mineralocorticoid receptor is found both in the cytoplasm and the nucleus in the inactivated form (6). The human androgen receptor (hAR) presents a more complicated cellular distribution. Immunohistochemical studies observing the endogenous hAR, and overexpression of hAR by transient transfection, provide conflicting data on receptor distribution in specific cell types (7).

The receptor becomes activated on binding of its cognate ligand, 5{alpha}-dihydrotestosterone (DHT), generated from testosterone by membrane bound 5{alpha}-reductase. The hAR is a cis-acting transcription factor essential for the growth and differentiation of cells within the prostate and male external genitalia. It is also a pivotal molecule in the development and progression of prostatic carcinoma.

Little is known about the mechanism of cytoplasmic translocation or associated molecules that coordinate movement of the activated hAR to the nucleus. Work with the hAR fused to a green fluorescent protein (GFP) reporter has allowed the study of the movement of the hAR in vivo. In Cos-7 cells, unliganded hAR is cytoplasmic and is fully translocated to the nucleus within 30 min of steroid addition (8). Although this study highlighted specific hAR kinetics, it did not reveal a molecular mechanism of receptor movement. Specific hAR kinetics has been linked to a bipartite nuclear targeting signal between the DNA binding domain and the hinge region of the hAR. A region of the hAR consisting of two clusters of basic amino acids, separated by 10 residues, is necessary for full receptor nuclear import, and is modulated by elements within the NH2 and carboxyl-terminal regions (9).

The role of the cytoskeleton in the trafficking of steroid hormone receptors has presented conflicting data. Studies involving a progesterone receptor mutant with an inactive karyophilic signal revealed that chemical disruption of the microtubule and actin network neither prevented nor delayed the hormone-dependent transfer of the PR mutant to the nucleus (10). Conversely, receptor trafficking studies involving the GR fused to a GFP highlighted the cytoskeletal network as an important structure in GR movement (11). Chemical disruption of the cytoskeleton by colcemid blocks the okadaic acid-dependent inhibition of hormone-dependent GR recycling to the nucleus. This implies that elements within the cytoskeleton are required for GR movement and that when the cytoskeleton is disrupted the normal shuttling of GR via cytoskeletal tracts utilizing cytoskeletal associating motor proteins is abrogated. A key molecule in this process is the cellular chaperone heat shock protein 90 (Hsp90) that forms a stable heterocomplex with the GR and has been shown to translocate to the nucleus with the receptor (12). The role of Hsp90 in this interaction may be to facilitate translocation by tethering the GR heterocomplex with the cytoplasmic movement machinery. Addition of okadaic acid may prevent the heterocomplex binding to these elements, allowing it to move to the nucleus via diffusion.

GFP-visualized movement of SHRs has also been performed previously for other members of the SHR superfamily (13, 14, 15, 16, 17). These dynamic studies have detailed the subcellular distribution of SHRs both before and after activation by ligand.

We have performed a yeast two-hybrid interaction trap assay (18) to isolate proteins that interact specifically with the hAR. The aim of this study was to identify novel hAR interacting proteins involved in either movement or signaling from the cytoplasm to the nucleus. A cDNA clone encoding a central portion of the cytoskeletal actin-binding protein filamin (ABP 280) was isolated as an hAR interacting protein. Using a GFP-hAR chimera, we have shown that hAR nuclear translocation is abolished in a cell line deficient in the cytoskeletal protein filamin, and hence that filamin appears to have a significant role to play in nuclear translocation of the hAR.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Actin-Binding Protein Filamin Is an hAR Interactor
The actin-binding protein filamin (ABP 280) was identified as interacting with the human hAR in a two-hybrid interaction trap assay using an hAR fragment comprising DNA-binding and ligand-binding domains as bait. The clone isolated in the screen spanned an area of 334 amino acids from amino acids 1,788 to 2,121, designated Fil1788-2121. This interaction is not dependent on ligand being present but is enhanced in its presence (Fig. 1Go). A separate two-hybrid assay (data not shown) also identified {gamma}-filamin (19) as an hAR-interacting protein. Both interacting clones span an area of high homology between the two isoforms overlapping highly conserved IgG-like repetitive regions. Androgen receptor constructs comprising either the DNA-binding domain or the ligand-binding domain produced only basal levels of ß-galactosidase activity. This suggests that the region of the AR involved with filamin interaction possibly requires the three-dimensional (3-D) structure created via the juxtaposition of the two adjacent domains.



View larger version (24K):
[in this window]
[in a new window]
 
Figure 1. Relative ß-Galactosidase Activities of Fil1788-2121 Cotransformed with hAR Deletions as Indicated, in a Yeast Two-Hybrid LacZ Assay

The hAR deletion construct comprising residues 559–918 presented the highest activity in the presence of DHT. A reduced interaction was observed with this construct in the absence of ligand. Other hAR constructs used in the assay did not present a significant interaction above that observed with empty vectors only. Bar chart shows the average activity observed from two independent experiments performed in triplicate.

