A G577R Mutation in the Human AR P Box Results in Selective Decreases in DNA Binding and in Partial Androgen Insensitivity Syndrome
Denis Nguyen,
Sergey V. Steinberg,
Etienne Rouault,
Samuel Chagnon,
Bruce Gottlieb,
Leonard Pinsky,
Mark Trifiro and
Sylvie Mader
Department of Biochemistry (D.N., S.V.S., E.R., S.C., S.M.),
Université de Montréal, Montréal, Québec H3C
3J7, Canada; Departments of Biology and Pediatrics (L.P.,), Human
Genetics (L.P., M.T.), and Medicine (M.T., S.M.), McGill University,
Montréal, Québec H3G 1Y6, Canada; Lady Davis Institute
for Medical Research (B.G., L.P., M.T.), Sir M. B. Davis-Jewish
General Hospital, Montréal, Québec H3T 1E2, Canada;
and McGill Center for Translational Research in Cancer (S.M.), McGill
University, Montréal, Québec H3G 1Y6,
Canada
Address all correspondence and requests for reprints to: Dr. Sylvie Mader, Département de Biochimie, Faculté de Médecine, Université de Montréal, C.P. 6128 Succursale Centre Ville, Montréal, Québec H3C 3J7 Canada. E-mail: sylvie.mader{at}umontreal.ca
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ABSTRACT
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We have characterized a novel mutation of the human AR,
G577R, associated with partial androgen insensitivity syndrome. G577 is
the first amino acid of the P box, a region crucial for the selectivity
of receptor/DNA interaction. Although the equivalent amino acid in
the GR (also Gly) is not involved in DNA interaction, the residue at
the same position in the ER (Glu) interacts with the two central base
pairs in the PuGGTCA motif. Using a panel of 16 palindromic
probes that differ in these base pairs (PuGNNCA) in gel
shift experiments with either the AR DNA-binding domain or the full
length receptor, we observed that the G577R mutation does not induce
binding to probes that are not recognized by the wild-type AR. However,
binding to the four PuGNACA elements recognized by the
wild-type AR was affected to different degrees, resulting in an altered
selectivity of DNA response element recognition. In particular,
AR-G577R did not interact with PuGGACA palindromes.
Modeling of the complex between mutant AR and PuGNACA
motifs indicates that the destabilizing effect of the mutation is
attributable to a steric clash between the Cß of Arg at position 1 of
the P box and the methyl group of the second thymine residue in the
TGTTCPy arm of the palindrome. In addition, the Arg side
chain can interact with G or T at the next position
(PuGCACA and PuGAACA elements, respectively).
The presence of C is not favorable, however, because of incompatible
charges, abrogating binding to the PuGGACA element.
Transactivation of several natural or synthetic promoters containing
PuGGACA motifs was drastically reduced by the G577R
mutation. These data suggest that androgen target genes may be
differentially affected by the G577R mutation, the first natural
mutation characterized that alters the selectivity of the AR/DNA
interaction. This type of mutation may thus contribute to the diversity
of phenotypes associated with partial androgen insensitivity
syndrome.
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INTRODUCTION
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ANDROGENS ARE NECESSARY for normal
prenatal male sexual development (masculinization) and for secondary
male sexual development around puberty (virilization). Mutations in the
X-linked AR can lead to a wide range of clinical conditions associated
with different degrees of androgen insensitivity (1).
Complete androgen insensitivity syndrome (CAIS) corresponds to subjects
with a male genotype (46,XY) but female external genitalia. Partial
androgen insensitivity syndrome (PAIS) regroups a range of phenotypes
with ambiguous external genitalia, and mild androgen insensitivity
syndrome is associated with infertile individuals with male external
genitalia (1).
The AR belongs to the superfamily of nuclear receptors, which includes
receptors for steroid hormones, RA, vitamin D3,
and thyroid hormone, as well as a large number of orphan receptors
(2, 3, 4). Nuclear receptors contain six regions of homology
organized in three main functional domains. Regions A-B and E-F, which
are poorly conserved and well conserved, respectively, within the
family, coincide with two transcriptional activation domains (AF1 and
AF2, respectively). Region E-F also contains a dimerization interface
and the ligand-binding pocket and binds coactivators in a
ligand-dependent manner (5, 6, 7). Region C, the most
conserved region, contains two zinc fingers and directs binding to
specific sequences of DNA, the hormone response elements. These
response elements are composed of PuGNNCA motifs, arranged
as palindromes in the case of the steroid receptors
(8, 9, 10, 11, 12).
PuGA/TACA motifs are
recognized by GRs, ARs, PRs, and MRs, whereas
PuGG/TTCA motifs are
selectively bound by ERs and receptors for nonsteroidal hormones
(12, 13, 14). Each motif is bound by one receptor, the two
zinc fingers of the DNA-binding domain (DBD) folding into a structural
unit that establishes an array of nonspecific contacts with phosphate
groups and a few specific contacts with base pairs of the response
motif via a DNA recognition helix in the first zinc finger (15, 16). Although some of these contacts are conserved in all
nuclear receptors, three amino acids in this helix (P box) differ
between ERs and other steroid receptors and are responsible for
discrimination between the PuGGTCA and PuGAACA
motifs (17, 18, 19). In addition, a dimerization interface in
the second zinc finger (D box) is important for recognition of the
spatial arrangement of these motifs as palindromes with a 3-bp spacer
(13, 14, 17, 19, 20).
