Disrupted Amino- and Carboxyl-Terminal Interactions of the Androgen Receptor Are Linked to Androgen Insensitivity
James Thompson,
Fahri Saatcioglu,
Olli A. Jänne and
Jorma J. Palvimo
Biomedicum Helsinki Institute of Biomedicine/Physiology (J.T.,
O.A.J., J.J.P.) Institute of Biotechnology (J.J.P.) and
Department of Clinical Chemistry (O.A.J.) University of
Helsinki FIN-00014 Helsinki, Finland
Biotechnology
Centre of Oslo (F.S.) University of Oslo N-0316 Oslo,
Norway
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ABSTRACT
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We have compared the functional consequences of
seven single-point mutations in the ligand-binding domain (LBD) of the
androgen receptor (AR). The mutations span helices 3 to 11 and are
present in patients suffering from androgen insensitivity syndromes
(AIS) and other male-specific disorders. The mutants, except M742V,
bound to androgen response elements in vivo and in
vitro and showed a testosterone-dependent conformational change.
With regard to functional activity, the mutant M742V had severely
blunted ability to transactivate or exhibit the androgen-dependent
amino/carboxyl-terminal (N/C) interaction; mutants F725L, G743V, and
F754L showed reduced transactivation potential and attenuated N/C
interaction; and mutants V715M, R726L, and M886V had minor functional
impairments. The mutants belonging to the first two groups also
displayed reduced response to coexpressed GRIP1. In addition, mutations
of amino acids M894 and A896 in the putative core activation domain 2
(AF2) in helix 12 confirmed that this helix is important for N/C
interactions. Thus, amino acids located between helices 3 and 4 (F725
and R726), in helix 5 (M742, G743, and F754), and in helix 12 (M894 and
A896) play critical roles in mediating the N/C interaction of AR. The
data also show that disrupted N/C interaction is a potential molecular
abnormality in AIS cases in which LBD mutations have not resulted in
markedly impaired ability to bind androgen.
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INTRODUCTION
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The development and maintenance of male and some female sexual
characteristics is dependent on the normal function of the androgen
receptor (AR). Upon binding the natural androgens,
5
-dihydrotestosterone (DHT) and testosterone (T), AR elicits through
a complex sequence of interactions the reproductive and homeostatic
functions of the male phenotype. Subtle disruptions of the molecular
structure and mechanisms of AR regulation can result in clinical
phenotypes that span from mild (MAIS) and partial (PAIS) to complete
androgen insensitivity syndromes (CAIS), and are also involved in the
development of prostate cancer (1). Most of these phenotypes and
syndromes result from single nucleotide substitutions in the AR gene
(2).
The steroid receptors belong to the nuclear receptor superfamily and
have a well characterized and conserved modular structure (3, 4). The
N-terminal domain (NTD) is the least conserved region among these
receptors and can vary in size from being <6% of the total protein
for the vitamin D receptor to >50% for AR. Within the AR NTD resides
the hormone-independent transcription activation function 1 (AF1)
(3, 4, 5). Also located within the AR NTD are conserved FXXLF and WXXLF
motifs (6) that form, in part, the interface for the interaction of NTD
with the hormone-dependent activation function 2 (AF2) located in the
C-terminal ligand-binding domain (LBD) (7). The AR LBD comprises 12
helices, and a ligand-binding pocket is formed by the helices 3, 4, 5,
7, 11, and 12 together with the ß-sheet preceding helix 6 (8, 9). The
LBDs of steroid receptors function not only to bind ligands but also to
stabilize homodimerization and orchestrate interactions with
coregulators (3, 4). Unlike many other steroid receptors, the AF2 of AR
is transcriptionally weak (7, 10). However, the ligand-dependent
interaction between the NTD and the LBD region is needed for optimal AR
function (6, 10, 11). Whether this N/C interaction is direct or
partially bridged via coregulators remains to be clarified. The closely
related p160 steroid receptor coactivators that include SRC-1,
GRIP1/TIF2, and AIB1/ACTR/TRAM-1 (reviewed in Refs. 12, 13, 14, 15) have
distinct regions that interact with the NTD and the LBD, thereby
potentially bridging the N/C interaction of AR (6, 10, 11). The three
conserved LXXLL motifs of p160 coactivators form amphipathic
-helices that bind to a hydrophobic groove formed by helices 3, 4,
5, and 12 of the LBDs, whereas regions outside these motifs are
involved in contacting the NTDs (16, 17, 18, 19).
In this report, we have elucidated the molecular consequences of seven
AR LBD mutations found in human patients (20). These amino acid
substitutions are localized in or around helices 3, 4, 5, and 11. Our
data indicate that many of these mutant ARs are defective in their N/C
interactions, even though the mutations have no severe effects on
ligand binding. In addition, by mutating AR amino acids M894 and A896,
we show that the integrity of helix 12 (21) is important for AR N/C
interaction.
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RESULTS
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Influence of the LBD Mutations on Transactivation by AR
Seven AR LBD point mutations that span helices 311 were selected
(20) and recreated into mammalian expression vectors to compare the
molecular mechanisms underlying some androgen-insensitive phenotypes
and other male-specific disorders (Table 1
). The AR mutants V715M, F725L, R726L,
G743V, and M886V are previously shown to possess normal equilibrium
dissociation constants for androgens, whereas M742V and F754L have 5-
and 2-fold reduced androgen-binding affinity, respectively (24, 27). Whole-cell binding assays in COS-1 cells using
[3H]mibolerone as a ligand showed that the
dissociation constant (KD) values of different AR
forms were indeed very similar, with the exception that the mutant
M742V exhibited a KD twice as high as that of
wild-type AR (data not shown). As a control, we used a helix
12-truncated version of AR that terminates at amino acid 889 (TRUNC)
and is unable to bind steroid (data not shown).
