Stepwise androgen receptor dimerization

Martin E. van Royen1, Wiggert A. van Cappellen2, Carola de Vos1, Adriaan B. Houtsmuller1 and Jan Trapman1,*1Department of Pathology, Josephine Nefkens Institute, Erasmus University Medical Center, PO-Box 2040, 3000 CA Rotterdam, The Netherlands2Department of Reproduction and Development, Erasmus University Medical Center, PO-Box 2040, 3000 CA Rotterdam, The Netherlands

*Author for correspondence (j.trapman@erasmusmc.nl)

Accepted 15 December 2011Journal of Cell Science 125, 1970–1979� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.096792

SummaryAndrogen-regulated gene expression is a highly coordinated dynamic process mediated by androgen receptor (AR) ligand binding andDNA binding, and by specific AR protein–protein interactions. The latter include DNA-binding domain (D-box) interactions in ARhomodimers, and the interaction of the FQNLF motif in the AR N-terminal domain and the coactivator groove in the ligand-bindingdomain (N/C interaction). We have studied these interactions in AR homodimerization using quantitative imaging techniques. We found

that the initial cytoplasmic intramolecular AR N/C interaction after ligand binding is followed by a D-box-dimerization-dependenttransition to intermolecular N/C interaction in a proportion of nuclear ARs. The consecutive steps leading to homodimerization areinitiated prior to DNA binding. Our data indicate the presence of nuclear pools of both AR homodimers and monomers. On the basis of

AR-regulated reporter assays we propose specificity in regulation of gene expression by AR homodimers and monomers mediated byAR domain interactions. Moreover, our findings elucidate important steps in the spatiotemporal organization of AR intra- and inter-molecular interactions.

Key words: Androgen receptor, Dimerization, N/C interaction, DBD, Quantitative live cell imaging, Target genes

IntroductionRegulation of gene expression is a dynamic process involving

many tightly orchestrated consecutive steps. Androgen-regulated

gene expression is mediated by the androgen receptor (AR). The

AR is a ligand-activated transcription factor and a member of the

steroid receptor (SR) subfamily of nuclear receptors (NRs). Like

all SRs, the AR has a modular structure composed of an N-terminal

domain (NTD), a conserved DNA-binding domain (DBD) and a C-

terminal ligand-binding domain (LBD) (Brinkmann et al., 1989).

Activated ARs regulate genes involved in the development and

maintenance of the male phenotype. AR is also a key factor in

prostate cancer. AR activity is not only regulated by ligand binding

and DNA binding but also by intramolecular interactions between

functional domains, by homodimerization and by interactions

with cofactors. The best-characterized interactions between AR

functional domains are the intra- and intermolecular NTD–LBD

interaction (N/C interaction) that is mediated by the FQNLF motif

in the NTD and the coactivator groove in the LBD, and

the intermolecular DBD–DBD interaction mediated by the

dimerization box (D-box) (Centenera et al., 2008). However,

the spatiotemporal relationship of the different intra- and

intermolecular AR domain interactions in androgen-regulated

gene expression is currently unknown.

Using fluorescence resonance energy transfer (FRET) and

combined fluorescence recovery after photobleaching (FRAP)

and FRET analysis, initial studies on the spatiotemporal

organization of AR protein–protein interactions have been

performed (Schaufele et al., 2005; van Royen et al., 2007).

FRET showed that in the cytoplasm the N/C interaction is in an

intramolecular conformation initiated directly after ligand-

binding and before translocation to the nucleus (Schaufele et al.,

2005; van Royen et al., 2007). In the nucleus, the intramolecular

N/C interaction is followed by an intermolecular N/C interaction

(Schaufele et al., 2005). The N/C interaction preferentially occurs

in mobile ARs and is lost when the AR is bound to DNA (van

Royen et al., 2007). These observations indicate that the AR itself

regulates the time and place of interactions with coregulators by

preventing untimely protein interactions when the AR is mobile,

and allowing coregulator binding when the AR is bound to DNA

(Dubbink et al., 2004; He et al., 2001; van Royen et al., 2007).

The intramolecular and intermolecular N/C interactions are

mediated by binding of the FxxLF peptide motif (FQNLF) in the

AR NTD to the ligand-induced cofactor binding groove in AR

LBD. The phenylalanine residues in the FxxLF motif are

essential for strong N/C interaction and bind deep into the

coactivator groove with van der Waals interactions, whereas

the leucine residue in the peptide motif lies in a shallow ridge on

the surface of the LBD, and the other two amino acid residues are

exposed to the solvent (Dubbink et al., 2004; Hur et al., 2004; van

de Wijngaart et al., 2006). In other SRs, N/C interactions are

absent or weak. In a homodimer, ARs also interact through their

D-boxes in the second zinc finger of the DBD. SR D-box

interactions are sustained by a network of hydrogen bonds

between individual amino acid residues in the D-box and by an

extensive complementary surface. In the AR DBD, a serine

residue at position 597 (S597), which is absent in other SRs,

forms a hydrogen bond and Van der Waals contacts with its

counterpart in the opposing D-box in an AR homodimer (Shaffer

et al., 2004). An additional pair of symmetrical hydrogen bonds is

formed between an alanine at 596 (A596) and a threonine at

602 (T602) in the opposing AR DBD and vice versa. These

interactions result in a relatively strong AR D-box dimerization

interface compared with those of other SRs (Shaffer et al., 2004).

The importance of the D-box in AR function is highlighted by the

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large number of mutations in this domain found in androgen

insensitivity syndrome (AIS) patients (Centenera et al., 2008)

(http://androgendb.mcgill.ca/).