Naturally occurring mutations in the AR are usually point
mutations that can affect any of the three main functional domains,
although most mutations cluster in the ligand-binding domain, resulting
in mutant receptors with altered ligand-binding properties (21, 22). Mutations in the DBD associated with androgen insensitivity
syndrome usually result in a partial or complete loss of DNA binding
(23, 24, 25, 26, 27), although defects in hormone binding have also
been described (24). Here we report a new mutation of the
AR that results in conversion of Gly 577 to Arg within the P box of
this receptor. We have investigated the effect of this mutation on
the affinity of the AR for its ligands and for its DNA response
elements. Because of the localization of this mutation within the AR P
box, we also investigated whether the G577R mutation altered the
selectivity of receptor/DNA interaction. Finally, we have assessed the
effect of this mutation on the transactivation of synthetic promoters
containing PuGNACA elements and natural promoters
containing imperfect palindromic elements.
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RESULTS
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Characterization of Mutation G577R in a Patient with PAIS
Genital skin fibroblasts were obtained from a patient with PAIS
(see Materials and Methods) when corrective surgery was
performed. AR sequences were amplified by PCR using exon-specific
probes and sequenced. A mutation converting a GGA triplet coding for
G577 to an AGA triplet (Arg) was detected in exon 2, which encodes the
first zinc finger of the AR DBD (Fig. 1
).
To ascertain that this mutation was not an artifact caused by PCR
amplification, exon 2 was reamplified by PCR and a second round of
sequencing was performed, confirming the presence of the mutation.

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Figure 1. The G577R Mutation Affects the First Residue of the
AR P Box
A, Autoradiogram of a sequencing reaction of the wild-type and
mutant ARs in the region surrounding position 577. The mutation (G to A
at the first position in the codon corresponding to Gly 577) is
indicated on the sequencing gel by an arrowhead. B,
Mutations in the AR DNA-binding region associated with defects in
androgen responsiveness are indicated by asterisks. The
DNA recognition helix is in bold, the P box amino acids
are circled, and the G577R mutation is
boxed.
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Scatchard analysis was then performed by incubating genital skin
fibroblasts with
[1,2,4,5,6,7-3H]5
-dihydrotestosterone
(DHT; 110 Ci/mmol) or two synthetic nonmetabolizable androgens,
[17
-methyl-3H]mibolerone (MB; 85 Ci/mmol)
and [17
-methyl-3H]methyltrienolone (86
Ci/mmol). Apparent dissociation constant (0.11, 0.09, and 0.07
nM) and maximum androgen-binding capacity (31, 37, and 28
fmol/g protein) values obtained were in the normal ranges. Because
androgen dissociation rates could not be repeatedly measured using
genital skin fibroblasts because of lack of material, the G577R
mutation was reintroduced in the wild-type receptor using site-directed
mutagenesis; lack of other mutations in the region amplified by PCR was
confirmed by sequencing. Expression vectors for wild-type or mutant ARs
were transiently expressed in HeLa or COS-1 cells, and Western blot
analysis of whole cell extracts indicated similar levels of expression
for both wild-type and mutant receptors in HeLa cells (Fig. 2A
) and COS-1 cells (data not shown).
Hormone dissociation analyses were performed at 37 C, with cells
transiently transfected with the mutant AR expression vector and
incubated with tritiated DHT and MB. Off rates obtained for these
hormones (6 and 3 x 10-3
min-1) were in the normal range (68 and
24 x 10-3 min-1,
respectively). Together, these results indicate that the G577R mutation
is not associated with detectable defects in AR hormone-binding
properties.

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Figure 2. Expression of the Wild-Type and Mutant
Full-Length ARs and the Corresponding DBDs
A, Expression vectors pSG5-AR (ARwt), pSG5-AR-G577R (AR-G577R), or the
parental pSG5 vector (0) were transiently transfected (15 µg each) in
HeLa cells. Whole cell extracts were analyzed for AR expression levels
by Western blot analysis using the F39.4.1 mouse monoclonal antibody at
a 1:10,000 dilution (58 ) (similar results were obtained in
transient transfections of COS-1 cells). B, Bacterial expression
vectors pET3-AR[DBD] (ARwt DBD), pET3-AR[DBD]G577R (AR-G577R DBD),
or the parental pET3 vector (0) were transformed into E.
coli BL21 DE3 cells. Aliquots (1 ml) of exponentially growing
cultures were centrifuged and resuspended in M9 medium containing each
amino acid except Met and Cys (0.01% wt/vol each). Bacteria were
incubated with rifampicin (200 µg/ml final concentration) and IPTG
(0.5 mM final concentration) for 30 min.
[35S]Met (10 µCi/ml) was then added, and cells were
further incubated at 37 C for 5 min. Bacteria were harvested by
centrifugation, resuspended in Laemmli buffer, and boiled for 5 min.
Labeled proteins were separated by electrophoresis on a 12%
polyacrylamide-SDS gel and revealed by fluorography.
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To investigate whether the mutant AR can direct transcriptional
activation of androgen-responsive genes, HeLa cells were transiently
cotransfected with varying amounts of expression vectors for wild-type
or mutant AR together with the pGRE5-CAT reporter vector. This reporter
vector contains five copies of the hormone response element present
in the rat tyrosine amine transferase (TAT) gene [Table 1
, rTAT glucocorticoid response
element (GRE)] upstream of the TATA box of the adenovirus major late
promoter (28). The TAT response element has been shown
previously to bind not only the GR but also the AR (29, 30). Chloramphenicol acetyl transferase (CAT) analysis of
extracts from transfected cells indicated that levels of
transcriptional activation observed with AR-G577R in the presence of MB
(2 nM) were not reduced compared with the wild-type
receptor (Fig. 3A). On the other
hand, neither the wild-type nor the mutant receptor activated
transcription from a reporter gene containing three consensus estrogen
response elements (EREs), which are not bound by ARs, confirming
that the activation observed with the pGRE5-CAT reporter is
mediated by the TAT response elements (Fig. 3B). Thus, these
results indicate that mutation G577R does not prevent transcriptional
activation of a reporter gene containing multimerized response
elements. However, cooperativity of DNA binding or transcriptional
activation could mask a reduced affinity for androgen response elements
(AREs). In addition, different response elements may be affected to
different degrees by the mutation.