The wild-type and mutant receptors were cotransfected into COS-1 cells
along with a probasin promoter (-285/+32)-driven luciferase reporter
to investigate the effects of the substitutions on
AR-dependent transcription in the presence of natural androgens, T
or DHT. With T, transcriptional activities of R726L and M886V were
slightly reduced, whereas mutants F725L, G743V, and F754L retained only
2030% of wild-type AR activity (Fig. 1A
). Interestingly, the mutant V715M was
approximately twice as active as wild-type receptor at 1 nM
T, and its activity was saturated already at 10 nM T. In
contrast, mutant M742V was practically inactive at all T concentrations
tested. Comparable results were obtained with a reporter driven
by two androgen response elements (AREs) in front of a minimal
TATA sequence (pARE2TATA-LUC) (data not
shown). When DHT was used as the androgen, transcriptional activities
of mutant receptors in relation to wild-type AR were generally 2030%
higher than in the presence of T (Fig. 1B
). It is of particular note
that the activity of M742V reached approximately 30% of that of
wild-type receptor at 100 nM DHT. The effects of synthetic
androgens, mibolerone and methyltrienolone, were also tested on the
mutants showing severely compromised transcriptional activity with T.
In the presence of the latter two steroids, the activities of F725L,
M742V, G743V, and F754L in relation to wild-type AR were higher than
those obtained in the presence of DHT (Fig. 1C
). In sum, androgens
activated all mutant receptors but the resulting activities varied
considerably with different androgens. M742V especially exemplifies
this phenomenon; it is almost inactive with T, but mibolerone can
rescue its transcriptional activity to
50% of that of wild-type AR.
Immunoblotting with an AR-specific antibody demonstrated that the
wild-type and mutant receptors were expressed at similar levels in
COS-1 cells (Fig. 1D
).

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Figure 1. Transcriptional Activation by Wild-Type and Mutated
ARs
COS-1 cells were transfected with the expression vectors encoding
wild-type and mutated ARs (2 ng) along with pPB(-285/+32)-LUC (200
ng), and pCMVß (50 ng) (see Materials and Methods).
A, Twenty-four hours after transfection, the cells received fresh
medium containing indicated concentrations of T for the subsequent
24 h. B, The same experiment as in panel A, except for DHT
replaced T. C, Transcriptional activities of wild-type AR and mutants
F725L, M742V, G743V, and F754L in the presence of 0.1, 1, and 10
nM T, mibolerone (MB), or methyltrienolone
(R1881). Luciferase activities of the cell extracts were adjusted to
the transfection efficiency using ß-galactosidase activity. In panels
A and B, the activity of wild-type AR in the presence of 100
nM T or 100 nM DHT,
respectively, is set as 100. In panel C, the activity of wild-type AR
in the presence of 10 nM T is set as 100. The
mean ± SD values from at least three
independent experiments are shown. D, An immunoblot analysis of mutant
ARs expressed in COS-1 cells. The cells on 12-well plates were
transfected with the expression vectors encoding wild-type and mutated
ARs (2 ng/well). Twenty-four hours after transfection, the cells
received fresh medium containing 100 nM T and
were cultured for an additional 24 h. Whole cell extracts were
prepared from cells pooled from three wells as described in
Materials and Methods. Extracts (20 µg protein/lane) were
subjected to electrophoresis under denaturing conditions followed by
immunoblotting with an AR-specific K333 antibody (56 ). (TRUNC, AR that
terminates at amino acid 889.)
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The effects of ectopic GRIP1 (glucocorticoid receptor interacting
protein 1) expression to the transcriptional activities of
mutant receptors were also examined. All the mutants responded to
coexpressed GRIP1 in the presence of 100 nM T; wild-type AR
and mutants V715M, R726L, and M886V displayed a
4-fold relative
increase in the transcriptional activity, whereas mutants F725L, M742V,
G743V, and F754L showed somewhat lower (
3-fold) coactivation by
GRIP1 (Fig. 2
). The helix 12-truncated AR
that is totally transcriptionally inactive failed to respond to GRIP1
coexpression.

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Figure 2. Influence of GRIP1 on Transcriptional Activation by
Wild-Type and Mutated ARs
COS-1 cells were transfected with the expression vectors for wild-type
or mutated ARs (2 ng), pPB(-285/+32)-LUC (200 ng), pCMVß (50 ng)
along with (+) or without (-) pSG5-GRIP1 (300 ng). The -GRIP1 cells
received 150 ng empty pSG5 and 150 ng pBluescript SK
(Stratagene). Twenty-four hours after transfection, the
cells received fresh medium with 100 nM T for the
subsequent 24 h. Luciferase activities in cell extracts were
normalized using ß-galactosidase activity. The activity of AR in the
presence of T without GRIP1 is set as 100, and the mean ±
SD values from at least three independent experiments
are shown. The fold increase in AR activity elicited by the presence of
GRIP1 is depicted above the bars.