 
Filamin and hAR Interact in Vitro
The interaction between filamin and hAR was analyzed using in vitro transcription and translation of the proteins labeled with 35S-methionine. Figure 2AGo represents in vitro transcribed/translated protein products before immunoprecipitation. A minimal domain of interaction was sought using hAR truncations corresponding to functional domains of the hAR. Figure 2BGo demonstrates that the filamin clone Fil1788-2121 interacts with the full-length hAR (amino acids 1–918), the DNA-binding domain and the steroid- binding domain (amino acids 559–918), and the steroid-binding domain alone (amino acids 624–918). No interaction was observed between Fil1788-2121 and the transactivation domain and the DNA-binding domain (amino acids 1–624). To further minimize this domain of interaction, hAR truncations encompassing the steroid-binding domain including the hinge region of the hAR were assessed for their ability to coprecipitate. Figure 2CGo shows that all the constructs containing the hinge region of the hAR coprecipitate with Fil1788-2121 including the hinge region of the hAR alone (amino acids 616–674).



View larger version (62K):
[in this window]
[in a new window]
 
Figure 2. In Vitro Interaction of Fil1788-2121 and hAR

A, Fil1788-2121, full-length hAR (AR1-918), and three hAR deletion constructs (hAR559-918, hAR1-624, hAR624-918) were transcribed and translated in vitro with 35S-methionine. Radiolabeled protein products were mixed and immunoprecipitated in the presence of DHT using an antibody directed against a penta-His motif on the filamin clone. B, Fil1788-2121 was observed to interact with the full-length hAR (hAR1-918) and constructs encoding the DNA-binding and steroid-binding domains (hAR559-918) and the steroid-binding domain alone (hAR624-918). No interaction was observed between Fil1788-2121 and the construct encoding the transactivation and DNA-binding domains of the hAR (hAR1-624). C, hAR constructs encoding N- and C-terminal deletions of hAR559-918 were used to further define the area of interaction with Fil1788-2121. hAR fragments containing the hinge region of the hAR (residues 616–674) showed an interaction with Fil1788-2121. Large arrows correspond to labeled Fil1788-2121.

 
The results obtained in two-hybrid interaction assay for the two proteins indicated that both the DNA-binding and the steroid-binding domains of the hAR were necessary for the filamin interaction. However, these constructs are fused to either the GAL4 activation or GAL4-binding domains, which may interfere with the correct folding of native proteins and thus alter potential sites of interaction. When the proteins are expressed in vitro, the interacting domains are more closely related, in terms of folding, to the in vivo state.

Filamin and hAR Interact in Vivo
Immunoprecipitation and subsequent Western analysis using hAR and filamin antibodies (Fig. 3Go) confirmed this interaction. A mouse monoclonal antibody was used to immunoprecipitate filamin protein from prostate LNCaP cells before detection of hAR by Western analysis. A 98-kDa band, corresponding to the hAR, was observed (lane 1), which was absent in control lanes omitting extract or antibody (lanes 2 and 3, respectively). The normal distribution of filamin has been well documented using immunohistochemistry, occurring predominantly along stress fibers and in the cell periphery (20). It is therefore likely that filamin-AR interaction occurs primarily within the cytoplasm.



View larger version (37K):
[in this window]
[in a new window]
 
Figure 3. Filamin and hAR Interact in Vivo

Protein extract from LNCaP cells was used to determine whether the hAR and filamin coprecipitate in vivo. Antifilamin antibody was used to immunoprecipitate filamin and associating proteins. Resulting protein complexes were resolved on a polyacrylamide gel and Western analysis was performed with an anti-hAR antibody as depicted above. Lane 1 demonstrates that the hAR associates with filamin in LNCaP cells. No coimmunoprecipitating hAR protein was observed in lane 2 (antibody in the absence of cell extract) or lane 3 (cell extract in the absence of antibody).

 
The Minimal Region of Interaction Spans a 150-Amino Acid Fragment of Filamin
The minimal domain of filamin required to interact with hAR was investigated by deleting residues from both the N- and C terminus of the Fil1788-2121 cDNA clone (Fig. 4AGo). Deletion constructs in pACT2 were tested for their ability to interact with hAR in yeast. The ß-galactosidase activity of these clones was assessed in comparison to the original filamin clone in pACT2. The relative ß-galactosidase activities of these clones were quantified in a liquid LacZ assay (Fig. 4BGo). The minimal domain of interaction was shown to lie within a 150-amino acid fragment encompassing repeats 18 and 19 of the filamin IgG-like repeats. This area of interaction is similar to that identified for the glycoprotein Ib{alpha}, which was found to interact between repeats 17 and 20. The majority of the other proteins identified as interactors of filamin appear to be focused around this C-terminal portion of the protein, indicating that elements within these IgG-like repeats are responsible for the specificity of interaction with these varied cellular molecules.



View larger version (23K):
[in this window]
[in a new window]
 
Figure 4. Minimal Domain Mapping of the Interaction of hAR on Filamin

Filamin deletion constructs were cotransformed into yeast strain PJ69–4A with hAR559-918 in pAS2–1. Resultant leu+, trp+ colonies were assayed for their ß-galactosidase activity. The relative ß-galactosidase activity compared with hAR559-918 was noted, as were the cotransformants’ ability to grow on his-, leu-, trp- media in the presence of 5 mM 3-AT, "+" denoting a comparable activity. A 400-bp filamin construct was observed to have ß-galactosidase activity comparable to that observed for the Fil1788-2121 construct. The graph represents the relative activities of the Filamin deletions compared with the original bait construct, Fil1788-2121. Data represents the average of three independent experiments performed in triplicate. Error bars represent the SD of three experiments.