To study the molecular mechanisms underlying AR

homodimerization and to investigate when and where domain

interactions take place, we used confocal microscopy and

quantitative microscopic techniques to examine cells expressing

functional, single and double YFP- and CFP-tagged wild-type ARs

and appropriate AR mutants. In addition, we investigated the role

that these molecular mechanisms have in differential target gene

expression.

ResultsInteractions of functional domains of single and double

YFP- and CFP-tagged ARs

YFP and CFP fluorescent proteins were fused to the N- and C-

terminus of a single AR (YFP–AR–CFP) or to separate ARs

(YFP–AR and AR–CFP) to study, by FRET and FRAP, AR

domain interactions and mobility in living human hepatoma

Hep3B cells (Fig. 1A). Western blot analysis showed that all

tagged ARs were of the expected size (Fig. 1B). All tagged ARs

were able to induce expression of a luciferase (Luc) reporter gene

driven by an androgen-regulated promoter, (ARE)2-TATA Luc

(supplementary material Fig. S1) (van Royen et al., 2007). In the

absence of hormone, double-tagged YFP–AR–CFP and single-

tagged YFP–AR and AR–CFP were mainly located in the

cytoplasm (Fig. 1C–E respectively, left panels). Upon hormone

addition, single- and double-tagged ARs rapidly translocated to

the nucleus (supplementary material Fig. S2). In the nucleus the

ARs were distributed in a typical punctate pattern (Fig. 1C–E

respectively, right panels). This punctate distribution pattern

correlates with a transient immobilization of the AR and partially

overlaps with sites of active transcription (Farla et al., 2005; van

Royen et al., 2007).

We then analyzed cells expressing either double-tagged or

single-tagged ARs by acceptor bleaching FRET (abFRET;

Fig. 1F). In abFRET, the relative increase of the donor

emission after acceptor photobleaching is a measure of the

interaction between the tagged molecules under surveillance

(Bastiaens and Jovin, 1996; Bastiaens et al., 1996; Karpova et al.,

2003; Kenworthy, 2001; van Royen et al., 2009a). In the presence

of the synthetic androgen, R1881, cells expressing double-tagged

AR and cells expressing a combination of single-tagged YFP–AR

and AR–CFP showed abFRET. FRET was not detected in the

nucleus or cytoplasm in the absence of hormone (supplementary

material Fig. S4). AbFRET was strongly reduced in double-

tagged N/C-interaction-deficient mutant ARs, in which the N-

terminal FQNLF motif was mutated to AQNAA (AR F23,27A/

L26A), and completely lost in single-tagged N/C-interaction-

deficient AR mutants (Fig. 1F). These results show that using

abFRET it is possible to quantitatively study inter- and

intramolecular N/C interactions in double-tagged ARs and

intermolecular N/C interaction specifically between single-

tagged ARs.

The AR N/C interaction is predominantly intermolecular

Confocal time-lapse microscopy showed that FRET, as measured

by the YFP/CFP ratio in double-tagged AR (YFP–AR–CFP)-

expressing cells increased rapidly after ligand addition, prior to

translocation to the nucleus (supplementary material Fig. S2A)

(Schaufele et al., 2005). By contrast, in cells expressing two

different single-tagged ARs (YFP–AR and AR–CFP) the YFP/

Fig. 1. Properties of double- and single-tagged

ARs. (A) Schematic representation of YFP–AR–

CFP, YFP–AR and AR–CFP [horizontal bars

represent a (Gly-Ala)6 spacer]. (B) Western blot of

the fusion proteins expressed in Hep3B cells. Lane 1:

YFP–AR–CFP; lane 2: YFP–AR (F23,27A/L26A)–

CFP; lane 3: YFP–AR; lane 4: YFP–AR (F23,27A/

L26A); lane 5: AR–CFP; lane 6: AR (F23,27A/

L26A)–CFP; lane 7: non-transfected cells. AR was

detected using mouse AR monoclonal antibody

(F39.4.1). (C–E) Confocal images of Hep3B cells

expressing double- and single-tagged AR (YFP–AR–

CFP, YFP–AR and AR–CFP) in the absence (left

panels) and presence (right panels) of 100 nM R1881.

Scale bars: 5 mm. (F) Acceptor photobleaching

FRET (abFRET) shows an interaction between the

N-terminal FQNLF motif and the AR-LBD (N/C

interaction) in YFP–AR–CFP and between YFP–AR

and AR–CFP. The N/C interaction is hormone

(100 nM R1881) induced (*P50.001610213 and

**P50.00961022, respectively, see Materials and

Methods) and dependent on the N-terminal FQNLF

motif (#P50.007610210 and ##P50.00761025,

respectively). Values are means 6 26standard error

of the mean (s.e.m.) of at least 25 cells measured in

two independent experiments.

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CFP ratio only increased following translocation of the tagged

ARs to the nucleus (supplementary material Fig. S2B). These

observations indicate that intramolecular N/C interactions were

initiated rapidly after hormone binding, followed by nuclear

translocation and initiation of intermolecular N/C interactions

(see also Schaufele et al., 2005).

To determine the ratio of intermolecular and intramolecular

interactions of nuclear ARs we co-transfected a YFP–AR–CFP

expression vector with increasing amounts of a vector expressing

untagged AR (Fig. 2A). In this setting, untagged AR competes

with YFP–AR–CFP for intermolecular N/C interactions, because

it will hetero-dimerize, resulting in a reduction of FRET. By

contrast, the untagged AR does not compete for intramolecular

N/C interactions, and therefore FRET of the intramolecular

N/C interaction will not be reduced. Indeed, with increasing

expression of untagged ARs the abFRET of YFP–AR–CFP

decreased proportionally (Fig. 2B, red squares). Comparing the

decrease of the experimental FRET data with theoretical FRET

efficiencies, based on complete intramolecular or complete

intermolecular N/C interactions (Fig. 2B, grey lines), showed

that in steady state AR N/C interactions were mostly

intermolecular. The curve of the experimental data dropped

more than halfway to the theoretical curve of sole intermolecular

FRET. The remaining clearly measurable AR fraction, estimated

to be 20–40%, showed intramolecular N/C interactions.