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Effects of LBD Mutations on the Conformation of AR
We have previously shown that the agonist-induced conformation of
AR protects the LBD from partial proteolysis, yielding 30-kDa trypsin-
or chymotrypsin-resistant fragments (30). In contrast, receptor
forms that are incapable of hormone binding are completely cleaved
under the same conditions. To study the influence of the mutations on
AR conformation, the mutants were translated in vitro in the
presence of [35S]methionine, incubated with or
without 100 nM T, and subsequently digested with
trypsin. All the mutants, except M742V, were resistant to digestion in
the presence of T and showed the same approximately 30 kDa-limit
fragments as the wild-type AR (Fig. 3A
),
indicating that the binding of T stabilizes their conformation. The
helix 12-truncated receptor that cannot bind androgens (data not shown,
Ref. 31) showed no resistance to trypsin (Fig. 3A
). The behavior of the
M742V mutant in this assay is consistent with the transactivation data,
suggesting that it binds T very weakly and/or its conformation in the
presence of T is distinct and less stable than those of the other AR
forms. Since M742V showed clearly detectable transcriptional activity
in the presence of both DHT and mibolerone, the influence of these two
steroids on the M742V conformation was studied. In agreement with the
ability of these androgens to rescue its transactivation, both DHT and
mibolerone were capable of conferring trypsin resistance on M742V (Fig. 3B
). However, the latter two androgens protected the wild-type AR
clearly more efficiently from trypsin than was the case with the M742V
mutant.

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Figure 3. Protease Resistance of Native and Mutated ARs
A, In vitro translation of wild-type and mutated AR
proteins was carried out in the presence of
[35S]methionine, and 25-µl portions of the translation
mixture were digested with trypsin (6 ng/µl) in the presence of 100
nM testosterone (+T) or vehicle (-T) for 30 min.
Upper panels, Labeled AR proteins at time point 0 min.
Lower panels, Resultant trypsin digestion pattern of
each mutant. B, Comparison of the ability of T, DHT, or mibolerone (MB)
(100 nM each) to confer trypsin resistance on the
M742V mutant and wild-type AR (WT). Reaction products were analyzed on
10% SDS-PAGE gels and visualized by fluorography.
Arrows depict the 30-kDa protease-resistant
fragments. Migration of molecular mass markers is shown on the
left.
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DNA Binding of AR Mutants
We next investigated whether the point mutations influence the
DNA-binding ability of the receptor forms. Mutated ARs were expressed
in COS-1 cells in the presence and absence of T, and whole cell
extracts were analyzed by electrophoretic mobility shift assay (EMSA).
The extracts were incubated with 32P-labeled
intronic ARE of the C3(1) gene, and receptor-DNA complexes were
resolved. In the absence of androgen in the culture medium, only
minimal binding of wild-type AR to the ARE was detectable, whereas
extracts derived from cells grown in the presence of T showed strong
AR-ARE complexes (Fig. 4A
). This was also
the case with mutants V715M, F725L, R726L, and M886V. In contrast, DNA
binding of M742V was barely stimulated by T. Inclusion of androgen had
a lesser effect on the DNA complex formation by G743V and F754L than
with the other mutants (Fig. 4A
). Helix 12-truncated AR did not bind to
ARE under these conditions. Immunoblotting of the cell extracts showed
that the less avid DNA binding of M742V, G743V, or F754L was not due to
a lower expression of these mutants in COS-1 cells (Fig. 4A
).

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Figure 4. Binding of Native and Mutant ARs to ARE
A, Upper panels, Equal amounts of whole cell extracts
(10 µg protein) from COS-1 cells expressing wild-type and mutated ARs
were preincubated with 100 nM testosterone (+T) or
vehicle (-T) at 22 C for 30 min before incubation (60 min) with
32P-labeled C3(1 )-ARE at 22 C. Protein-DNA complexes were
separated on 4% nondenaturing polyacrylamide gels and detected by
autoradiography. Lower panels, Immunoblot analysis of
the cell extracts subjected to EMSA analysis; K333 (56 ) was used as the
primary antibody. B, ARs (7 µl) synthesized by in
vitro translation in reticulocyte lysates were preincubated
with 100 nM testosterone (+T) or vehicle (-T) and
subjected to EMSA analysis as described in panel A. Note that although
somewhat variable amounts of incubation mixtures were loaded, as
illustrated by the nonspecific bands (NS) migrating between the AR-ARE
complex and free probe, it does not hamper interpretation of the data.
An arrowhead depicts the positions of specific AR-ARE
complexes and F refers to the free probe. The experiments were repeated
twice with essentially identical results.
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We have previously shown that in vitro-translated
holo-AR-DNA complex migrates on EMSA gels faster than that of apo-AR,
which is interpreted to reflect a more compact structure of the holo-AR
(32). To complement the experiments performed with COS-1 cell extracts,
receptors were produced in rabbit reticulocyte lysates and subjected to
EMSA analyses. The in vitro-translated receptors were
preincubated in the presence of T or vehicle before the addition of
32P-labeled ARE. In agreement with our previous
results (30, 32), receptors produced in the reticulocyte lysate
displayed a considerable DNA-binding activity even without androgen
(Fig. 4B
). Whether this is due to residual androgen(s) in the lysates
or to some other factors is not clear at this time. In any event,
differences between the mobility of holo- and apo-AR complexes were
detectable for wild-type AR and mutants V715M, R726L, and M886V. T
elicited a less pronounced or no effect on the migration of protein-DNA
complexes formed by F725L, M742V, G743V, and F754L. The helix
12-truncated AR form was again severely compromised in its ability to
interact with DNA in this assay. These data show collectively that
mutants with a weak DNA-binding activity in COS-1 cells also have
diminished conformational change when expressed in reticulocyte lysates
and exposed to T, probably reflecting the same phenomenonan improper
folding of the LBD in response to androgen.