 
Mutant Filamin Inhibits hAR-Dependent Transactivation
A transient transfection reporter gene assay was performed to investigate the influence of Fil 1788-2121 on the transactivation activity of hAR (Fig. 5AGo). COS-7 cells were transfected with plasmids encoding wild-type hAR (50 ng) and Fil1788-2121 (0–800 ng), together with a luciferase reporter plasmid containing an enhancer element comprising three androgen responsive elements (AREs), p (ARE)3-Luc. As shown in Fig. 5Go, hAR is activated by the synthetic androgen ligand, mibolerone. Increasing amounts of Fil1788-2121, in the presence or absence of androgen, cause a dramatic reduction in the level of hAR transactivation. Parallel experiments, using wild-type filamin in place of Fil1788-2121, only caused a small reduction in the mibolerone-induced AR activation, observed at the highest doses of filamin (data not shown). A negative control promoter (1,659 bp) derived from the matrix metalloproteinase 2 (MMP2) promoter, which lacks defined SHR-responsive DNA elements and is known to be unresponsive to androgens (our unpublished data) was used to demonstrate that Fil1788-2121 had no effect on the inhibition of transactivation via a nonspecific promoter element (Fig. 5BGo). These results suggest that the truncated form of filamin, Fil1788-2121, is acting as a dominant negative protein by binding hAR and preventing activation of target genes.



View larger version (26K):
[in this window]
[in a new window]
 
Figure 5. Fil1788-2121 Inhibits hAR-Dependent Transactivation of a Target Gene but Has No Effect on a Non-AR-Responsive Promoter Element

COS-7 cells were transfected with wild-type hAR and increasing amounts of pCMV-Fil1788-2121 together with p(ARE)3-Luc or p(MMP2 RE)3-Luc. In the absence of Fil1788-2121, the hAR is activated by the presence of 10 nM mibolerone. The addition of increasing amounts of Fil1788-2121 results in a rapid decrease in the transactivational activity of the hAR (A). A non-AR-responsive MMP2 promoter is unaffected by increasing addition of Fil1788-2121 (B).. Results shown are the average of three independent experiments performed in triplicate. Error bars represent the SD of three experiments.

 
The ability of filamin to inhibit the activation potential of a target gene has been reported previously (21). Tumor necrosis factor receptor-associated factor (TRAF) 2 and tumor necrosis factor-{alpha} activation of nuclear factor-{kappa}B and TRAF2 activation of the JNK/SAPK (c-jun N-terminal kinase/stress-activated protein kinase) pathway is inhibited by the addition of increasing amounts of filamin added in a similar reporter gene assay. Interestingly, the filamin clone used encompassed amino acids 1644–2118 of the full cDNA sequence, a fragment overlapping the one identified in our study as an hAR interactor. It would be tempting to speculate that the mechanism of filamin-induced inhibition of target gene expression is similar in both systems.

A GFP-hAR Chimera Remains Cytoplasmic in a Filamin-Negative Cell Line
To assess the functional significance of the filamin-hAR interaction, the movement of the hAR in response to ligand was analyzed in a cell line deficient in filamin. Previous reports regarding the GR highlighted the importance of the cytoskeleton in the recycling of the GR to the nucleus. A GFP-hAR fusion was used to visualize hAR localization. An hAR construct was generated which lacked the N-terminal transactivation domain (GFP-hAR559-918), previously shown to be constituitively nuclear in the absence of ligand (22), to act as a positive control. A recent investigation with an AR-GFP chimera reported that unliganded AR was cytoplasmic but rapidly translocated to the nucleus upon addition of ligand (8). In the filamin-deficient parental cell line (M2FIL-) we observed the hAR to be cytoplasmic in location and remained cytoplasmic even after prolonged exposure to synthetic ligand (Fig. 6Go, A and B). However, in a stably transfected derivative of this cell line expressing filamin (A7FIL+), the normal reported movement of the hAR in response to ligand was observed. Unliganded cytoplasmic hAR quickly translocated to a nuclear site upon addition of ligand (C and D). Interestingly, the GFP-hAR559-918 protein remained in the cytoplasm (E) in the presence or absence of ligand in M2FIL- cells. In contrast unliganded GFP-hAR559-918 was constituitively nuclear in A7FIL+ cells (F), as previously reported (22). We also observed a shift in morphology in the A7FIL+ cells, where the cells moved along their axes to become more spherical in response to ligand. This effect has been reported previously for the GR (23). In accordance with this study, there appears to be an underlying nuclear organization of GFP-hAR accumulation. A known nuclear localizing protein, Tip60 (tat interacting protein 60) (24) was fused to a GFP to analyze the movement of a non-steroid hormone receptor construct in the M2 and A7 cell lines. No aberrant localization of this construct was observed in the absence of filamin in the absence or presence of mibolerone (G and H). Taken together, these results suggest that the cytoskeletal protein filamin is important in the nuclear translocation of the activated hAR.



View larger version (19K):
[in this window]
[in a new window]
 
Figure 6. hAR Shuttling in a Cell Line Deficient in Filamin Expression

Confocal micrographs represent GFP-hAR expressing cells. To analyze the functional significance of the hAR-filamin interaction, hAR nuclear translocation was studied in a cell line deficient in filamin expression, denoted (M2FIL-), and the corresponding cell line with filamin was stably transfected at near wild-type levels (A7FIL+). In the absence of steroid, the cellular localization of GFP-hAR1-918 in M2FIL- and A7FIL+ cell lines is predominantly cytoplasmic with background nuclear fluorescence (A and C, respectively). After 20 min exposure to mibolerone, the cellular localization of the GFP-hAR1-918 construct remains predominately cytoplasmic in M2FIL- cells (B). Conversely, in filamin-containing cells (A7fil+) the cellular location of GFP-hAR1-918 is predominately nuclear (D). The GFP-hAR559-918 construct, known to be constituitively nuclear even in the absence of ligand, was predominately cytoplasmic in the M2FIL- cell line (E). The nuclear translocation of this construct was again restored in the filamin containing A7FIL+ cell line in the presence of ligand (F). A control trafficking protein, Tip60, known to be constituitively nuclear in localization, was unaffected by the absence of filamin (M2 Fil-) in the absence (G) or presence (H) of ligand. Scale bar represents 25 µm. Similar results were observed in two further experiments.