Mutation G577R Alters the Affinity and Selectivity of AR
Interaction with AREs
The G577R mutation is located in the P box of the AR, a region
shown previously to play a key role in discriminating between EREs and
GREs/AREs (17, 18, 19). These two types of receptor
recognition motifs differ by the two central base pairs in the
PuGNNCA motifs (GT in EREs, AA or TA in AREs; Table 1
). To
investigate whether mutation G577R affected the DNA-binding affinity
and/or selectivity of the AR, we examined the binding profiles of
bacterially expressed wild-type and mutant AR DBDs to a panel of 16
palindromic probes containing a repeat of each possible
PuGNNCA motif. The wild-type and mutant AR DBDs (amino
acids 550656) were expressed to similar levels, as assessed by
labeling with [35S]Met of a fraction of the
bacterial population used for preparation of whole cell extracts (Fig. 2B
). The wild-type AR DBD bound specifically to four probes,
corresponding to the consensus PuGNACA (Fig. 4A
, lanes 1, 5, 9, 13; the position of
the specific complexes is indicated by the arrowhead). The mutant AR
DBD did not bind to probes that were not bound by the wild-type DBD
receptor (Fig. 4B
). Complexes formed with three of the
PuGNACA probes, corresponding to the AA, CA, and TA motifs,
were weaker than those formed with the wild-type DBD (Fig. 4
, C and D).
Binding to the GA probe by the mutant DBD was undetectable (Fig. 4
, B
and D). Note that the GA and CA probes were bound to similar extents by
the wild-type DBD (Fig. 4
, A and C) but that the mutant DBD only bound
the latter probe (Fig. 4
, B and D). In addition, although the wild-type
DBD bound preferentially to the AA and TA elements, the mutant DBD
preferred AA and CA elements (Fig. 4
, C and D). Thus, the G577R
mutation differentially affected binding to the four palindromic
PuGNACA elements. In addition, the same differential effect
was observed with elements containing a consensus PuGAACA
motif and a variable PuGNACA motif (data not shown).

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Figure 4. The Wild-Type and Mutant AR DBDs Recognize
PuGNACA Elements with Different Specificities
A and B, Gel shift analysis was performed using extracts from
E. coli BL21 DE3 cells expressing the ARwt DBD (A) or
the AR-G577R DBD (B) with a panel of 16 probes that differ by the two
central base pairs in the PuGNNCA motif. The sequence of
the probes used for each lane is indicated above the autoradiogram of
the gel. The position of the AR-probe complexes is indicated by an
arrowhead. The four PuGNACA probes bound by
ARwt DBD are underlined. C and D, The bands
corresponding to the bound and free probe fractions were quantitated by
manual excision and scintillation counting for the four
PuGNACA elements. The percentage of probe bound by the
wild-type or mutant AR DBDs is shown. Results are an average of seven
experiments.
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Binding selectivity of the full length wild-type and mutant receptors
was also investigated using whole cell extracts from HeLa cells
transiently transfected with expression vectors for either receptor or
with the parental pSG5 expression vector. Nonspecific bands were
observed when extracts from cells transfected with the pSG5 vector were
used (Fig. 5C
). Similar to what was
observed with the isolated wild-type AR DBD, the full length wild-type
AR bound specifically only to PuGNACA motifs, with a
preference for AA and TA elements (Fig. 5A
, lanes 1, 5, 9 and 13; the
position of the specific complexes is indicated by the closed
arrowhead). Specific complexes were also observed with extracts
from cells transfected with AR-G577R on probes containing AA, CA, and
TA elements (Fig. 5B
, lanes 1, 5, and 13) but not with the probe
containing GA motifs (Fig. 5B
, lane 9). In addition, the mutant
receptor bound preferentially to the AA and CA elements. These changes
in the DNA selectivity of the mutant receptor are similar to those
observed with the bacterially expressed DBDs. Note that in this
experiment, higher concentrations of mutant AR protein extract were
used to achieve detectable binding.

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Figure 5. The Wild-Type and Mutant Full-Length ARs Recognize
PuGNACA Elements with Specificities Similar to Those of the
Corresponding DBDs
HeLa cells were transiently transfected with 15 µg of expression
vectors for the wild-type AR (ARwt), the mutant AR (AR-G577R), or the
parental pSG5 expression vector (0). Whole cell extracts were used in a
gel shift assay with the 16 PuGNNCA probes. A closed
arrowhead indicates the position of the AR-containing
complexes. An open arrowhead indicates the position of a
nonspecific complex that is observed also in non-AR-expressing cells
(C) and migrates close to the specific band. The four
PuGNACA probes bound by wild-type AR are
underlined. Note that higher concentrations of mutant AR
protein were used to achieve detectable binding. Similar patterns of
gel shifts were observed in three independent experiments.