The Function of AR LBD in Isolation
The AR LBD contains an AF2 domain that functions in yeast but is
barely active in mammalian cells (7, 10). To investigate the
consequences of the single amino acid substitutions in more detail, we
inserted each mutation into the AR LBD fused in-frame to Gal4
DNA-binding domain (Gal4). COS-1 cells transfected with the Gal4-LBD
constructs and immunoblotting of the cell extracts with Gal4 antibody
revealed that the encoded proteins were expressed to similar levels
(data not shown). Transcriptional activities of the Gal4-LBD proteins
were examined in COS-1 cells using a Gal4 response element-driven
luciferase reporter (pG5-LUC). The effect of cotransfected GRIP1 was
also investigated. In agreement with our previous results (7, 10), the
AF2 of wild-type LBD remained very weak in the presence of T, and none
of the mutant LBDs showed transcriptional activity in this assay (Fig. 5
, and data not shown). Coexpression of
GRIP1 in the absence of androgen did not influence the activities of
the Gal4-LBDs (data not shown), whereas in the presence of hormone,
GRIP1 activated wild-type AF2 as well as V715M, R726L, and M886V by
28-fold. In contrast, mutants F725L, M742V, and G743V responded only
marginally to coexpressed GRIP1 (5- to 7-fold activation), and F754L
showed an intermediate level (
16-fold) of activation (Fig. 5
).

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Figure 5. The Effect of GRIP1 on the Transcriptional
Activation by Wild-Type and Mutated LBDs
COS-1 cells were transfected with the expression vectors encoding
wild-type and mutated LBDs fused to Gal4 DBD (100 ng), pG5-LUC
reporter (200 ng), pCMVß (50 ng) in the presence (+) or absence (-)
of pSG5-GRIP1 (120 ng). For -GRIP1 cells, 60 ng of pSG5 plus 60 ng of
pBluescript SK were added. Twenty-four hours after transfection, the
cells received fresh medium with 100 nM T, and the
culture was continued for additional 24 h. LUC activities,
normalized by the ß-galactosidase activity, are expressed relative to
that of wild-type LBD in the presence of androgen but without GRIP1 (WT
LBD + testosterone = 1). The mean ± SD
values from at least three independent experiments are shown. The fold
increase in the AR LBD activity elicited by GRIP1 is depicted
above the bars.
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In addition to the patient mutations, M894 and A896 in helix 12 were
mutated to investigate their role in the function of AR LBD. The
nonpolar amino acid residue Met at codon 894 was changed either to an
acidic (Asp) or to a small nonpolar residue (Ala), and Ala at codon 896
was substituted with bulkier residues, either Val or Leu. M894A and
M894D mutants exhibited no response to coexpressed GRIP1, and both
A896L and A896V were activated only by about 5-fold, in comparison to
the approximately 30-fold activation of wild-type LBD (Fig. 5
). Thus,
the LBD mutants fall into three categories in terms of their responses
to GRIP1: 1) very little or no activation (mutants F725L, M742V, G743V,
M894A, M894D, A896L, and A896V); 2) partial activation (F754L); and 3)
activation comparable to wild-type AR LBD (V715M, R726L, and
M886V).
The Effects of the LBD Mutations on the N/C Interaction
The N- and C-terminal regions of AR interact in an
androgen-dependent fashion to coordinate molecular events that are
important for the function of the receptor (6, 10, 33). It is possible
to reconstitute the AR N/C interaction by coexpressing a Gal4
DNA-binding domain (DBD) fused to the AR LBD and a fragment containing
AR NTD fused to the VP16 activation domain (AD) (10). Using this
two-hybrid system in COS-1 cells, we investigated how substitutions in
the AR LBD influence the N/C interaction and what is the effect of
coexpressed GRIP1.
In the absence of coexpressed GRIP1, mutants V715M, R726L, and M886V
showed N/C interaction similar to that of wild-type AR (Fig. 6
, open bars). In contrast,
mutants G743V and F754L had 2030% and mutants F725L and M742 had
only
10% of the activity of wild-type receptor LBD. Of the helix 12
mutants, M894A showed 50% of wild-type N/C interaction, whereas
mutants A896L and A896V had only
25% of the wild-type LBD activity.
Interestingly, conversion of M894 to Asp totally abolished N/C
interaction as assessed by the two-hybrid system (Fig. 6
). Coexpression
of GRIP1 enhanced the NTD interaction of all the receptor LBDs by
approximately 3- to 8-fold, except for M894D that showed no rescue of
N/C interaction. Differences in the GRIP1 interactions of the mutants
as isolated LBDs and when coexpressed with the NTD are highlighted by
the helix 12 mutant M894A; GRIP1 failed to activate its LBD in
isolation, but M894A maintained approximately 50% of the wild-type
activity to interact with the NTD, which was rescued to the level of
the wild-type LBD by coexpressed GRIP1 (Fig. 6
). Mutants F725L, M742V,
G743V, and F754L showed severely reduced responses to GRIP1 as isolated
LBDs; nevertheless, GRIP1 stimulated their N/C interaction by a factor
that was similar to wild-type LBD, suggesting that the reduced
transcriptional activity of the mutants is mainly due to compromised
N/C interaction rather than impaired GRIP1 response. These results also
indicate that amino acids in the loop region between helices 3 and 4,
together with those in helices 5 and 12, play important roles in the
N/C interaction.