 
Filamin Deficiency Affects Ligand-Induced hAR Transcriptional Activity
The transcriptional activity of GFP-hAR was investigated in filamin-deficient (M2FIL-) and proficient (A7FIL+) cell lines (Fig. 7Go). Transient transfection of GFP-hAR and p(ARE)3-Luc into M2FIL- cells failed to induce a response to the synthetic ligand, Mibolerone. In contrast, a similar transfection into A7FIL+ cells resulted in a 4.5-fold increase in luciferase activity in response to ligand. These results demonstrate that GFP-hAR is functional and that the inability of GFP-hAR to translocate to the nucleus in filamin-deficient cells results in lower transcriptional activity.



View larger version (11K):
[in this window]
[in a new window]
 
Figure 7. Filamin-Deficient Cells Exhibit Reduced hAR Transcriptional Activity

Transient transfection of the GFP-hAR and p(ARE)3-luc into filamin-deficient M2 cells failed to induce a response to synthetic ligand, mibolerone. Conversely, similar transfections into filamin-positive A7 cells exhibited a 4.5-fold increase in luciferase activity in response to ligand. Graph represents data from three independent experiments performed in triplicate. Error bars represent the SD of three experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study, we have identified the actin-binding protein filamin as interacting with the hAR. Filamin, a 280-kDa protein, was originally identified as a structural component of the cell cytoskeleton governing the solation-gellation status of the cytoplasm at the cell membrane (25). This is facilitated by filamin’s ability to cross-link F-actin fibers into orthogonal arrays, thereby defining the cells 3-D structure and motility. Filamin consists of three distinct domains: 1) an N-terminal actin-binding domain homologous to those found in other actin-binding proteins such as dystrophin, vinculin, and talin, 2) a central rod-like domain consisting of 24 repeat regions that forms the ß-sheet backbone of the protein with domains highly conserved in filamin isoforms, 3) a C-terminal domain containing the 24th repeat motif that defines a self-association domain forming the interface of the filamin dimer, creating a leaf spring-like molecule (26).

Filamin consists of three main isoforms, assigned {alpha}, ß, and {gamma}. All three share a high degree of sequence homology due to their role as F-actin cross-linking proteins within the cell. ABP 280 or {alpha}-filamin is the original isoform and is located telomeric to the color vision locus (R/GCP) and centromeric to G6PD in Xq28 (27).

Filamin has previously been identified as interacting with the ß1-integrins, transmembrane molecules involved in cell-cell contact and transmembrane signaling (28). The integrins are also critical in the formation of focal adhesions, local areas on the cell membrane implicated in signal transduction events from the extracellular matrix to the cell nucleus. The structure of focal adhesions also links the integrins with F-actin and actin-associating proteins such as talin, vinculin, and filamin.

Another interacting partner of filamin is the stress-activated protein kinase SEK-1, responsible for signal transduction to downstream molecules JNK and c-Jun affecting cellular responses such as growth and differentiation (29). It is unclear as to the role of filamin in this signal transduction pathway, whether filamin itself is phosphorylated or is acting as an anchoring protein similar in function to JIP-1 (JNK inhibiting protein), which also allows cross-talk between the mitogen-activated protein kinase (MAPK) and SAPK pathways (30), remains to be determined.

More recent studies have identified caveolin-1, a cholesterol-binding integral membrane protein, as an interacting partner for filamin (20). The association between androgen-independent prostate cancer and high expression of caveolin-1 has been documented (31, 32). The identification of filamin as an hAR interactor highlights a potential link between caveolin-1, filamin, and the hAR in androgen-independent prostate cancer.

The melanoma cell line deficient in filamin expression has been well characterized elsewhere (33, 34). The majority of work on this cell line analyzed the cell plasma membrane activity relating to filamin expression. Cells deficient in filamin expression display an increased occurrence of membrane blebbing due to a decrease in the local actin polymerization rate. The retraction of these cell surface protrusions is dependent upon the establishment of a stable actin network, and the prolonged blebbing displayed by the filamin-deficient cell line is due to the local rate of actin polymerization being outpaced by the fluid-driven expansion of the cell membrane. This prolonged blebbing is abolished when filamin is reexpressed to wild-type levels and increases the rate of actin polymerization leading to increased protrusive activity, thus increasing the motility of the cell. The importance of filamin-dependent motility is highlighted in an X-linked male lethal condition known as periventricular heterotopia (PH). This condition arises due to a mutation in the filamin gene resulting in aberrant neural cell migration within the neural network (35; reviewed in Ref. 36).