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Mutation G577R Compromises Transcriptional Activity on Response
Elements Containing PuGGACA Elements and on Imperfect
Response Elements
Although transactivation of the GRE5-TATA promoter, which contains
elements composed of one TA and one AA motif (Table 1
, rTAT GRE), was
not affected by the G577R mutation, results from the DNA-binding
experiments suggested that promoters containing PuGGACA
elements may not be activated by the mutant receptor because of lack of
binding. To test this prediction, we introduced oligonucleotides
containing two PuGNACA response elements upstream of the
TATA box of the adenovirus major late promoter and of the CAT gene. The
resulting reporter genes were transiently transfected into HeLa cells
together with increasing concentrations of expression vectors for
wild-type or mutant AR. A dose-dependent increase in transcriptional
activation was observed for all four response elements with the
wild-type receptor, with comparable levels of peak transcriptional
activity (Fig. 6A
). Expression of the
reporter genes containing AA, CA, and TA elements was also increased
with transfection of progressively higher concentrations of expression
vector for the mutant AR (Fig. 6B
). However, no transactivation was
observed using PuGGACA motifs with the mutant receptor
(Fig. 6B
).

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Figure 6. AR-G577R Does Not Transactivate a Reporter Vector
Containing Two Copies of the PuGGACA Elements
HeLa cells were transiently cotransfected with varying concentrations
of the expression vectors for the wild-type AR (ARwt) or the mutant AR
(AR-G577R), with CAT reporter vectors containing two copies of
PuGNACA palindromes (AA, TA, CA, or GA) inserted upstream
of the adenovirus major late promoter TATA box (2 µg), and with the
internal control pCMV-ßGal (2 µg). After the calcium-phosphate
precipitate was removed, cells were treated with MB (2 nM)
for 24 h. CAT activity was measured in whole cell extracts. The
experiment was repeated four times. Results of a typical experiment are
shown.
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We also tested the effect of the G577R mutation on transactivation from
natural AREs containing PuGGACA or TGTCCPy
motifs. Both the element present in the prostatic binding protein C1
gene (31) and the element present in the androgen receptor
gene (ARE1; see Table 1
; 32) mediated transactivation by
the wild-type receptor (Fig. 7
, C and E)
but not detectably by the mutant AR (Fig. 7
, D and F), whereas both
receptors transactivated PuGAACA palindromes to similar
levels under the same conditions (Fig. 7
, A and B).

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Figure 7. Transactivation by AR-G577R Is Impaired with
Response Elements Containing GA Motifs
HeLa cells were transiently cotransfected with 0.25, 1.25, or 6.25 µg
of expression vectors for the wild-type AR (ARwt) or the mutant AR
(AR-G577R), with 2 µg of reporter vectors containing two copies of
the PuGAACA palindrome (A and B), the C1 ARE (C and D), or
the human AR (hAR) ARE1 (E and F), and with the internal control
pCMV-ßGal (2 µg). Cells were treated or not with MB (2
nM) for 24 h after the calcium-phosphate precipitate
was removed. CAT activity was measured in whole-cell extracts after
standardization for ß-galactosidase activity. Results are an average
of three experiments.
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In addition to using synthetic minimal promoters, we investigated the
effect of the G577R mutation on the transactivation of natural
promoters by androgens. The mouse mammary tumor virus (MMTV) promoter
is a viral promoter that responds to glucocorticoids, androgens, and
progesterone in transient transfections and also when incorporated into
minichromosomes or into the cellular genome, although the specificity
of the hormonal response can be restricted depending on the site of
integration (33, 34, 35). MMTV contains imperfect palindromic
elements composed of one PuGAACA arm each (Table 1
), the
other motif being in both cases related to the PuGTACA
motif (PuTTACA and PuGTATC for the upstream and
the downstream elements, respectively; 36, 37, 38, 39). Both
AR-G577R and the wild-type receptor transactivated the MMTV reporter
vector efficiently (20- to 30-fold, Fig. 8
, A and B). The probasin promoter
(40), which is specifically activated by AR but not by GR
(29, 30, 41, 42, 43), is composed of two imperfect elements,
one of which contains a variant TA motif (Table 1
). With this promoter,
5-fold reduction in transactivation levels was observed with the
mutant receptor, down to
3-fold activation (Fig. 8
, C and D; note
that different scales are used in C and D). Finally, the
prostate-specific antigen (PSA) promoter (44), which
contains two imperfect response elements, one of which has a
degenerated GA motif, was transactivated by the wild-type receptor,
whereas hormone addition had little effect on transcriptional activity
in the presence of the G577R AR mutant (Fig. 8
, E and F). These results
demonstrate that transactivation of natural promoters by the AR is
affected by the G577R mutation to different degrees.

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Figure 8. Transactivation by AR-G577R Is Impaired with the
Probasin and PSA Promoters
A and B, COS-1 cells were transiently cotransfected with 0.33, 1, or 3
µg of the expression vectors for the wild-type AR (ARwt) or the
mutant AR (AR-G577R), with the pMMTV-hGH reporter vector (3 µg), and
with the internal control pRSV-ßGal (2 µg). Cells were treated or
not with MB (2 nM) for 24 h after the
calcium-phosphate precipitate was removed. The human GH activity was
measured in cell supernatants using an hGH-ELISA kit according to the
manufacturers instructions (Medicorp). Human GH activity was adjusted
according to ß-galactosidase activity. C and D, COS-1 cells were
transiently cotransfected as described above except that the reporter
vector contained the rat probasin promoter (pPB-hGH). Note that
different scales are used in C and D. E and F, COS-1 cells were
transiently cotransfected as described above except that the reporter
vector contained the human PSA promoter (PSA-hGH). Results are an
average of three experiments.