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Figure 6. The Influence of Point Mutations in the AR LBD on
the N/C Terminal Interaction
COS-1 cells were transfected with the expression vectors for Gal4 DBD
fusions of wild-type and mutated LBDs (100 ng), pVP16-rAR-(5538) (100
ng, an AR NTD expression vector), pG5-LUC reporter (200 ng), pCMVß
(50 ng), and with (+) or without (-) pSG5-GRIP1 (120 ng) as depicted.
The total amount of DNA transfected was balanced by adding empty
expression vectors when appropriate. For -GRIP1 cells, 60 ng of pSG5
plus 60 ng of pBluescript SK were added. The indicated AR LBDs were
present both in the presence and absence of coexpressed GRIP1.
Twenty-four hours after transfection, the cells received fresh medium
with 100 nM T, and the culture was continued for
24 h. Normalized LUC activities are expressed relative to that of
wild-type LBD in the presence of androgen but without GRIP1 (WT LBD +
NTD = 100). The mean ± SD values from at
least three independent experiments are shown. The fold increase in the
N/C interaction activity elicited by GRIP1 is depicted above the
bars.
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The Influence of LBD Mutations on AR-Mediated Repression of Nuclear
Factor (NF)-
B
Activation of AR by ligand binding can also lead to
transcriptional repression of AR target genes (34, 35, 36). Even though
transrepression does not usually involve the interaction of AR with
specific DNA elements (34, 35, 36), our previous data show that
transactivation and transrepression are influenced in a dissimilar
fashion by AIS mutations in the AR DNA-binding domain (36). One well
characterized group of target genes that are repressed by AR are those
under the positive control by members of the nuclear factor-
B
(NF-
B) family. We have shown that AR attenuates transactivation by
RelA in a dose- and androgen-dependent fashion (34, 36). To
investigate whether the LBD mutants differ in their abilities to
inhibit RelA-dependent transcription, COS-1 cells were cotransfected
with the expression vectors for wild-type or mutated ARs along with
RelA and a NF-
B-regulated reporter
(p
B6tk-LUC). All the AR mutants, except the
helix 12-truncated form and M742V, were similar to the wild-type
receptor in their ability to repress RelA-activated transcription in a
T-dependent fashion (Fig. 7
). It is of
note that even though T could not stimulate M742V in transactivation
assays, it still partially activated the mutants transrepressive
function (Fig. 7
), which was approximately 50% of that of wild-type
AR. These results agree with our previous data showing that
transactivation and transrepression are separate functional entities
and that AR LBD plays a minor role in the inhibition of RelA function
(34, 36).

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Figure 7. The Ability of AR Mutants to Repress RelA-Induced
Transactivation
COS-1 cells were transfected with the expression vectors encoding
full-length wild-type and mutated ARs (20 ng) along with 30 ng RelA
(pCMV-p65), p B6tk-LUC reporter (150 ng), and pCMVß (50
ng). All transfections contained equal amounts of DNA. Twenty-four
hours after transfection, the cells received fresh medium with (+) or
without (-) 100 nM T for the subsequent 24 h.
Normalized LUC activities are expressed relative to reporter gene
activity elicited by RelA alone (RelA = 100). The mean ±
SD values from at least three independent experiments
are shown.
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DISCUSSION
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We have evaluated the molecular consequences of seven AR point
mutations linked to patients with AIS or prostate cancer,
which did not exhibit a markedly impaired androgen-binding activity.
The mutants F725L and M742V were found in PAIS patients (1, 24). These
residues are conserved among the steroid receptors, except that there
is a Leu in the estrogen receptor sequence at the position
corresponding to M742 (Fig. 8
). Both
mutants are severely impaired in their N/C interactions and ability to
transactivate. The effects of the F725L substitution on AR activity and
domain interaction are comparable to those observed in a recent
study (11). F725 resides in a loop region between helices 3 and 4,
and the corresponding residue F367 in ER
is reported to form part of
the interface for the recognition by GRIP1 nuclear receptor box II
peptide (37, 38). F725 might be involved in helix positioning, and
changing Phe to Leu reduces structural rigidity, and the coordination
of the helices 3, 4, and 5 is compromised. Interestingly, a mutation in
a neighboring amino acid (N727K) was recently shown to lead to a
similar disruption between receptor domains and GRIP1/TIF2 (39).

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Figure 8. Alignment of LBD Sequences that Correspond to
Steroid Receptor Helices 35 and Helices 1112
AR amino acid residues mutated in this investigation are indicated by
the bold text above the sequence alignments. Mutations,
except for those in helix 12, were selected from the AR mutations
database (20 ). (ER, Estrogen receptor; GR, glucocorticoid receptor; PR,
progesterone receptor; MR, mineralocorticoid receptor.)