This study implicates filamin in the cytoplasmic trafficking of the hAR. There has been evidence to suggest that SHRs interact with components of the cytoskeletal architecture, in particular the studies carried out on the GR (11) that demonstrated an intact cytoskeletal network is important in the shuttling function of the receptor. Disruption of the cyto-architecture rendered the GR unable to shuttle between the cytoplasm and the nucleus. This implies that there is a regulatory component within the cytoskeleton essential for the trafficking of molecules to the nucleus. Earlier work (37) presented the first evidence that SHRs associate with the actin cytoskeleton. This study demonstrated that the inactive 8S GR contacts the actin filaments via its binding to Hsp90. Hsp90, similarly to filamin, also has the ability to cross-link actin filaments (38). Upon hormone binding, the Hsp90-receptor complex dissociates and the receptor transforms into the activated complex able to bind glucocorticoid-responsive elements on target genes within the nucleus. The conclusions from this study were that the GR (and possibly other SHRs) bind the actin fibers via their Hsp90 moieties, and this binding anchors the inactive receptor and prevents translocation to the nucleus. Later work, again studying GR translocation, implicated the microtubule network as important in the mechanism of action of GR hormones (39).

The function of filamin, in the context of hAR cytoplasmic trafficking, may involve the disruption of the high-affinity association between Hsp90 and the receptor. In the absence of filamin, the receptor-Hsp90 complex may remain anchored in an inactive state to the actin filaments even in the presence of steroid and an available nuclear localization sequence on the receptor. It is tempting to speculate that filamin may be acting as a mediator between the receptor and the molecular chaperone Hsp90 controlling the release of activated receptor after ligand binding. Interaction between heat shock proteins and filamin has been detailed (40). The small stress protein cvHsp has been reported to interact with the C-terminal tail of {alpha}-filamin in a yeast two-hybrid assay. It is plausible that filamin may interact with Hsp90 in a similar fashion in complex with the hAR.

This study has shown that the filamin-hAR interaction is not steroid dependent, suggesting receptor anchoring in the absence of ligand. In the presence of ligand the affinity of the interaction is increased and, after Hsp90 dissociation, filamin may be acting as a molecular chaperone, maintaining the active hAR in a stable conformation and fulfilling its other role as a signaling molecule interacting with components of the SAPK and MAPK pathways.

This study also has clinical implications. A prostate cancer susceptibility locus, termed HPCX, has recently been identified in the region of Xq27–28, where the filamin locus maps (41). Linkage analysis in 153 prostate cancer families over a 30 centimorgan (cM) region of HPCX has provided additional support for the existence of a prostate cancer susceptibility locus at Xq28 in a separate study (42). Our investigations highlight an altered response of the hAR to steroid in a cell line lacking expression of filamin, which is restored in its presence. This work provides an insight into hAR movement. Further investigation is required to ascertain whether this interaction extends to other members of the SHR superfamily and the significance of this interaction in clinical specimens.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Yeast Two-Hybrid Analysis
Two-hybrid methodology, bait use, and hAR primer design were performed as described previously (43). An hAR fragment comprising residues 559–918, encompassing the DNA-binding and ligand-binding domains, was used as bait in the assay. Positive clones were isolated, sequenced, and submitted into the GenBank database. One positive clone, 1.002 kb in size, gave a 100% sequence homology to the actin binding protein filamin (ABP-280, accession no. X53416).

The 1.002-kb filamin clone (hereafter designated Fil1788-2121) in pACT2 was transformed back into the yeast strain PJ69–4A as previously described (44) with truncated forms of the hAR in pAS2–1. Transformants were grown on media lacking leucine and tryptophan, and resultant colonies were grown in selective media overnight at 30 C in the presence of 1 µM dihydrotestosterone. Samples were diluted to A600 0.2 and grown to an A600 of 0.6–0.8 and split into three 1-ml samples. Samples were subjected to liquid LacZ assays as described previously (45). Individual experiments were performed in triplicate.

In Vitro Interaction of hAR and Filamin
Fil1788-2121cDNA was excised with EcoRI and HindIII digestion from pAS2–1 and directionally cloned into pT7–7 or pRSETC (Invitrogen, San Diego, CA) via EcoRI and HindIII sites. hAR fragments used in the yeast two-hybrid analysis were excised from pAS2–1 with EcoRI and cloned into the EcoRI site of pRSETC. Constructs were sequenced to confirm the maintenance of the open reading frame.

Template DNA was subjected to Geneclean (Anachem, Luton, UK) to remove any contaminant RNase, and the coupled Transcription/Translation Kit (Promega Corp., Madison, WI) was used for in vitro interaction analysis. Templates were labeled with 35S-methionine and the reaction was performed according to the manufacturer’s guidelines. After the 90-min incubation, samples were split and mixed equally, and 1 ml of reaction lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.2 mM Na3VO4, 0.5% Nonidet P-40, 1 mM phenylmethlsulfonyl fluoride, 1 mM dithiothreitol, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 25 µg/ml pepstatin) was added. Interacting proteins were precipitated and visualized as previously described (43).