The intermolecular N/C interaction is driven by the ARD-box interaction

The second domain enabling an intermolecular interactionbetween two ARs is the dimerization box (D-box) in thesecond zinc-finger of the AR DBD (Fig. 3A). Three residues in

the D-box interact with their counterpart in the corresponding ARDBD in an AR dimer (A596 with T602, S597 with S597 andT602 with A596; Fig. 3B). We introduced a combination of two

D-box mutations that affect AR activity in individuals withpartial androgen insensitivity syndrome (AIS), A596T andS597T, in single- and double-tagged ARs to study the role of

the D-box in AR dimerization and in N/C interaction. Westernblot analysis showed that the tagged mutant proteins were of theexpected sizes (Fig. 3C–E). Mutating both residues (S597T andA596T) in YFP and CFP single-tagged ARs, abolishing all three

hydrogen bonds between the D-boxes, resulted in a complete lossof FRET efficiency, indicating absence of N/C interaction(Fig. 3F). The complete absence of N/C interaction in the

double D-box mutant strongly suggested that the AR DBD–DBDinteraction drives the intermolecular N/C interaction. In otherwords, although the N/C interaction requires binding of the

FQNLF motif in the AR NTD to the coactivator groove in theLBD, the D-box interaction is essential for intermolecular N/Cinteraction.

Substitution of the same D-box amino acid residues in YFP–AR–

CFP hardly affected the FRET efficiency (Fig. 3G). As shownabove, in wild-type YFP–AR–CFP most N/C interactions areintermolecular (Fig. 2). Together, our data indicate that an

expected drop in FRET in double-tagged D-box mutants, becauseof the loss of intermolecular N/C interactions, was compensated byintramolecular N/C interactions. In conclusion, the intramolecular N/

C interaction is independent of D-box dimerization, and moreimportantly, D-box dimerization is an essential step in intermolecularN/C interaction, possibly because of a conformational change in theAR induced by DBD–DBD interaction.

Stable DNA binding is not essential for AR dimerization

To study the role of DNA binding in AR dimerization we introduceda mutation in the a-helix in the first zinc-finger of the DBD, which

binds the major groove of the androgen response element (ARE)half-sites. The arginine residue at position 585 (R585) within thishelix and directly flanking the defined P-box makes base-specific

van der Waals contacts with the thymine residue in a consensusARE (Shaffer et al., 2004). Western blot analysis of double- andsingle-tagged ARs in which the arginine was replaced by either a

lysine (R585K) as is found in complete AIS (Sultan et al., 1993) orby alanine (R585A; which has a more subtle effect because itprobably retains the tertiary structure of the DBD) showed that theexpressed YFP–AR–CFP, YFP–AR and AR–CFP mutants were of

the expected size (Fig. 4A–C, respectively). High resolutionimaging of Hep3B cells expressing the AR mutants showed ahomogeneous nuclear distribution unlike the speckled pattern found

for wild-type AR (Fig. 4D) and very similar to a previouslypublished DNA-binding-deficient AR mutant (A573D) (Farla et al.,2004). Moreover, FRAP analysis (supplementary material Fig. S5)

of both AR mutants (R585K and R585A) showed a recovery ofintensity that was very similar to that of AR A573D (Fig. 4E,F).Previously it was shown that AR A573D lacked the transient

immobilization that is caused by binding to chromatin as shown bywild-type AR that binds to DNA (Farla et al., 2004; Farla et al.,2005). As expected from DNA-binding-deficient AR mutants, the

Fig. 2. AR N/C interaction in the nucleus is mainly intermolecular.

(A) Western blot analysis of lysates of Hep3B cells co-transfected with a

construct expressing YFP–AR–CFP and increasing amounts of constructs

coding for untagged AR in the transfection ratios 1:0, 4:1, 2:1, 1:1, 1:2 and

1:4 (YFP–AR–CFP: AR); lane 1–6, respectively. Lane 7 contained a lysate of

control cells not expressing ARs. Plasmid samples were corrected for the total

DNA content and total CMV-promoter content. AR was visualized using

mouse monoclonal antibody F39.4.1. (B) Acceptor bleaching FRET analysis

on Hep3B cells coexpressing YFP–AR–CFP and increasing amounts of

untagged AR (see A). Increasing quantities of untagged AR results in a lower

FRET efficiency in cells expressing YFP–AR–CFP. Grey lines indicate the

stochastic FRET efficiencies if only intermolecular N/C interaction (100%

intermolecular N/C interaction, lower line) up to 100% of the intramolecular

N/C interaction (0% intermolecular N/C interaction, top line) occurs in YFP–

AR–CFP. Values are means 6 26s.e.m. of at least 60 cells measured in four

independent experiments.

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AR mutants (R585K and R585A) were unable to induce luciferase

expression from the transiently transfected reporter (ARE)2TATA–

Luc (supplementary material Fig. S3) (Sultan et al., 1993).