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M742 resides in helix 5 and, according to the AR LBD crystal structure,
is directly involved in forming the ligand-binding pocket and
contacting the ligand (9). Although Val at codon 742 fits into this
pocket, the mutated residue enlarges the pocket and thereby affects
ligand binding (9). Interestingly, our results show that the
transcriptional activity of M742V differs significantly with different
androgens; the mutant is not activated by T, whereas the bulkier
androgens, DHT and mibolerone, can markedly rescue its transcriptional
activity. Consistent with these data, DHT and mibolerone, but not T,
are able to elicit a conformational change in the receptor. Thus, our
functional data showing that the M742 residue is one of the amino acid
residues critical in distinguishing between different androgens agree
well with the AR LBD structural model (9). The M742 mutant also binds
poorly to DNA. Since F725L binds efficiently to ARE under the same
conditions, even though it shows very little N/C interaction, the lack
of association between LBD and NTD does not alone explain the inability
of M742V to bind DNA.
The mutants G743V and F754L reside in helix 5 (Fig. 8
), and they have
been reported to cause PAIS/CAIS and PAIS, respectively (25, 26, 27). The
latter mutation has also been identified in prostate cancer (28).
Neither G743 nor F754 residues are conserved among the nuclear
receptors, suggesting that they possess an AR-specific role. Both
mutants showed severely compromised N/C interactions and a reduced
ability to transactivate from the probasin promoter. The latter result
with G743V is in agreement with previous data obtained using the mouse
mammary tumor virus promoter (25). G743 and F754 bound to AREs more
efficiently than the M742V mutant, albeit their receptor-DNA complex
formation was weaker than that of wild-type AR in the presence of T.
The compromised N/C interaction of the helix 5 mutants is probably due
to distortions in helix-helix associations within the LBD. The
substitutions in helix 5 may perturb its conformation in such a way
that they weaken the helix 3-helix 5 interaction, which appears to be
important for the bending of helix 3 and receptor activation (40).
Structural models predict that the bent helix 3 is involved together
with helix 12 in the formation of a surface(s) for coactivator binding
(4). Taken together, our data indicate that helix 5 of the AR LBD plays
a central role in N/C contacts. Our results also suggest that the LBD
surfaces participating in interactions with the AR NTD resemble those
shown to be involved in LBD-coactivator interactions of other
receptors.
GRIP1 is able to bind through its three nuclear receptor boxes (LXXLL
motifs) to AR LBD (18) and, independently of these motifs, to the AF1
region of AR (17, 41). However, in comparison to other steroid
receptors, AR shows clear preference for certain LXXLL motifs (42).
Mutations of the nuclear receptor boxes eliminated completely the
coactivator function of GRIP1 on many nuclear receptors, such as
thyroid hormone receptor, but they reduced coactivation on AR only by
about 40% (17). Although some mutations in the AR LBD, such as those
in helix 5, reduced significantly the response of Gal4-LBDs to GRIP1,
the coactivator was nevertheless able to enhance the N/C interaction of
the mutant receptors as well as their transcriptional activity as
full-length proteins. These data together with previous reports (11, 16, 17, 43) indicate that AR LBD plays a minor role in GRIP1
interactions.
V715M and R726L have been identified in prostate cancer (22, 23), and
M886V is associated with oligospermic infertility (29), a condition
regarded as mild androgen insensitivity. V715M resides in helix 3,
R726L in the loop between helices 3 and 4, and M886V in helix 11 (Fig. 8
). The mutations did not affect the stability of receptor-DNA
complexes, all three showed N/C interactions similar to that of
wild-type AR, and the AF2s of the three mutants were activated by GRIP1
as well as or better than that of wild-type receptor. The
transcriptional activities of M886V and R726L as full-length receptors
on the probasin promoter were, however, slightly reduced. Unlike in a
previous report (29), we detected no reduction in the ability of M886V
to interact with AREs, nor was there impairment in its N/C
interactions. V715M is reported to be activated by adrenal androgens
and progesterone more efficiently than the native receptor (22), which
may have some bearing on the progression of prostate cancer. With
regard to predisposition to prostate cancer, our data add an important
feature to V715M function, in that this substitution renders the
receptor more sensitive to low androgen concentrations. Even though the
R726L mutant had in this work properties very similar to those of
wild-type AR, others have shown it to be activated by estradiol to a
greater extent than the wild-type receptor (23), which is perhaps the
reason for the association of this mutation to a 6-fold increased risk
of prostate cancer (44). Some other mutants found in prostate cancer,
such as T877A and L701H (45, 46), also alter the ligand-binding
specificity of AR, but no detailed structural and/or functional
characterization of the mutants has been carried out.
In addition to V903M mutation in a person with PAIS, five other
mutations (M895T, I898T, P904S, P904H, L907F) within helix 12 of AR
have thus far been reported in patients with CAIS (2, 47, 48, 49, 50).
Mutations at codon 894 demonstrated that Met is involved in the N/C
interaction and also in the interaction with GRIP1; M894D showed no
association with the NTD, and it was not activated by GRIP1 in either
the N/C interaction assay or as Gal4-LBD. As a full-length receptor,
the corresponding mutant is transcriptionally inactive (43). Even
though the LBD containing the M894A substitution was totally
unresponsive to GRIP1, it retained about one-half of the wild-type N/C
interaction. This result is in line with the finding that the
transcriptional activity of the corresponding full-length form is
reduced only by 20% (43). Bevan et al. (51) have recently
shown that mutation of both M894 and the neighboring M895 to Ala
abolishes the activity of AR. However, mutagenesis of single amino acid
residues was not performed in that study. Mutation of A896 to Leu or
Val reduced the N/C terminal interactions to one third of wild-type
level. As LBDs, both A896L and A896V harbored very little response to
GRIP1, but similar to M894A, their interaction with NTD was rescued by
coexpressed GRIP1. In agreement with our results, He et al.
