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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-0057
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 1 507-515
Copyright © 2005 by The Endocrine Society

Functional Analysis of a Novel Androgen Receptor Mutation, Q902K, in an Individual with Partial Androgen Insensitivity

Arzu Umar1, Cor A. Berrevoets1, N. Mai Van, Marije van Leeuwen, Michael Verbiest, Wim J. Kleijer, Dennis Dooijes, J. Anton Grootegoed, Stenvert L. S. Drop and Albert O. Brinkmann

Departments of Reproduction and Development (A.U., C.A.B., N.M.V., M.v.L., M.V., J.A.G., A.O.B.) and Clinical Genetics (W.J.K., D.D.), Erasmus MC, University Medical Center Rotterdam, 3000 DR Rotterdam, The Netherlands; and Division of Endocrinology (S.L.S.D.), Sophia Children’s Hospital, 3000 CB Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Dr. Albert O. Brinkmann, Department of Reproduction and Development, Erasmus MC, University Medical Center Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: a.brinkmann{at}erasmusmc.nl.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Androgen insensitivity syndrome (AIS) is caused by defects in the androgen receptor (AR) that render the AR partially or completely inactive. As a result, embryonic sex differentiation is impaired. Here, we describe a novel mutation in the AR found in a patient with partial AIS. The mutation results in a substitution of a glutamine (Q) by a lysine (K) residue at position 902, Q902K. The AR Q902K mutation was investigated in vitro with respect to its functional properties. The equilibrium dissociation constants (Kds) of AR Q902K in the presence of either the synthetic androgen R1881 or the natural ligand DHT were slightly elevated. The R1881 dissociation rate (t1/2) was increased 3-fold for AR Q902K compared with wild type. Transcriptional activity was decreased to 85% of wild type, and the dose-response curve revealed that the sensitivity to hormone was decreased due to the mutation. Furthermore, the 114-kDa androgen-induced phosphorylated AR protein band was not detectable in genital skin fibroblasts. However, it could be detected in transfected CHO cells expressing the mutant receptor in the presence of 10 and 100 nM R1881. Functional interaction assays and a GST pull-down assay showed that the interaction between the NH2 and COOH terminus of AR Q902K was reduced to 50% of wild type. Furthermore, the transactivation by the coactivator TIF2 (transcriptional intermediary factor 2) was decreased 2- to 3-fold. The half-maximal response in both assays was shifted to a higher hormone concentration compared with wild type. These results indicate that residue Q902 is involved in TIF2 and NH2/COOH interaction and that the Q to K mutation results in a mild impairment of AR function, which can explain the partial AIS phenotype of the patient.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
ANDROGEN INSENSITIVITY SYNDROME (AIS) is a disorder that results from mutations in the X-linked androgen receptor (AR) gene (1, 2). Several hundred different mutations in the AR gene have been reported (www. mcgill.ca/androgendb), which result in a wide spectrum of clinical phenotypes (1, 3, 4). Phenotypes of 46,XY individuals with AIS can vary from completely female, complete AIS (CAIS), to male and all possible stages in between, the latter being referred to as partial AIS (PAIS).

The AR belongs to the superfamily of nuclear receptors that is well characterized and conserved in structure. These receptors function as ligand-inducible transcription factors (5, 6, 7, 8). Like other steroid hormone receptors belonging to the same family, the AR is composed of distinct domains (9). The variable NH2-terminal domain (NTD) is mainly involved in transcription activation through the activation function-1 (AF-1) region. The activation of AF-1 is ligand dependent. The COOH-terminal region harbors the moderately conserved ligand binding domain (LBD), which is involved in ligand-dependent transcription activation through AF-2, and functional interaction with nuclear receptor coactivators and corepressors (10, 11, 12).

Crystal structure of AR LBD revealed that the LBD is constituted of nine {alpha}-helices, two 310 helices (together termed helix 1–12), and four short ß-strands associated in two antiparallel ß-sheets, all arranged as a helical sandwich (11, 13). An AF-2 activation domain (AD) core region, conserved throughout the nuclear receptor family, was identified in helix 12, at the C-terminal end of the LBD between residues 893 and 900 (14). Upon ligand binding, helix 12 repositions, providing an interaction surface that is suitable for coactivator interaction and thereby generating transcriptional activity of AF-2 (11, 15, 16, 17). Various mutations introduced in the AF-2 AD core decrease transcription activation but not necessarily ligand binding (16, 17, 18). Coactivators like TIF2 and SRC1 have been shown to functionally interact with AF-2 AD core via their conserved LXXLL leucine motifs (16, 17, 18, 19) and enhance AR transactivation. Furthermore, the NTD also functionally interacts with AF-2 (the so-called N/C interaction) in a ligand-dependent manner, and mutational analysis showed that AF-2 AD core plays an important role in this interaction (17, 20, 21, 22). The N/C interaction with AF-2 occurs via an FXXLF motif present in the AR N terminus (23, 24). Several mutations found in the AR of AIS patients appear to affect either coactivator binding or N/C interaction or both; therefore, such functional analyses are very useful in determining AR function after mutation and provide detailed information on the mechanism of AR transcription activation.