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DISCUSSION
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Mutations in the AR associated with androgen resistance cluster
mostly in the ligand-binding domain and, to a lesser extent, in the
DBD. All of the mutations found in the DBD that correspond to deletions
or stop mutations result in CAIS. Missense mutations are associated
either with CAIS (currently 15 mutations affecting 11 different amino
acids in the AR gene mutation database) or PAIS (currently 18 point
mutations affecting 13 different amino acids in the AR gene mutation
database). Although analysis of the functional properties of the
resulting mutant receptors is often not available, previous mutagenesis
analysis of steroid receptors together with structural data resulting
from the crystallographic analysis of the ER and GR DBDs in complex
with their response elements provide explanations for the phenotypes
associated with some of these mutations. For instance, it is not
surprising that mutations C559Y, C576R, C576F, C579Y, C579F, C601F, and
C611Y are associated with CAIS, given the stringent requirement that is
well documented in the glucocorticoid receptor (45) for
Cys residues to coordinate zinc atoms in the two zinc fingers. Other
mutations affect residues that in the GR structure are involved in the
dimerization interface (A596T), in contacts with phosphate groups
(Y571C, R585K, R608K, R615H/P) or with base pairs (K580R, V581F, R585K)
[see the AR gene mutation database (21, 22) for
references]. On the other hand, mutation G577R affects an amino acid
that is not known to be involved in any of these functions. However,
G577 belongs to the DNA-binding helix of the AR and is part of the P
box, previously defined as crucial for discriminating between EREs and
GREs (17, 18, 19). The corresponding amino acid in the human
ER
, E203, makes specific contacts with the 2 bp that differ
between an ERE and an ARE (16). This suggested that
mutation G577R has the potential of affecting AR DNA-binding affinity
and/or selectivity.
We used a panel of 16 probes differing by the two central bases
of the recognition motif (PuGNNCA) to investigate whether
the G577R mutation allows binding to motifs not bound by the wild-type
receptor or modulates binding to the sites recognized by the wild-type
receptor. The wild-type AR bound specifically the four elements
corresponding to the PuGNACA palindromes (Figs. 4
and 5
).
This result is consistent with the crystallographic analysis of the
GR-GRE structure (15). Indeed, although G at position -5,
T at position +3, and G at position +2 are contacted by a lysine (K580
in AR), a valine (V581 in AR), and an arginine (R585 in AR) in the DNA
recognition helix, respectively, no contact involving the bases at
position -4 or +4 was observed. The G577R mutant did not bind to
response elements in the panel of 16 probes that were not recognized by
the wild-type receptor. The possibility that mutant ARs may bind to ERE
had been previously suggested for mutations associated with breast
cancer (46); however, no evidence of such alteration in
DNA-binding selectivity has been found to date. The G577R mutant did
not bind the ERE in vitro, and a reporter vector containing
multimerized EREs was not transactivated in transient transfections.
This result is consistent with previous studies using derivatives of a
GR mutant containing the ER P box and therefore capable of binding to
an ERE. Although it was found that not only Glu but also other amino
acids (Trp, Tyr, Phe, Asn, and His) at the first position of the P box
can retain binding to an ERE, replacement of Glu by Arg considerably
weakened binding to an ERE (47). Similarly, we observed
that although E203 in the ER could be replaced by other amino acids
such as Asn and His without abrogating binding or transactivation of an
ERE, replacement of Glu by Arg in the ER generated a receptor that did
not bind the ERE or any of the 16 PuGNNCA palindromes
(Rouault, E., D. Ngugen, and S. Mader, unpublished
data). Thus, Arg at this position in the P box does not seem
compatible with DNA binding in the context of the ER. However, we
observed here that introducing Arg at position 577 of the AR still
allowed binding to three of the four PuGNACA palindromes
recognized by the wild-type receptor. The fact that the mutant receptor
only bound to elements recognized by the wild-type receptor suggests
that Val 581 is still the main determinant for discrimination between
the two central base pairs of the motif, although its function is
modulated by the presence of Arg at position 577.
We observed that the G577R mutation resulted in a general weakening of
the interactions between AR and the PuGNACA palindromes.
Although no structure is available for the AR DBDs complexed to its
response element, the conservation of the DNA recognition helix with
that of GR (12 of 13 amino acids are identical, with one Val-to-Ala
substitution) suggests a very similar mode of recognition of the
response element motifs. Replacing Gly by Arg in the GR P box would
result in a steric clash at the level of the Arg Cß with the methyl
group of the T at position +3 in the TGTTCPy motif (Fig. 9
). Any amino acid other than Gly would
lead to a similar incompatibility. This is consistent with the
observation that replacement of the first amino acid in the GR P box
(Gly) by the corresponding amino acid in the ER (Glu) repressed
transactivation from a glucocorticoid-responsive promoter
(48), although this effect was not seen by others
(19). The crystal structure of a noncognate complex
between a GR mutant carrying both the ER P box and the TR D box
(E/TRGR) and a consensus GRE is also available (49).
Superimposition of wild-type GR and the E/TRGR structures indicated a
shift in the position of the DNA recognition helix of more than 2 Å
(Fig. 9
), resulting in a structure devoid of steric conflict between
the Cß atom at the first position of the P box (Glu in the E/TRGR
structure) and the thymine at position +3. The position of the mutant
GR DBD allowed the presence of five additional fixed water molecules in
the protein/DNA interface, imposing a potential entropic burden on the
stability of the complex and leading to an estimated 10-fold
reduction in the stability of the complex (49). The E/TRGR
receptor does not contain a Val but an Ala in its P box, preventing
contacts with T at position +3. However, replacement of Ala by Val in
that structure followed by energy minimization indicates that this van
der Waals contact was possible within that structure, increasing
stability with response elements containing T at position +3 (Fig. 10
). Thus, this structural model
accounts for both the selectivity for PuGNACA elements
retained by the mutant G577R receptor and the overall decrease in the
stability of these complexes.