(11) have recently demonstrated that several AR LBD substitutions, such
as I898T (helix 12) and K720A (helix 3), weaken the interaction of LBD
with both GRIP1/TIF2 and NTD. I898T exhibits attenuated N/C interaction
but interacts well with GRIP1/TIF2, whereas K720A has lost the ability
to bind the p160 coactivator but retained the domain interaction
activity (11). The behavior of the latter mutant is similar to that of
M894A in this study. Thus, the surfaces of AR LBD for GRIP1/TIF2 and
NTD interaction overlap but are not identical.
The data of this work, together with the results obtained by
mutagenesis of other amino acids of AR LBD found in AIS (11, 52),
support the notion that disrupted N/C interaction is a potential
molecular defect in many AIS patients, in whom the LBD mutations have
not resulted in severe impairment in androgen-binding properties. Many
of these substitutions may also alter the ability of AR to interact
with coactivators such as those of the p160 family. It should be
pointed out, however, that defect(s) in coactivator protein(s) can also
lead to CAIS, as illustrated in a recent study (53).
 |
MATERIALS AND METHODS
|
---|
Plasmid Constructions
For studies using the mammalian two-hybrid system, the pM-LBD
that expresses Gal4 DBD fusion of wild-type human AR LBD (amino acids
624919) has been described (7). The individual point mutations were
incorporated into the LBD using the Quick Change Site-Directed
Mutagenesis Kit (Stratagene, La Jolla, CA). The following
upper and lower oligonucleotides (mutated codons underlined)
were used:
PCR was performed for 12 cycles in accordance with the
manufacturers instructions. Mutation incorporation was checked by
sequencing both strands using the Pharmacia ALFexpress DNA sequencing
system (Amersham Pharmacia Biotech, Buckinghamshire,
UK). As a negative control we created an AF2 region deletion
mutant termed pM-LBD-TRUNC (residues 624889) that was created
using PCR and the following primers: upper,
5'-CGTAGGATCCGGATG624ACTCTGGGAGCCCGGA-3';
lower,
5'-TGACGTCGAC-TCA889CACGCTCACCATGTG-3'.
Upper primer creates a BamHI site at amino acid residue 624
(underlined) and lower primer creates a SalI site
at amino acid residue 889 (underlined). The PCR fragments
were inserted into BamHI/SalI-cleaved pM vector
(CLONTECH Laboratories, Inc., Palo Alto, CA). The LBD
helix 12 (core AF2) mutants fused to GAL4 DBD were created using
single-stranded mutagenic primers corresponding to amino acid residues
890902 of AR (43). The pVP16-rAR-(5538), which expresses amino acid
residues 5538 of rAR, has been described previously (10). Full-length
AR mutants were created by digesting the pM-LBD mutants with
Tth111I and XbaI and inserted the fragment into
the Tth111I/XbaI site of full-length human AR
expression vector pCMV-hAR (54). For in vitro studies,
full-length mutant sequences were transferred into pSG5 vector
(Stratagene) by removing a
Tth111I/BamHI sequence from a pSG5-hAR construct
(32) and ligating the corresponding restriction fragment from the
pCMV-hAR mutant forms. All sequences were confirmed by Pharmacia
ALFexpress DNA sequencing system. pPB(-285/+32)-LUC containing
nucleotides -285 to +32 of the rat probasin promoter driving firefly
luciferase coding region has been described (34). pCMV-RelA and
p
B6tk-LUC were kindly provided by Dr. Patrick
Baeuerle (55). pG5-LUC was obtained from Promega Corp.
(Madison, WI) and pSG5-GRIP1 was a gift from Dr. Michael Stallcup.
Cell Culture and Transfections
COS-1 cells (from American Type Culture Collection,
Manassas, VA) were maintained in DMEM containing penicillin (25 U/ml),
streptomycin (25 U/ml), and 10% FBS. Transfections were performed
using FuGENE 6 reagent (Roche Molecular Biochemicals,
Mannheim, Germany) according to the manufacturers instructions. In
brief, 30 x 103 COS-1 cells were seeded on
12-well plates 24 h before transfection. Four hours before
transfection cells received fresh medium containing 10%
charcoal-stripped FBS. One hundred fifty nanograms of reporter plasmid,
50 ng of pCMVß (CLONTECH Laboratories, Inc.), and
indicated amounts of expression constructs were transfected. Eighteen
hours after transfection, the cells received fresh medium containing
2% charcoal-stripped FBS and 100 nM T or vehicle. After a
30-h culture, the cells were harvested, and then lysed in reporter
lysis buffer (Promega Corp.), after which the luciferase
(LUC) and ß-galactosidase activities were assayed as described
previously (10).