AbFRET analysis of single YFP- and CFP-tagged DNA-

binding-deficient AR mutants (R585K and R585A) showed that

loss of DNA binding did not abolish the intermolecular N/C

interactions although the FRET value for the mutants was

somewhat lower than that of wild-type AR (Fig. 4G). Together

with our findings shown in Fig. 3 and described above, this

observation indicates that the majority of D-box interactions

occur prior to DNA binding of AR homodimers. AbFRET

analysis of double-tagged ARs showed that the DNA-binding-

deficient AR mutants were not diminished in their total intra- and

intermolecular N/C interactions (Fig. 4H). Therefore, to

summarize, transcriptionally inactive, DNA-binding-deficient

AR mutants, mutated in an amino acid residue directly

involved in AR–DNA contact, are able to show both intra- and

more importantly intermolecular N/C interaction, the latter

involving also the D-box interaction.

Transactivation capacity of AR dimerization mutants ispromoter dependent

High-resolution confocal images of Hep3B cells expressing wild-

type and mutant ARs show a typical speckled pattern for the

wild-type AR but a more homogeneous pattern is found of the

AR D-box double mutant (Fig. 5A). As we previously showed

using FRAP, this speckled pattern is always accompanied by a

reduced mobility of the AR due to transient immobilization

(Farla et al., 2005). Here we applied the strip FRAP procedure to

study the mobility of the D-box mutants (Houtsmuller, 2005; van

Fig. 3. Mutations in the AR D-box inhibit the intermolecular but not the intramolecular N/C interaction. (A) Schematic representation of the AR DBD. The

DBD consists of two zinc fingers. The amino acid residues in the D-box (red) and the residues in the P-box (green), responsible for the interaction with DNA, are

indicated. (B) Structure of the dimer-interface (D-box) in a fragment of the second zinc-finger of the DBD. Major interactions between the two AR D-boxes are

indicated (A596 and T602, S597 and S597, and T602 and A596). The structure was modified from PBD-file 1R4I (www.pdb.org) (Shaffer et al., 2004).

(C–E) Western blot analysis of YFP and CFP single- and double-tagged wild-type AR and D-box mutants. AR was detected using a mouse AR monoclonal

antibody (F39.4.1). Single- and double-tagged wild-type AR and the A596T/S597T double D-box mutant were expressed in Hep3B cells. All fusion proteins had

the expected size. (F) Acceptor bleaching FRET analysis of Hep3B cells coexpressing single-tagged wild-type YFP–AR and AR–CFP and the D-box mutant. AR

D-box mutant (A596T/S597T) shows a complete lack of FRET efficiency in the presence of 100 nM R1881 (*P50.00161026), indicating complete loss of the

intermolecular N/C interaction. Values are means 6 26s.e.m. of at least 40 cells measured in three or more independent experiments. (G) Acceptor bleaching

FRET on Hep3B cells expressing YFP and CFP double-tagged wild-type AR and the AR D-box mutant in the presence of 100 nM R1881. The D-box mutation did

not result in a lower FRET efficiency in the double-tagged ARs (**P50.005), indicating that lower intermolecular N/C interaction is compensated by higher

intramolecular N/C interaction. Values are means 6 26s.e.m. of at least 30 cells measured in at least two independent experiments.

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Royen et al., 2009b). There was a rapid redistribution of the D-

box mutant (A596T/S597T; Fig. 5B, blue curve), similar to that

of the DNA-binding-deficient mutant AR A573D (Fig. 5B, black

curve), indicating that the D-box mutant is much more mobile

than wild-type AR and that it lacks the relatively long transient

immobilization of wild-type AR (Fig. 5B, grey curve). This

finding correlates with less efficient target gene expression, but,

as we have shown previously, such an association is not absolute

(see Farla et al., 2004).

As a first step in exploring the role of the D-box dimerization in

individual AR-regulated gene expression, we studied, using 1 and

100 nM R1881, the activity of AR mutants on transiently

transfected luciferase reporter genes driven by minimal

promoters containing different types of ARE. The D-box amino

acid substitutions differentially affected AR transactivation

capacity on different promoters in this assay (Fig. 5C,D). Both

wild-type AR and the D-box mutant were able to activate a

minimal promoter composed of two high-affinity AREs. The

mutant AR showed a somewhat lower relative activity on a single

ARE promoter (Fig. 5C, top panel for wild type and bottom panel

for D-box mutant). Although the double-mutant AR (A596T/

S597T) showed activity similarly to wild-type AR on a minimal

promoter composed of two high-affinity consensus AREs

[(ARE)2TATA-Luc; ARE sequence: 59-TGTACAnnnTGTTCT-

39; Fig. 5C)], it was much less active on a minimal promoter

driven by two probasin AREIIs (sequence: 59-AGTA-

CTnnnAGAACC-39), or other weak AREs: SARG (specifically

androgen regulated gene) AREs (sequence: 59-TGTGC-

TnnnTGTTCT-39) and TMPRSS2 (transmembrane protease,

serine 2) AREs (sequence: 59-AGGACAnnnCACTCT-39;

Fig. 5D, bottom panel) (Denayer et al., 2010). By contrast, wild-

type AR was similarly active on all promoters and even somewhat

more active on the promoter driven by two TMPRSS2 AREs

(Fig. 5D, top panel). Also, complete lack of D-box interaction did

not substantially affect AR activity on the MMTV LTR promoter

that is composed of multiple AREs with variable AR affinity

(Fig. 5E). In summary, complete loss of D-box interactions leads

to lower AR activity or has no effect on activity. Importantly, the

differential effects are ARE dependent.