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Figure 9. Scheme of Modeled Protein-DNA Contacts in the
Half-Complex between AR-G577R and the Consensus ARE
The last 5 bp of the TGTTCT half-site are shown. Note that the DNA is
drawn underwound for reasons of clarity. The position of the DNA
recognition helix in the GR-GRE complex (15 ) used as model
for the complex between AR wild-type and the consensus ARE is indicated
by an interrupted line. G indicates the position of the
Gly residue at the first position of the P box. The position of the
AR-G577R DNA recognition helix is based on the structure of the
noncognate complex between the GR mutant E/TRGR (49 ) and
is drawn in bold. The position of the mutant Arg (R)
residue and its contacts with the T at position +4 are indicated. Note
the steric hindrance that would result between Arg 577 Cß and the
methyl group of T+3 (drawn as a black circle) in the
absence of a shift of the recognition helix.
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Figure 10. Stereo View of the Contacts between the AR-G577R
DNA Recognition Helix and a PuGAACA Half-Site in a Model
Derived from the E/TRGR-GRE Complex
Modeling of the complex between the AR-G577R DBD and the
PuGAACA element was performed as described in
Materials and Methods. Arrows point to
the Ts at positions +3 and +4 and the G at position -5, as well as to
the Arg (R) and Val (V) at positions 577 and 581, respectively. Arg
interacts with the O4 of T+4, and with the O6 of G-5. Val makes a van
der Waals contact with the methyl group of T+3.
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Our results indicated in addition that the decrease in binding levels
was not proportional on all of the PuGNACA elements.
Indeed, TA elements were more compromised than CA and AA elements, and
GA elements were not bound under the condition of our gel shift assays,
with protein extracts containing either the mutant full length AR or
its isolated DBD. This correlated also with the absence of
transactivation of a reporter gene containing two copies of the GA
element by the mutant receptor. Therefore, we examined whether Arg at
position 577 of the AR could discriminate between different bases at
position +4, resulting in the observed preference for G and T (C and A
at position -4) and in potential conflict with C (G at position -4).
Modeling of the Arg chain into the E/TRGR structure indicated a
favorable influence of negatively charged chemical groups on the side
of the +4 base exposed in the major groove. Both T and G present only
negatively charged groups in the major groove. Arg can establish a
hydrogen bond with the O4 of T at position +4 and interacts also with
the O6 of G at position -5 (Figs. 9
and 10
). Interactions between the
Arg amino groups and O6 and N7 of G are also frequently found in
protein/DNA complexes (50, 51) and could also be modeled
in this complex with G at position +4 (data not shown). An A residue
would be less favorable because of the presence of both a positively
charged group (the N6 amine) and a negatively charged group (N7).
Finally, a C residue leads to unfavorable electrostatic interactions
because of the presence of only a positively charged group (the N4
amine). This model, therefore, is consistent with our experimental
results.
The effects of mutation G577R on DNA binding are likely to result in
different transcriptional activation properties of the mutant receptor
compared with the wild-type AR. Accordingly, synthetic reporter genes
containing GA palindromes were not transactivated by the mutant
receptor. To test the effect of mutation G577R on transactivation
mediated by natural AREs, we introduced two copies of response elements
found in the regulatory sequences of the androgen-responsive C1 and AR
genes upstream of a minimal promoter. The elements chosen all contained
one GA motif, in addition to other base replacements with respect to
the consensus motif. Note that our in vitro binding assays
indicated that GA motifs severely compromise binding by the mutant
receptor even when the other motif is the strong PuGAACA
motif (data not shown). The resulting reporter genes were all
stimulated by MB with the wild-type receptor, but the mutant
receptor was essentially inactive on these elements.
Although we observed a loss of binding efficiency on all
PuGNACA palindromes with the mutant AR, we did not observe
a reduction in transcriptional activity on reporter vectors containing
two copies of the AA, TA, or CA elements. This could be attributable to
cooperativity of DNA binding or transactivation on the two elements,
but the lack of transactivation observed with one response element in
front of the minimal promoter did not allow us to test the effect of
single mutated elements on transcription. Thus, it remains possible
that the differences in DNA binding observed in vitro may
not drastically affect transcriptional activity from
PuGNACA palindromes when N is A, C, or T. However, the
sequences of the few natural AREs characterized to date (Table 1
) often
deviate from the consensus palindromic sequence at one or several
positions, likely weakening interactions established by K580, V581, or
R585. The destabilizing effect of the G577R mutation may be more acute
in that context, depending on the nature of the substitutions. In
agreement with that prediction, transactivation from promoters that can
mediate androgen responsiveness, such as the MMTV promoter, and from
promoters of natural androgen target genes, such as the probasin and
PSA genes, all of which contain response elements diverging from the
consensus at several positions, was affected to various degrees by the
AR-G577R mutation. Note in particular that transcription from the
probasin promoter, which does not contain GA motifs, was reduced about
5-fold. Thus, the presence of the G577R mutation in the AR is likely to
affect the regulation of a large number of androgen-responsive genes
depending on the type of PuGNACA motif and the presence of
variations from this consensus motif in one or both repeats of the
motif. The finding that several synthetic promoters (minimal promoters
containing two PuGNACA palindromes) or natural promoters
(MMTV) are still well transactivated by the mutant receptor is
consistent with the phenotype of partial androgen insensitivity
associated with the mutation. Furthermore, our results suggest that the
wide range of phenotypes associated with PAIS may be explained not only
by the partial loss of AR function but also by the differential effect
of some mutations on specific subsets of androgen-responsive genes.
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MATERIALS AND METHODS
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Patient
The subject (patient no. 12694) was diagnosed at birth with
severe penile/scrotal hypospadias with bifid scrotum and retractable
testis that could not be brought all the way down. His karyotype was
46,XY, and his levels of T and LH were increased (345 ng/dl and 14.47
IU/liter, respectively, compared with average values of 150 ng/dl and 5
IU/liter in that age group). The patient was treated with T enanthate,
which resulted in a significant increase in penis size. These clinical
observations are indicative of PAIS. It is of note that a PAIS was
diagnosed in a maternal uncle of the patient, who was born with
undescended testicles, microphallus, and a vaginal opening but did not
undergo genetic evaluation.