Immunoblot Analysis of Extracts from Transfected Cells
Transfected COS-1 cells were washed twice with PBS and the
pellets were resuspended in SDS-PAGE sample buffer, heated at 95 C for
5 min, and analyzed on 10% or 12% SDS-polyacrylamide gels. The
proteins were transferred onto a nitrocellulose membrane
(Amersham Pharmacia Biotech). Membranes were then
immunoblotted with a mouse monoclonal Gal4 DBD antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or with an antiserum
K333 raised against full-length rat AR (56) as needed. The membranes
were subsequently incubated with either a horseradish
phosphatase-conjugated goat antimouse or antirabbit IgG antibody
(Zymed Laboratories, Inc., South San Francisco, CA), and
immunocomplexes were detected using the ECL Western blot reagents
(Amersham Pharmacia Biotech) according to the
manufacturers instructions.
Partial Proteolytic Digestion of in Vitro-Translated
Human AR Mutant Constructs
[35S]Methionine-labeled ARs were
translated using 0.5 µg cDNA in pSG5 backbone and the TNT T7 Quick
Coupled Transcription/Translation System (Promega Corp.)
according to the manufacturers instructions in the presence of 25
µM ZnCl2 at 30 C for 90 min.
Twenty-five microliters of the resulting proteins were incubated for 10
min in the presence of 100 nM T or vehicle at 30 C before
being digested with trypsin (6 ng/µl). Samples were taken at time
points 0 min and 25 min. The reactions were stopped by adding 1.5 µl
of the digestion mixture to 23.5 µl of 2 x SDS-PAGE sample
buffer, followed by a 5-min incubation at 95 C. Samples were analyzed
on 10% SDS-PAGE gels. After the electrophoresis, the gels were fixed
in methanol (45%, vol/vol)-acetic acid (10%, vol/vol), treated with
Amplify (Amersham Pharmacia Biotech), dried, and
visualized by fluorography.
EMSA
For preparation of whole cell extracts, 100 x
103 COS-1 cells on six-well plates were
transfected with 1.5 µg of wild-type or mutated AR cDNAs (pCMV-hAR)
using the FuGENE 6 reagent. Twenty-four hours after transfection, the
cells received fresh medium with or without 100 nM T and
were grown for additional 24 h. Subsequently, the cells were
washed twice with 2 ml of ice-cold PBS and harvested, after which
whole-cell extracts were prepared by using a buffer containing 20
mM Tris-HCl (pH 7.5), 400 mM KCl, 15%
(vol/vol) glycerol, 1 mM EDTA, 2 mM
dithiothreitol, and 1:200 protease inhibitor cocktail (P-8340,
Sigma, St. Louis, MO ) with or without
10-7 M T. Aliquots of cell extracts
(10 µg of protein) were taken for DNA-binding reactions performed at
22 C as described previously (57). Alternatively, the different AR
forms were produced by translation in vitro as described
above. After a 30-min incubation at 22 C in the absence or presence of
100 nM T, 32P-labeled
C3(1)-ARE element (32) was added to reactions, and incubations were
continued for 60 min. The mixtures were then loaded onto a 4%
nondenaturing polyacrylamide gel (29:1), which had been prerun at 200V
for 30 min. Electrophoresis was carried out in 0.25 x Tris-borate
EDTA (TBE) at 22 C for 135 min. The gel was subsequently dried
and subjected to autoradiography.
Whole-Cell Steroid Binding Assay
For whole-cell steroid-binding assays, 30 x
103 COS-1 were seeded and transfected with 0.4
µg of each pCMV-hAR mutant expression vector. Twenty-four hours after
transfection, cells received fresh DMEM medium containing 10%
charcoal-stripped FBS and were grown for an additional 24 h.
Subsequently, the cells were washed once with PBS and the medium
changed to DMEM without serum containing a range of
[3H]mibolerone concentrations (0.160
nM). For measurements of nonspecific binding, duplicate
wells contained [3H]mibolerone with a 200-fold
molar excess of nonradioactive mibolerone. After 2 h of incubation
at 37 C and 5% CO2, medium was collected for
radioactivity measurements, and the cells were washed three times with
ice-cold PBS (1 ml/well), harvested, and collected by centrifugation at
2,000 x g for 10 min at 4 C. The cell pellets were
resuspended in 0.5 M NaOH and incubated at 56 C
for 15 min. The radioactivity of the solubilized pellets and collected
media was measured, and the method of Scatchard was employed to
determine binding constants essentially as described by Isomaa et
al. (58).
 |
ACKNOWLEDGMENTS
|
---|
The excellent technical assistance of Leena Pietilä,
Birgit Tiilikainen, Seija Mäki, Kati Saastamoinen, and Pirjo
Kilpiö is gratefully acknowledged. We thank Thomas Slagsvold for
AF2 constructs and Michael Stallcup for the GRIP1 expression
plasmid.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jorma J. Palvimo, Ph.D., Biomedicum Helsinki, Institute of Biomedicine/Physiology, P.O. Box 63 (Haartmaninkatu 8), University of Helsinki, FIN-00014 Helsinki, Finland. E-mail: jorma.palvimo{at}helsinki.fi
This work was supported by grants from the Medical Research Council
(Academy of Finland), the Finnish Foundation for Cancer Research,
Norwegian Cancer Society, the Sigrid Jusélius Foundation,
Biocentrum Helsinki, the Helsinki University Central Hospital, and the
Association for the Cure of Cancer of Prostate (CaP CURE).
Received for publication December 27, 2000.
Accepted for publication February 27, 2001.
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