DiscussionTranscription activity of SRs is regulated not only by ligand

binding and DNA binding but also by multiple protein–protein

interactions including homodimerization and interactions with

Fig. 4. DNA-binding-deficient AR mutants show inter- and

intramolecular N/C interaction. (A–C) Western blot analysis of

YFP and CFP single- and double-tagged wild-type AR and DNA-

binding-deficient mutants. AR was visualized with antibody

F39.4.1. Single- and double-tagged wild-type and DNA-binding

mutant ARs were expressed in Hep3B cells. All fusion proteins

had the expected size. (D) High-resolution confocal images of

Hep3B cells expressing YFP and CFP double-tagged wild-type

AR and DNA-binding-deficient mutants. The AR mutants

(R585K and R585A) have a homogeneous distribution. Scale bar:

5 mm. (E,F) Strip-FRAP analysis on YFP emission of YFP and

CFP double-tagged AR DNA-binding mutants in the presence of

100 nM R1881. The AR mutants (R585K and R585A) showed a

rapid recovery, similar to the DNA-binding-deficient mutant AR

A573D. Curves represent the mean of at least 25 cells.

(G) Acceptor bleaching FRET analysis on co-transfected wild-

type YFP–AR and AR–CFP and DNA-binding-deficient mutants.

AR R585K and R585A retained most of the intermolecular N/C

interaction (*P50.017 and **P50.002, respectively). Values are

means 6 26s.e.m. of at least 30 cells measured two or more

independent experiments. (H) Acceptor bleaching FRET analysis

on Hep3B cells expressing wild-type YFP–AR–CFP and the

DNA-binding-deficient mutants in the presence of 100 nM

R1881. The N/C interaction was not diminished in either of the

DNA-binding-deficient mutants (#P50.232 and ##P50.235,

respectively). Values are means 6 26s.e.m. of at least 30 cells

measured in two or more independent experiments.

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transcriptional coregulators (reviewed by Rosenfeld et al., 2006).

A subgroup of SR coregulators interacts with a hydrophobic cleft

in the LBD through LxxLL-like motifs (Dubbink et al., 2004;

Hur et al., 2004). Unlike other SRs, the deep cofactor groove

of AR preferentially binds bulky FxxLF motifs, enabling

interactions with cofactors containing FxxLF-like motifs. The

D-box in the second zinc finger of the DBD is a well-

characterized dimerization interface of DNA-bound SRs

(Dahlman-Wright et al., 1991). The AR contains the unique

FQNLF sequence in the NTD as a second homodimerization

motif that can bind to the coactivator groove in the LBD leading

to AR N/C interaction. However, AR N/C interaction not only

occurs intermolecularly, but also intramolecularly, so either

between an FQNLF motif and a cofactor groove in the same AR

molecule or between two ARs (Schaufele et al., 2005). In the

present study we investigated the spatiotemporal association

between D-box–D-box interaction and the N/C interaction in AR

dimerization, by using quantitative live-cell imaging of cells

expressing YFP and CFP single- and double-tagged wild-type

and mutant ARs (Fig. 1A). Moreover, we investigated the role of

AR monomers and AR dimerization in DNA binding. On the

basis of our findings we propose a model for the dynamics of AR

protein–protein interactions (Fig. 6). In the model, AR D-box

interaction is an essential step between intra- and intermolecular

AR N/C interaction.

Previously, we and others found that the intramolecular N/C

interaction, but not the intermolecular N/C interaction is initiated

rapidly after hormone binding before the AR translocates to the

nucleus (Schaufele et al., 2005; van Royen et al., 2007). These

findings are summarized in part I of the proposed model (Fig. 6).

Only after nuclear translocation is the intramolecular N/C

interaction followed by an intermolecular N/C interaction

(supplementary material Fig. S2; Fig. 6) (Schaufele et al.,

2005). In an in vivo abFRET-based competition assay where we

added increasing amounts of untagged AR to YFP–AR–CFP, we

showed that in steady state the majority of FRET in YFP–AR–

CFP is by this intermolecular N/C interaction. This suggests that

AR homodimers are the preferred conformation of AR in the

nucleus. However, we calculated that a substantial percentage

(20–40%) of nuclear AR shows an intramolecular N/C interaction

(Fig. 2). This observation can hardly be explained as a transient

intermediate population prior to AR intermolecular N/C

interaction. Our AR N/C and D-box mutant analyses suggest a

dynamic equilibrium between intra- and intermolecular N/C

interaction in mobile AR. This raises the question of whether

ARs with intramolecular N/C interaction are homodimers that are

stabilized by D-box interaction. However, because stable AR D-

box interaction has never been observed in AR DBDs in solution

(see above) we favor the alternative explanation that the

intramolecular N/C interaction in nuclear ARs represents a

stable monomer subpopulation (Fig. 6, II).

Based on crystal structures of DBDs complexed with DNA,

DBD–DBD dimerization through the D-box has been established

as an important protein–protein interaction interface of SRs

(Luisi et al., 1991; Roemer et al., 2006; Schwabe et al., 1993a;

Shaffer et al., 2004). The most prominent amino acid residues

involved in the AR dimerization in this complex are A596, S597

and T602 (Fig. 3B) (Shaffer et al., 2004). We showed here that

Fig. 5. Loss of stable DNA-binding of AR D-

box mutants and differential effects on

different types of AR-regulated reporter genes.