Coimmunoprecipitation
Approximately 107 cells from an LNCaP, hAR-positive cell line were washed in cold PBS, harvested, and lysed in reaction lysis buffer on ice for 30 min. Lysates were centrifuged at 14,000 x g at 4 C for 10 min. Supernatants were precleared with 25 µl protein G sepharose (PGS) after three washes in reaction lysis buffer and rotated at 4 C for 4 h. PGS and nonspecific bound protein were removed by centrifugation at 14,000 x g for 5 min. Immunoprecipitations were performed with 4 µg of mouse monoclonal antifilamin antibody (Chemicon Intl. Inc., Temecula, CA) or 4 µg of rabbit polyclonal C-terminal AR antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and incubated overnight at 4 C with rotation. After incubation, a further 25 µl of PGS were added to immunoprecipitate samples and returned to 4 C for 1 h with rotation. PGS with bound protein complexes was recovered by centrifugation at 14,000 x g for 5 min, and samples were washed once in wash buffer A (PBS, 0.2% Triton-X-100, 350 mM NaCl) and twice in wash buffer B (PBS, 0.2% Triton-X-100). Sample buffer containing 10% ß-mercaptoethanol was added to recovered fractions. Samples were resolved on 10% denaturing polyacrylamide gels for 30 min and transferred to nitrocellulose filters. The membrane was probed with polyclonal AR (1:500) or monoclonal filamin (1:1000) antibodies. Immunoreaction was visualized using enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech, Arlington Heights, IL) and developed on radiographic film.

Deletion Constructs of Filamin Clone
Three deletion constructs of the Fil1788-2121 in pACT2 were constructed via PCR. Primers used to generate deletion constructs were: Fil1788-2081, pACT2FSP (CLONTECH Laboratories, Inc., Palo Alto, CA), 2R-5'-TGA GCC CAC CAT AGC CTG C-3'; Fil1930-2081, 1F-5'-GGG ACT ACA GCA TTC TAG TC-3', 2R-5'-TGA GCC CAC CAT AGC CTG C-3'; Fil1788-1930, pACT2FSP (CLONTECH Laboratories, Inc.), 1R-5'-GAC TAG AAT GCT GTA GTC CC-3'. Amplified inserts were cloned into pCR2.1 (Invitrogen) and subcloned into pACT2 via the EcoRI site. Samples were sequenced to verify the open reading frame. Deletion constructs were cotransformed with AR559-918 construct in pAS2–1 into yeast strain PJ69–4A, and resultant transformants were assayed for ß-galactosidase activity as described previously (45).

Transient Transfection
COS-7, M2, and A7 cells were grown in steroid-depleted RPMI 1640 medium containing 10% FCS 48 h before transfection. Cells were transfected with either pCDNA3-AR, pEGFP-Tip60, pEGFP-hAR, pCMV-Fil1788-2121, or pMMP2-Luc (donated by Dr. Y. Sun, Parke-Davis, Ann Arbor, MI) and p(ARE)3-Luc (provided by Dr. D. Gioeli, University of Virginia, Charlottesville, VA), as indicated, using Superfect reagent (QIAGEN, Chatsworth, CA). A total of 1.5 µg DNA was used per 35- mm well. Transfected cells were cultured in steroid-depleted medium with or without synthetic androgen, Mibolerone, where indicated. After 48 h incubation, cells were harvested, and luciferase activities were determined using a luciferase reporter system (Promega Corp.). pCMV-ß-gal was used as an internal control for normalization of transfection efficiency.

Microscopic Analysis of GFP-hAR Chimera in M2 and A7 Cell Lines
Full-length hAR (corresponding to residues 1–918) was cloned into the XbaI site of the GFP plasmid pEGFP-C1 (CLONTECH Laboratories, Inc.) to generate GFP-hAR1-918. Residues 559–918 of the hAR comprising DNA-binding and ligand-binding domains of the hAR were cloned into the EcoRI site of pEGFP-C2 (CLONTECH Laboratories, Inc.) to produce GFP-hAR559-918. Full- length Tip60 was isolated as described previously (43) and cloned into the BamHI site of pEGFP-C2 (CLONTECH Laboratories, Inc.). Human epithelial melanoma cell lines, M2 (filamin -ve) and A7 (filamin +ve) (a kind gift from Dr. T. Stossel, Harvard University, Cambridge, MA), were used in the GFP-hAR translocation assays. Cell lines were grown overnight on 22 x 22 mm microscope cover slips in six-well cell culture plates. Cells were transfected with 2 µg of GFP-hAR1-918 or GFP-hAR559-918 construct using Superfect and cultured in RPMI 1640 medium for 24 h. Cells were then placed in steroid-depleted media for a further 24 h and exposed to 10 nM Mibolerone for 0, 5, and 20 min. Coverslips were washed with sterile PBS and fixed in 100% methanol for 30 min at -20 C, dried, inverted on microscope slides, and mounted in antifading medium (Shandon Southern Instruments, Inc., Sewickley, PA). Slides were analyzed on an MRC 600 scanning laser confocal microscope (Bio-Rad Laboratories, Inc., Richmond, CA).


    ACKNOWLEDGMENTS
 
We would like to thank Dr. Trevor Booth for his technical assistance in obtaining the confocal images, Dr. Tom Stossel for providing the M2 and A7 cell lines, and Drs. Yi Sun and Dan Gioeli for the pMMP2-Luc and p(ARE)3-Luc plasmids, respectively.


