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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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.

 
Scatchard analysis was then performed by incubating genital skin fibroblasts with [1,2,4,5,6,7-3H]5{alpha}-dihydrotestosterone (DHT; 110 Ci/mmol) or two synthetic nonmetabolizable androgens, [17{alpha}-methyl-3H]mibolerone (MB; 85 Ci/mmol) and [17{alpha}-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. 2AGo) 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 (6–8 and 2–4 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.

 
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 1Go, 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.


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Table 1. Consensus and Natural AREs

 
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 1Go). 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 550–656) 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. 2BGo). The wild-type AR DBD bound specifically to four probes, corresponding to the consensus PuGNACA (Fig. 4AGo, 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. 4BGo). 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. 4Go, C and D). Binding to the GA probe by the mutant DBD was undetectable (Fig. 4Go, B and D). Note that the GA and CA probes were bound to similar extents by the wild-type DBD (Fig. 4Go, A and C) but that the mutant DBD only bound the latter probe (Fig. 4Go, 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. 4Go, 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.

 
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. 5CGo). 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. 5AGo, 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. 5BGo, lanes 1, 5, and 13) but not with the probe containing GA motifs (Fig. 5BGo, 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.

 
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 1Go, 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. 6AGo). 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. 6BGo). However, no transactivation was observed using PuGGACA motifs with the mutant receptor (Fig. 6BGo).



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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.

 
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 1Go; 32) mediated transactivation by the wild-type receptor (Fig. 7Go, C and E) but not detectably by the mutant AR (Fig. 7Go, D and F), whereas both receptors transactivated PuGAACA palindromes to similar levels under the same conditions (Fig. 7Go, 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.

 
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 1Go), 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. 8Go, 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 1Go). With this promoter, ~5-fold reduction in transactivation levels was observed with the mutant receptor, down to ~3-fold activation (Fig. 8Go, 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. 8Go, 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 manufacturer’s 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}, 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. 4Go and 5Go). 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. 9Go). 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. 9Go), 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. 10Go). 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.

 
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. 9Go and 10Go). 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 1Go) 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}-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 2–8 were used to amplify the coding portion of each exon in regions C–F 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{phi}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{phi}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 550–656 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 manufacturer’s 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.

 

    ACKNOWLEDGMENTS
 
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
 
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{alpha}-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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