(A) High resolution confocal images of Hep3B

cells expressing YFP and CFP double-tagged

wild-type and mutant ARs. The AR D-box mutant

(A596T/S597T) showed a more homogeneous

fluorescence. Scale bars: 5 mm. (B) Strip-FRAP

analysis on YFP emission of YFP and CFP

double-tagged AR D-box mutant (A596T/S597T)

in the presence of 100 nM R1881. Loss of the

ability to dimerize results in a rapid recovery of

fluorescence similar to the DNA-binding-

deficient mutant [AR (A573D)] indicating the

loss of stable DNA binding of the AR (A596T/

S597T) mutant. Curves represent the mean of at

least 25 cells. (C–E) Normalized transcriptional

activity of wild-type AR (top panel) and D-box

AR mutant (bottom panel) in the presence of

1 nM or 100 nM R1881 measured on luciferase

reporters driven by a transiently transfected

minimal promoter containing a single or two

high-affinity AREs [(ARE)2-TATA Luc;

Pwt,0.002 and PD-box mut,0.002; C], a series of

naturally occurring AREs, two probasin AREIIs

[(PB-AREII)2-TATA Luc], two SARG AREs

[(SARG-ARE)2-TATA Luc] or two TMPRSS2

AREs [(TMPRSS2-ARE)2-TATA Luc; all

Pwt.0.003 and PD-box mut,0.002; D] or the

complex MMTV promoter (MMTV Luc; E).

Values are means 6 26s.e.m. of at least three

independent experiments.

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mutation of two of these three amino acid residues completely

abolished intermolecular N/C interaction, most probably because

of complete absence of the D-box interaction. The mutations had

no effect on intramolecular N/C interaction. In fact, in the

absence of intermolecular N/C interaction in D-box mutants, an

increased intramolecular N/C interaction was observed

(Fig. 3F,G). These findings strongly suggest that D-box to D-

box interactions drive the transition from intramolecular AR N/C

interaction to intermolecular N/C interaction in nuclear AR

(Fig. 6, III).

It is not known whether peptide motif interactions other than

D-box interactions and N/C interactions can play a prominent

role in AR dimerization. For AR the evidence for LBD–LBD

interactions, as documented for other SRs, is limited. Although

amino acid residues involved in glucocorticoid receptor (GR)

LBD–LBD interactions are conserved in AR (Centenera et al.,

2008), in crystallographic studies, the isolated AR LBD is present

as a monomer in solution, in contrast to GR, progesterone

receptor (PR) and estrogen receptor (ER) LBDs (Bledsoe et al.,

2002; Matias et al., 2000; Sack et al., 2001; Tanenbaum et al.,

1998; Williams and Sigler, 1998). However, a (weak)

dimerization function in the hinge region as suggested for GR,

or in the C-terminal extension of the AR DBD cannot be

completely excluded (Centenera et al., 2008; Haelens et al., 2003;

Savory et al., 2001).

It has long been disputed whether AR dimerization occurs

before or after DNA binding (Centenera et al., 2008). We

previously showed that the N/C interaction occurs predominantly

when the ARs are mobile and is lost when the ARs are bound to

chromatin (van Royen et al., 2007). Combined with the present

observation that D-box interaction drives the intermolecular N/C

interaction (Fig. 3) this indicates that the D-box interaction

occurs before DNA binding (Fig. 6). These findings are in

contrast to theories based on crystallographic studies which

suggest that separate SR DBDs are monomeric in solution and

show cooperative dimerization when bound to DNA (Freedman

et al., 1988; Hard et al., 1990a; Hard et al., 1990b; Luisi et al.,

1991; Schwabe et al., 1993a; Schwabe et al., 1993b; Shaffer et al.,

2004). However, AR dimerization before DNA binding was

confirmed by experiments carried out with the DNA-binding-

deficient mutants (Fig. 4). Possibly, the stronger D-box to D-box

interaction in AR, compared with other SRs, combined with the

intramolecular N/C interaction, are of crucial importance in this

regard.

The relatively strong dimerization of the AR enables activation

of promoters containing different types of ARE (Fig. 5)

(reviewed by Centenera et al., 2008; Claessens et al., 2008;

Denayer et al., 2010; Shaffer et al., 2004). We showed that ARs

without appropriate D-box interaction cannot activate promoters

driven by two probasin AREIIs, SARG AREs or TMPRSS2

AREs, although a promoter with high affinity AREs can be

stimulated (Fig. 5C–E) (Denayer et al., 2010). On the basis of our

findings it is tempting to speculate that promoters with high

affinity AREs can be activated both by AR homodimers and by

consecutive binding of AR monomers that subsequently dimerize

on the DNA. By contrast, promoters with low-affinity AREs

would preferentially be activated by AR homodimers (Fig. 6,

IV). If this is the case then AR monomers in the nucleus are of

functional importance.

Recently, genome-wide chromatin immunoprecipitation

(ChIP) approaches indicated the presence of thousands or even

tens of thousands of AR binding sites in the human genome (Jia

et al., 2008; Takayama et al., 2011; Wang et al., 2007; Wang

et al., 2009; Yu et al., 2010). Interestingly, the majority of the AR

binding regions found in these studies, and AR binding sites

identified by ChIP in promoter and enhancer regions of

Fig. 6. Schematic representation of the spatiotemporal organization

of domain interactions in AR function. In the absence of ligand the AR

is predominantly located in the cytoplasm. Hormone-binding by the AR

results in a rapid initiation of the intramolecular N/C interaction followed

by nuclear translocation (I). In the nucleus both AR monomers and

dimers form a stable population (II), but the majority of ARs dimerize

through the D-box interaction that drives a transition from intra- to

intermolecular N/C interaction (III). The indicated intermediate

conformations, the AR homodimer with intramolecular N/C interaction

or without the N/C interaction, might be rapidly processed. The AR

dimer is able to stably bind to either a high-affinity ARE (light-green

rectangles) or a more selective low-affinity ARE (dark-green rectangles)

in promoter or enhancer regions of target genes. Two AR monomers are

proposed to bind consecutively to a high-affinity ARE where they

dimerize, but not to a low-affinity ARE (IV). In the DNA-bound AR the

N/C interaction is lost, allowing interactions with coactivators to

initiate transcription.