    FOOTNOTES
 
Address requests for reprints to: Dr. C. N. Robson, Prostate Research Group, School of Surgical and Reproductive Sciences, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, England. NE2 4HH. E-mail: c.n.robson{at}ncl.ac.uk

Received for publication March 31, 2000. Revision received June 12, 2000. Accepted for publication July 3, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Evans RM 1988 The steroid and thyroid receptor superfamily. Science 240:889–895[Medline]
  2. O’Malley BW 1990 The steroid receptor superfamily: more excitement predicted for the future. Mol Endocrinol 4:363–369[Medline]
  3. King WJ, Greene GL 1984 Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells. Nature 307:745–747[Medline]
  4. Perrot-Applanat M, Logeat M, Groyer-Picard MT, Milgrom E 1985 Immunocytochemical study of mammalian progesterone receptor using monoclonal antibodies. Endocrinology 116:1473–1484[Abstract]
  5. Wikstrom AC, Bakke O, Okret S, Bronnegard M, Gustafsson JA 1987 Intracellular localization of the glucocorticoid receptor: evidence for cytoplasmic and nuclear localization. Endocrinology 120:1232–1242[Abstract]
  6. Lombes M, Binart N, Delahaye F, Baulieu EE, Rafestin-Oblin ME 1994 Differential intracellular localization of human mineralocorticosteroid receptor on binding of agonists and antagonists. Biochem J 302:191–197[Medline]
  7. Jenster G, Trapman J, Brinkmann AO 1993 Nuclear import of the human androgen receptor. Biochem J 293:761–768[Medline]
  8. Georget JM, Lobaccaro B, Terouanne B, Mangeat P, Nicolas JC, Sultan C 1997 Trafficking of the androgen receptor in living cells with fused green fluorescent protein-androgen receptor. Mol Cell Endocrinol 129:17–26[CrossRef][Medline]
  9. 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-terminaland carboxyl-terminal sequences. J Biol Chem 269:13115–13123[Abstract/Free Full Text]
  10. Perrot-Applanat M, Lescop P, Milgrom E 1992 The cytoskeleton and the cellular traffic of the progesterone receptor. J Cell Biol 119:337–348[Abstract]
  11. Galigniana MD, Housley PR, DeFranco DB, Pratt WB 1999 Inhibition of glucocorticoid receptor nucleocytoplasmic shuttling by okadaic acid requires intact cytoskeleton. J Biol Chem 274:16222–16227[Abstract/Free Full Text]
  12. Kang KI, Devin J, Cadepond F, Jibard N, Guiochon-Mantel A, Baulieu EE, Catelli MG 1994 In vivo functional protein-protein interaction: nuclear targeted hsp90 shifts cytoplasmic steroid receptor mutants into the nucleus. Proc Natl Acad Sci USA 91:340–344[Abstract]
  13. Fejes-Toth G, Pearce D, Naray-Fejes-Toth A 1998 Subcellular localisation of mineralocorticoid receptors in living cells: effects of receptor agonists and antagonists. Proc Natl Acad Sci USA 95:2973–2978[Abstract/Free Full Text]
  14. Zhu XG, Hanover J, Hager GL, Cheng SY 1998 Hormone-induced translocation of thyroid hormone receptors in living cells visualized using a receptor green fluorescent protein chimera. J Biol Chem 273:27058–27063[Abstract/Free Full Text]
  15. Htun H, Holth L, Walker D, Davie JR, Hager GL 1999 Direct visualization of the human estrogen receptor alpha reveals a role for ligand in the nuclear distribution of the receptor. Mol Biol Cell 10:471–486[Abstract/Free Full Text]
  16. Michigami T, Suga A, Yamazaki M, Shimizu C, Cai G, Okada S, Ozono K 1999 Identification of amino acid sequence in the hinge region of human vitamin D receptor that transfers a cytosolic protein to the nucleus. J Biol Chem 274:33531–33538[Abstract/Free Full Text]
  17. Lim CS, Baumann CT, Htun H, Xian W, Irie M, Smith CL, Hager GL 1999 Differential localisation and activity of the A-and B-forms of the human progesterone receptor using green fluorescent protein chimeras. Mol Endocrinol 13:366–375[Abstract/Free Full Text]
  18. Fields S, Song O 1989 A novel genetic system to detect protein-protein interactions. Nature 340:245–246[CrossRef][Medline]
  19. Xu WF, Xie Z, Chung DW, Davie EW 1998 A novel human actin-binding protein homologue that binds to platelet glycoprotein Ib{alpha}. Blood 92:1268–1276[Abstract/Free Full Text]
  20. Stahlhut M, van Deurs B 2000 Identification of filamin as a novel ligand for caveolin-1: evidence for the organization of caveolin-1-associated membrane domains by the actin cytoskeleton. Mol Biol Cell 11:325–337[Abstract/Free Full Text]
  21. Leonardi A, Ellinger-Ziegelbauer H, Franzoso G, Brown K, Siebenlist U 2000 Physical, functional interaction of filamin (actin-binding protein-280), tumor necrosis factor receptor-associated factor 2. J Biol Chem 275:271–278[Abstract/Free Full Text]
  22. 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 266:510–518[Abstract/Free Full Text]
  23. Htun H, Barsony J, Renyi I, Gould DL, Hager GH 1996 Visualisation of the glucocorticoid receptor translocation and intranuclear organisation in living cells with a green fluorescent protein chimera. Proc Natl Acad Sci USA 93:4845–4850[Abstract/Free Full Text]