Androgen-Binding Properties of the AR in Skin
Fibroblasts
Androgen-binding studies were performed on cultured genital skin
fibroblasts obtained from skin biopsies of the patient. The
androgen-binding properties of the AR were determined according to
standard techniques using tritiated hormones (52). The
maximum androgen-binding capacity and the apparent dissociation
constant of the AR were determined from Scatchard analysis using
[17
-methyl-3H]methyltrienolone (86 Ci/mmol),
DHT (110 Ci/mmol), and MB (85 Ci/mmol).
Dissociation kinetics were determined by incubating COS-1 cells
transiently transfected by electroporation (52) with
expression vectors for wild-type and mutant ARs (1 µg) in the
presence of 3 nM tritiated DHT and MB (NEN Life Science Products, Boston, MA) for 2 h. Cells were then
incubated with a 200-fold excess of nonlabeled hormones for different
times (30, 60, 90, or 120 min). Cells were harvested by trypsinization
and lysed in 0.5 M NaOH. Protein concentration of the cell
extract was quantitated by Lowry assay, and radioactivity was measured
by scintillation counting.
DNA Amplification and Sequencing of the AR from Skin
Fibroblasts
Synthetic oligonucleotides corresponding to the borders of exons
28 were used to amplify the coding portion of each exon in regions
CF of the AR (53). The resulting products were
sequenced, and the presence of the G577R mutation in exon 2 was
confirmed by an independent round of PCR amplification and
sequencing.
Plasmids
pSG5-AR was constructed by subcloning the AR cDNA from
pSVhAR-BHEX (49) into the pSG5 expression vector
(54). pSG5-AR-G577R was constructed by site-directed
mutagenesis of the AR cDNA using PCR amplification and by cloning a
HindIII-XhoI fragment containing the mutation
into pSVhAR-BHEX, followed by subcloning of a
KpnI-XhoI fragment into pSG5-AR. The entire
HindIII-XhoI fragment was sequenced to exclude
artifactual mutations that may have been introduced during PCR.
The pGRE5-TATA-CAT and pMMTV-hGH reporter vectors have been
described previously (28, 52). pPB-hGH was constructed by
transferring the androgen-responsive region in the probasin promoter
from p(-285)PB-CAT (40) in the p
GH vector
(Nichols Institute Diagnostics, San Juan Capistrano,
CA). pPSA-hGH was constructed by transferring the
androgen-responsive region in the PSA promoter from pPA2-CAT
(55) in the p
GH vector.
The bacterial expression vectors pET3-AR[DBD] and pET3-AR[DBD]G577R
were constructed by PCR amplification of the cDNA fragment
corresponding to amino acids 550656 and subcloning between the
KpnI and XhoI sites of the pET31 vector
(20). The limits of the AR DBD were chosen to match those
of the previously described ER DBD (20).
The pRE2-CAT reporter vectors were prepared by replacing the three EREs
in pERE3-TATA-CAT (56) by double-stranded oligonucleotides
containing two response elements flanked by BamHI and
BglII sites. The sequence of the top strand oligonucleotide
was
5'-GATCCAAATGTCAGNNCACAGTGNNCTATCTAATAAAGTAGCTAGNNCACAGTGNNC-TAAGA-3'.
The ARE C1 and ARE1 human AR reporter vectors were constructed as
described for the pRE2-CAT reporter vectors, except that
double-stranded oligonucleotides containing two copies of the C1 ARE or
the human AR ARE1 flanked by BamHI and BglII
sites were used.
Escherichia coli Expression of the AR DBD
The wild-type and G577R AR DBDs were expressed by transformation
of E. coli BL21 DE3 cells with pET3-AR[DBD] or
pET3-AR[DBD]G577R bacterial expression vectors and induction of
exponentially growing cultures with isopropylthio-ß-galactoside
(IPTG; 0.5 mM final concentration) for 1 h.
Whole bacterial extracts were prepared by sonication in extraction
buffer (25 mM Tris-HCl, pH 7.4; 0.1
mM EDTA, pH 8.0; 400 mM
NaCl; 10% glycerol; 1 mM dithiothreitol; 1
mM phenylmethylsulfonyl fluoride; and protease
inhibitors) and centrifugation (10,000 x g, 30 min).
Aliquots (1 ml) of each culture reaction were taken before IPTG
induction, centrifuged, and resuspended in M9 medium containing each
amino acid except Met and Cys (0.01% wt/vol each). Rifampicin was
added (200 µg/ml final concentration) to inhibit bacterial RNA
polymerase, and expression of the T7 polymerase was induced with IPTG
(0.5 mM final concentration) for 30 min.
[35S]Met (10 µCi/ml) was then added, and
cells were further incubated at 37 C for 5 min. Bacteria were then
harvested by centrifugation, resuspended in Laemmli buffer, and boiled
for 5 min. Labeled proteins were separated by electrophoresis on a 12%
polyacrylamide-SDS gel and revealed by fluorography.
Transient Transfection, CAT, and GH Assays
HeLa and COS-1 cells were maintained in DMEM supplemented with
5% FBS. For transient transfection, cells were switched to medium
containing 5% FBS pretreated with activated charcoal to remove traces
of hormones. Transient transfections were performed using the
calcium-phosphate coprecipitation method as described previously
(57). Essentially, HeLa cells were transfected with
varying concentrations of expression vectors for wild-type AR or
AR-G577R with pCMV-ßGal (2 µg), pRE2-TATA-CAT (2 µg), and
pBluescribe-M13+ (to 15 µg total). Precipitates were washed after
16 h, and MB was added (final concentration, 2 nM).