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androgen-regulated genes, apparently contain ARE half-sitemotifs, low-affinity AREs or ARE half-sites with suboptimal

spacing and not obvious high-affinity AR binding motifs (Massieet al., 2007; Wang et al., 2007). Loss of AR dimerization mightdirectly result in less AR binding to these sites (Fig. 5B).

Genome-wide ChIP-seq approaches combined with global geneexpression profiling in cells that exclusively express AR

monomers compared with cells that contains AR mainly in thehomodimer conformation would provide more detailedinformation on the role of monomers in AR-regulated gene

expression. The AR monomer to dimer ratio in a nucleus mightbe a mechanism of regulation of specificity in gene expression.

One obvious parameter that affects the dimerization status of theactivated nuclear AR population, is its concentration. Thishypothesis can well be extended to a role for specific cofactors

that differentially interact with AR monomers and dimers orcofactors that regulate AR dimerization (Bai et al., 2005).

In summary, previous data on the AR intra- and intermolecularN/C interaction lead to a model in which the intramolecular N/Cinteraction is initiated in the cytoplasm directly after hormone

binding, followed by intermolecular N/C interaction in the nucleus(Schaufele et al., 2005). Using quantitative imaging techniques, weelucidated the essential role of D-box dimerization in the transition

from intramolecular to intermolecular N/C interaction (Fig. 6).The D-box dimerization and the shift from intramolecular to

intermolecular N/C interaction might occur as one event or twoseparate events, but both independent of DNA binding. Togetherwith our observations showing that the AR N/C interaction is lost

in DNA-bound AR enabling cofactor interactions (van Royen et al.,2007), data in the present study elucidated the spatiotemporal

relationship of the consecutive AR intra- and intermoleculardomain interactions in living cells (Fig. 6). Moreover, the modelproposes a dynamic equilibrium of AR homodimers and

monomers in the nucleus, which can be an important mechanismof AR-regulated gene expression.

Materials and MethodsConstructs

In all constructs expressing AR fusion proteins the AR was separated from thefluorescent tag by a flexible (Gly-Ala)6 spacer (Farla et al., 2004) indicated by asingle dash. Constructs coding for wild-type and A573D variants of YFP–AR–CFPand AR–CFP were generated as previously described (van Royen et al., 2007). Theconstruct expressing N-terminally YFP-tagged AR was generated by replacingEGFP in pGFP-AR (Farla et al., 2004) with EYFP-C1 (Clontech Laboratories, Inc.,Mountain View, CA). The construct expressing untagged AR was obtained byinserting the AR cDNA from pAR0 (Brinkmann et al., 1989) into pEGFP-C1 fromwhich EGFP was deleted. The F23,27A/L26A mutation of YFP–AR–CFP, AR–CFP and untagged AR was introduced using the QuikChange mutagenesis kit(Stratagene, La Jolla, CA). In YFP–AR–CFP an LBD–CFP fragment was replacedwith an AR–LBD fragment from YFP–AR to obtain YFP–AR (F23,27A/L26A).The DBD mutations R585K, R585A and A596T/S597T were introduced byQuikChange mutagenesis in pYFP-AR-CFP. The LBD mutation E897A inuntagged AR was also generated with QuikChange mutagenesis. Formutagenesis primers see supplementary material Table S1. To generate thesingle-tagged DBD mutant ARs, the AR DBDs of pYFP-AR and pAR-CFP werereplaced with a pYFP-AR-CFP fragment containing the mutant DBD.

The (ARE)2-TATA Luc reporter, containing two high-affinity AREs(underlined in the following sequence: 59-CCGGGAGCTTGTACAGGATG-TTCTGCATGCTCTAGATGTACAGGATGTTCTGGTA-39) was a gift from G.Jenster (Rotterdam, Netherlands). The other reporters were generated by swappingthe ARE fragment in (ARE)2-TATA Luc with a fragment containing a single highaffinity ARE as present in the (ARE)2-TATA Luc reporter (59-CCG-GGAGCTTGTACAGGATGTTCTGCATGCTCTAGAGGTA-39), two probasinAREIIs (59-CCGGGAGCTAGTACTGGAAGAACCGCATGCTCTAGAAGTA-CTGGAAGAACCGGTA-39), two SARG AREs (59-CCGGGAGCTTGTGC-TGGATGTTCTGCATGCTCTAGATGTGCTGGATGTTCTGGTA-39), or twoTMPRSS2 AREs (59-CCGGGAGCTAGGACAGGACACTCTGCATGCTCTAG-AAGGACAGGACACTCTGGTA-39). The MMTV-Luc reporter construct was

described previously (de Ruiter et al., 1995). All new constructs were verified bysequencing. Sizes of expressed ARs were verified by western blotting.

Cell culture, transfection and luciferase assay

For 2 days before microscopic analyses, Hep3B cells, lacking endogenous ARexpression, were grown on glass coverslips in six-well plates in a-MEM(Cambrex, East Rutherford, NJ) supplemented with 5% fetal bovine serum(FBS; HyClone), 2 mM L-glutamine, 100 IU/ml penicillin and 100 mg/mlstreptomycin. At least 4 hours before transfection, the medium was replacedwith medium containing FBS stripped with 5% dextran-coated charcoal (DCC-FBS). Transfections were performed with 1 mg/well AR expression constructs or0.5 mg/well empty YFP or CFP expression vector in FuGENE6 (Roche MolecularBiochemicals, Indianapolis, IN) transfection medium. Four hours aftertransfection, the medium was replaced with 5% DCC-FBS with or without100 nM R1881. In the abFRET competition experiments 1 mg YFP–AR–CFP wasco-transfected with increasing amounts of untagged AR (ratio YFP–AR–CFP: AR1:0, 4:1, 2:1, 1:1, 1:2 and 1:4). Different vector sizes were taken into account. Theamounts of CMV promoters and total transfected DNA were corrected by co-transfecting pcDNA3 (CMV) and pTZ19 vectors.