  24. Yamamoto T, Horikoshi M 1997 Novel substrate specificity of the histone acetyltransferase activity of HIV-1 tat interactive protein Tip60. J Biol Chem 272:30595–30598[Abstract/Free Full Text]
  25. Koteliansky VE, Shirinsky V, Gneushev GN, Smirnov 1981 Filamin, a high relative molecular mass actin-binding protein from smooth muscles, promotes actin polymerization. FEBS Lett 136:98–100[CrossRef][Medline]
  26. Gorlin JB, Yamin R, Egan S, Stewart M, Stossel TP, Kwiatkowski DJ, Hartwig JH 1990 Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring. J Cell Biol 111:1089–1105[Abstract]
  27. Gorlin JB, Henske E, Warren ST, Kunst CB, D’Urso M, Palmiera G, Hartwig JH 1993 Actin-binding protein (ABP-280) filamin gene (FLN) maps telomeric to the color vision locus (R/GCP) and centromeric to G6PD in Xq28. Genomics 17:496–498[CrossRef][Medline]
  28. Loo DT, Kanner S, Aruffo A 1998 Filamin binds to the cytoplasmic domain of the beta1-integrin Identification of amino acids responsible for this interaction. J Biol Chem 273:23304–23312[Abstract/Free Full Text]
  29. Marti A, Luo Z, Cunningham C, Ohta Y, Hartwig J, Stossel TP, Kyriakis JM, Avruch J 1997 Actin-binding protein-280 binds the stress-activated protein kinase (SAPK) activator SEK-1 and is required for tumor necrosis factor-{alpha} activation of SAPK in melanoma cells. J Biol Chem 272:2620–2628[Abstract/Free Full Text]
  30. Pedram A, Razandi M, Levin ER 1998 Extracellular signal-regulated protein kinase/jun kinase cross-talk underlies vascular endothelial cell growth factor induced endothelial cell proliferation. J Biol Chem 273:26722–26728[Abstract/Free Full Text]
  31. Yang G, Truong L, Wheeler TM, Thompson TC 1999 Caveolin-1 expression in clinically confined human prostate cancer: a novel prognostic marker. Cancer Res 59:5719–5723[Abstract/Free Full Text]
  32. Nasu Y, Timme T, Yang G, Bangma CH, Li L, Ren C, Park SH, DeLeon M, Wang J, Thompson TC 1998 Suppression of caveolin expression induces androgen sensitivity in metastatic androgen-insensitive mouse prostate cancer cells. Nat Med 4:1062–1064[CrossRef][Medline]
  33. Cunningham CC, Gorlin JB, Kwiatkowski DJ, Hartwig JH, Janmey PA, Byers HR, Stossel TP 1992 Actin-binding protein requirement for cortical stability and efficient locomotion. Science 255:325–327[Medline]
  34. Cunningham CC 1995 Actin polymerization and intracellular solvent flow in cell surface blebbing. J Cell Biol 129:1589–1599[Abstract]
  35. Fox JW, Lamperti E, Eksioglu YZ, Hong SE, Feng Y, Graham DA, Scheffer IE, Dobyns WB, Hirsch BA, Radtke RA, Berkovic SF, Huttenlocher PR, Walsh CA 1998 Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia. Neuron 21:1315–1325[Medline]
  36. Fox JW, Walsh C 1999 Periventricular heterotopia and the genetics of neuronal migration in the cerebral cortex. Am J Hum Genet 65:19–24[CrossRef][Medline]
  37. Miyata Y, Yahara I 1991 Cytoplasmic 8S glucocorticoid receptor binds to actin filaments through the 90-kDa heat shock protein moiety. J Biol Chem 266:8779–8783[Abstract/Free Full Text]
  38. Koyasu S, Nishida E, Kadowaki T, Matsuzaki F, Iida K, Harada F, Kasuga M, Sakai H, Yahara I 1986 Two mammalian heat shock proteins, HSP90 and HSP100, are actin-binding proteins. Proc Natl Acad Sci USA 83:8054–8058[Abstract]
  39. Akner G, Wikstrom A, Gustafsson JA 1995 Subcellular distribution of the glucocorticoid receptor and evidence for its association with microtubules. J Steroid Biochem Mol Biol 52:1–16[CrossRef][Medline]
  40. Krief S, Faivre J, Robert P, Le Douarin B, Brument-Larignon N, Lefrere I, Bouzyk MM, Greller LD, Tobin FL, Souchet M, Bril A 1999 Identification and characterization of cvHsp A novel human small stress protein selectively expressed in cardiovascular and insulin-sensitive tissues. J Biol Chem 274:36592–36600[Abstract/Free Full Text]
  41. Xu J, Meyers D, Freije D, Isaacs S, Wiley K, Nusskern D, Ewing C, Wilkens E, Bujnovszky P, Bova GS, Walsh P, Isaacs W, Schleutker J, Matikainen M, Tammela T, Visakorpi T, Kallioniemi OP, Berry R, Schaid D, French A, McDonnell S, Schroeder J, Blute M, Thibodeau S, Trent J, et al. 1998 Evidence for a prostate cancer susceptibility locus on the X chromosome. Nat Genet 20:175–179[CrossRef][Medline]
  42. Lange EM, Chen H, Brierley K, Perrone EE, Bock CH, Gillanders E, Ray ME, Cooney KA 1999 Linkage analysis of 153 prostate cancer families over a 30-cM region containing the putative susceptibility locus HPCX. Clin Cancer Res 5:4013–4020[Abstract/Free Full Text]
  43. Brady ME, Ozanne DM, Gaughan L, Waite I, Cook S, Neal DE, Robson, CN 1999 Tip60 is a nuclear hormone receptor co-activator. J Biol Chem 274:17599–17604[Abstract/Free Full Text]
  44. Gietz RD, St-Jean JA, Woods RA, Schiestl RH 1992 Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425[Medline]
  45. Gietz RD, Triggs-Raine B, Robbins A, Graham KC, Woods RA 1997 Identification of proteins that interact with a protein of interest: applications of the yeast two-hybrid system. Mol Cell Biochem 172:67–79[CrossRef][Medline]