Cells were harvested after 24 h, and extracts were prepared by
freeze thawing in Tris-HCl, pH 8.0 (0.25 M). CAT assays
were performed after standardization with ß-galactosidase activity as
described previously (56).
For GH assays, COS-1 cells were transfected with varying concentrations
of expression vectors for wild-type AR or AR-G577R with pRSV-ßGal (2
µg), pPB-hGH, pMMTV-hGH, or pPSA-hGH (3 µg), and pBluescribe-M13+
(to 15 µg total). Precipitates were washed after 16 h, and MB
was added (final concentration, 2 nM). Cell supernatants
were harvested after 24 h and assayed for human GH concentration
using an hGH-ELISA kit according to the manufacturers instructions
(Medicorp, Montréal, Canada). Cells were also harvested,
and ß-galactosidase activity was measured to control for transfection
efficiency.
Immunoblot Analyses
Protein concentrations of whole-cell extracts from HeLa or COS-1
cells transiently transfected with 15 µg of expression vectors for
the wild-type or mutant AR were estimated using a Bradford assay.
Protein extracts (4 µg) in Laemmli buffer were heat denatured, loaded
onto an 8% polyacrylamide-SDS gel, and electrophoresed at 125 V. After
transfer to a polyvinylidene difluoride membrane and incubation of the
membranes in blocking solution (1x Tris-buffered saline, 0.05% Tween
20, 3% BSA) for 20 min, receptor bands were revealed using the F39.4.1
mouse monoclonal antibody at a 1:10,000 dilution (58), a
horseradish peroxidase-coupled secondary antibody, and the Renaissance
enhanced chemiluminescence detection kit (NEN Life Science Products).
Gel Shift Assays
Whole bacterial extracts containing the wild-type or mutant AR
DBD were diluted to 80 mM NaCl and incubated on ice with 2
µg of poly(dI·dC) for 15 min. After the addition of
32P-labeled, double-stranded oligonucleotide
probes (50,000 cpm/sample), reactions were incubated at 25 C for 15
min, terminated by the addition of 2 µl of dye mix (0.1% bromophenol
blue, 60% glycerol), and loaded onto a 7% polyacrylamide gel in
0.25x TBE (22.5 mM Tris-HCl, 22.5 mM boric
acid, 0.5 mM EDTA). Complexes were separated by
electrophoresis, and gels were dried and autoradiographed. For
quantitation of complexes, both the bands corresponding to the complex
and the free probes were excised and counted by scintillation counting,
and the percentage of shifted probe was calculated.
Gel shift assays with whole-cell extracts from HeLa cells transiently
transfected with AR expression vectors (15 µg/9-cm dish) were
performed essentially as described above except that extracts were
diluted to a final KCl concentration of 125 mM and loaded
onto a 5% polyacrylamide gel.
Modeling of the Interaction between the AR-G577R DBD and the
PuGAACA Element
Modeling of the complex between the AR-G577R DBD and the
PuGAACA element was performed in the Insight II-97
environment using the refined structure of the mutant GR DBD E/TRGR
(1LAT.PDB, 49) by replacement of P box amino acids Glu by Arg and Ala
by Val, followed by manual adjustments and energy minimization in the
AMBER force field to avoid steric clashes (software from
Accelrys, Inc.).

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Figure 3. Both Wild-Type AR and AR-G577R Transactivate
GRE5-TATA-CAT but Not ERE3-TATA-CAT
A and B, Variable amounts (0.25, 1.25, or 6.25 µg) of expression
vectors pSG5-ARwt (A) or pSG5-AR-G577R (B) were transiently transfected
in HeLa cells together with pGRE5-CAT reporter vector (2 µg) and the
internal control vector pCMV-ßGal (2 µg) using the calcium
phosphate method. After removing the precipitate, the cells were
incubated in the presence or absence of MB (2 nM) for
24 h. CAT activity was quantitated in the corresponding whole-cell
extracts, and the ratio of the transcriptional activity in the presence
vs. the absence of hormone is indicated. C and D, HeLa
cells were transiently transfected as described above except that the
pERE3-CAT vector was used instead of pGRE5-CAT. Results are the average
of three experiments.
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ACKNOWLEDGMENTS
|
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Our thanks to Dr. Robert J. Matusik (Vanderbilt University
Medical Center, Nashville, TN) for providing us with the probasin and
PSA promoters. We are grateful to Sunita de Tourreil and Rose
Lumbroso for technical assistance and to Dr. Lenore K. Beitel (Lady
Davis Institute) for background information and review of the
manuscript.
 |
FOOTNOTES
|
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This work was supported by a grant from the Natural Science and
Engineering Research Council of Canada to S.M. S.V.S. received an
operating grant from the Canadian Institute of Health Research. S.V.S.
and S.M. are chercheurs-boursiers of the Fonds de Recherche en
Santé du Québec.
Abbreviations: ARE, Androgen response element; CAIS,
complete androgen insensitivity syndrome; CAT, chloramphenicol
acetyl transferase; DBD, DNA-binding domain; DHT,
[1,2,4,5,6,7-3H]5
-dihydrotestosterone; ERE,
estrogen response element; GRE, glucocorticoid response element;
IPTG, isopropylthio-ß-galactoside; MB, mibolerone; MMTV,
mouse mammary tumor virus; PAIS, partial androgen insensitivity
syndrome; PSA, prostate-specific antigen; TAT, tyrosine amine
transferase.
Received for publication October 17, 2000.
Accepted for publication June 18, 2001.
 |
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