For the AR transactivation experiments, Hep3B cells were cultured in 24-wellplates in a-MEM supplemented with 5% DCC-FBS in the absence or presence ofR1881 (1 or 100 nM) and transfected using 50 ng AR expression construct and100 ng luciferase reporter construct. After 24 hours, cells were lysed andluciferase activity was measured in a luminometer (GloMax Microplateluminometer; Promega Corporation, Madison, WI).

Western blot analysis

Hep3B cells were cultured and transfected in 6-well plates. After 24 hours, cellswere washed twice in ice-cold PBS and lysed in 200 ml Laemmli sample buffer(50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 10 mM DTT and 0.001%Bromophenol Blue). After boiling for 5 minutes, a 5-ml sample was separated on a10% SDS-polyacrylamide gel and blotted to Immobilon-P transfer membrane(Millipore, Billerica, MA). Blots were incubated with anti-AR (1:2000; mousemonoclonal antibody F39.4.1) and subsequently incubated with HRP-conjugatedgoat anti-mouse antibody (DakoCytomation, Glostrup, Denmark). Protein bandswere visualized using Super Signal West Pico Luminol solution (Pierce ChemicalCo., Rockford, IL), followed by exposure to x-ray film.

Confocal imaging, YFP/CFP ratio imaging and abFRET analysis

Immunofluorescence imaging of Hep3B cells expressing tagged ARs wasperformed using a confocal laser-scanning microscope (LSM510; Carl ZeissMicroImaging, Inc., Gottingen, Germany) equipped with a Plan-Neofluar 406/1.3NA oil objective (Carl Zeiss MicroImaging, Inc.) at a lateral resolution of 100 nm.An argon laser was used for excitation of CFP and YFP at 458 and 514 nm,respectively. In all quantitative imaging experiments cells with a physiologicallyrelevant expression level of tagged ARs were selected for analysis (van Royenet al., 2007; van Royen et al., 2009a).

N/C interactions of double-tagged YFP–AR–CFP, or co-transfected YFP–ARand AR–CFP were assessed using YFP/CFP ratio imaging and acceptorphotobleaching FRET (abFRET) (van Royen et al., 2009a and referencestherein). In YFP/CFP ratio imaging cells expressing YFP and CFP double-tagged AR or a combination of YFP–AR and AR–CFP with initially similar signalratios to YFP–AR–CFP were imaged with an interval of 30 seconds using a458 nm excitation at low laser power to avoid monitor bleaching. YFP and CFPemissions were detected using a 560 nm longpass emission filter and a 470–500 nm bandpass emission filter, respectively. The AR N/C interaction wasinitiated by adding R1881 to the cell culture. After subtraction of backgroundFRET was calculated as: IYFP/ICFP. The relative nuclear intensity was determinedsimultaneously using the YFP emission and was calculated as: Inucleus/(Inucleus+Icytoplasm).

In abFRET, YFP and CFP images were collected sequentially beforephotobleaching of the acceptor. CFP was excited at 458 nm at moderate laserpower, and emission was detected using a 470–500 nm bandpass emission filter.YFP was excited at 514 nm at moderate laser power, and emission was detectedusing a 560 nm longpass emission filter. After image collection, YFP in the nucleuswas bleached by scanning a region of ,100 mm2 25 times at 514 nm at high laserpower, covering almost the complete nucleus. After photobleaching, a secondYFP and CFP image pair was collected. Apparent FRET efficiency wasestimated (correcting for the amount of YFP bleached) using the equationabFRET5[(CFPafter2CFPbefore)6YFPbefore]6[(CFPafter6YFPbefore)2 (CFPbefore6YFPafter)]

21, where CFPbefore and YFPbefore are the mean prebleach fluorescenceintensities of CFP and YFP, respectively, in the area to be bleached (after subtractionof background), and CFPafter and YFPafter are the mean postbleach fluorescenceintensities of CFP and YFP, respectively, in the bleached area (Dinant et al., 2008).The apparent FRET efficiency was finally expressed relative to control measurementsin cells expressing either free CFP and YFP (abFRET0) or the CFP–YFP fusionprotein (abFRETCFP–YFP fusion): apparent FRET efficiency5(abFRET2abFRET0)6

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(abFRETCFP-YFP fusion2abFRET0)21. For statistical analysis, the abFRET data sets

were compared using the one-tailed Student’s t-test.

FRAP

The mobility of interacting proteins was studied using FRAP (supplementarymaterial Fig. S5) (van Royen et al., 2009b). A narrow strip spanning the nucleuswas scanned at 458 nm excitation (because of simultaneous CFP recording inFRET FRAP) (van Royen et al., 2007) using short intervals (100 ms) at low laserpower (YFP is sufficiently excited at this wavelength) (van Royen et al., 2007).Fluorescence intensity of YFP was recorded using a 560-nm longpass filter. After40 scans, a high-intensity, 100-ms bleach pulse at 514 nm was applied tophotobleach YFP inside the strip. Subsequently, scanning of the bleached strip wascontinued at 458 nm at low laser intensity. The curves were normalized using theequation Inorm5(Iraw2I0)/(Ipre2I0), where Ipre and I0 are the fluorescent intensitiesbefore and immediately after the bleach, respectively.

FundingThis work was supported by the Dutch Cancer Society [grant numberDDHK 2002-2679 to M.v.R.]; and the European Science Foundation[grant number 03-DYNA-F-18 to M.v.R.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.096792/-/DC1

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