Herein, we report a novel AR mutation resulting in a Q to K substitution at position 902 in a patient with PAIS. We have performed functional analyses on the AR to determine the effect of the Q902K mutation on TIF2-enhanced transcription activation and N/C interaction to explain the phenotype and potential functional activity.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Clinical data

The patient was referred to the clinic at the age of 2 yr because of hypospadias. He is the first child of unrelated parents born after an uneventful term pregnancy. The 46,XY boy had proximal shaft hypospadias and a bifid scrotum. Because of a positive family history of genital malformations (uncle and nephew of the mother were known with hypospadias, gynecomastia, and infertility), the AR gene was screened for a mutation. Single-strand conformation polymorphism and DNA sequencing analysis identified a point mutation in exon 8 of the AR gene (CAA -> AAA) at amino acid residue 902 (numbering according to http://www.mcgill.ca/androgendb/) (25) leading to an amino acid substitution of glutamine (Q) to lysine (K). This Q902K mutation was also identified in the mother, grandmother, and great-grandmother of the patient (Fig. 1Go). The boy’s uncle and great-uncle have a reported positive history of genital malformation. At the age of 3 yr, the boy underwent a chordectomy and hypospadias repair. During this procedure, a genital skin biopsy was obtained for genital skin fibroblast (GSF) culture. Informed consent was given by the parents before the chordectomy and the hypospadias correction.



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  		FIG. 1. Pedigree of family with AR Q902K mutation. Square symbol, male; circle, female. The arrow indicates the index patient described in this study.

 
Scatchard analysis

GSFs were cultured in minimal essential medium containing 1% nonessential amino acids (Invitrogen, Carlsbad, CA), 10% fetal calf serum (FCS; Hyclone, Logan, UT), 100 IU/ml penicillin, and 100 µg/ml streptomycin (BioWhittaker, Vervier, Belgium). For ligand binding characteristics, a whole-cell binding assay was performed as previously described (26). Briefly, GSFs were cultured to confluency, washed once with PBS, and subsequently placed overnight on medium without serum. The cells were than incubated for 1 h at 37 C with increasing concentrations of radioactive ligand (0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 nM 3H-R1881 (NEN Life Science Products, Boston, MA; or 0.03, 0.1, 0.3, 1.0, 3.0, and 10 nM 3H-DHT, Amersham Biosciences, Piscataway, NJ) in the absence or presence of a 200-fold nonradioactive R1881/DHT. Cells were then placed on ice, washed four times with ice-cold PBS, and subsequently lysed in 0.5 M NaOH. 3H-activity in the lysate was measured in a scintillation counter. Scatchard analysis was carried out to determine the equilibrium dissociation constant (Kd) using the Kell software package (Radlig, Biosoft, Ferguson, MO). Hormone dissociation rates were determined by incubating GSFs with 3 nM 3H-R1881 for 2 h at 37 C, followed by a chase with 6 µM nonradioactive R1881 for different time periods (0, 15, 30, 60, 90, and 150 min) (27). Cells were then washed, lysed, and radioactivity was determined as described above. Data are represented as the natural logarithmic ratio (ln) of radioactivity bound (B) and bound at t = 0 (B0).

Site-directed mutagenesis and construction of AR expression vectors

The Q902K mutation was introduced into the AR using site-directed mutagenesis. The human wild-type AR cDNA expression plasmid pAR0 (28) was used to generate pARQ902K in two separate PCR amplification steps, using Pfu polymerase. Sense and antisense Q902K primers containing the mutation, which is depicted in lowercase lettering, were combined with the sense EQ1 and antisense EQ4 primers previously described by Berrevoets et al. (17). Q902K sense (5'-ATCTCTGTGaAAGTGCCCA-3') was combined with EQ4 antisense (5'-CAAGGGGCTTCATGATGTCC-3'), and Q902K antisense (5'-TGGGCACTTtCACAGAGAT-3') was combined with EQ1 sense (5'-ACAGCCAGTGTGTCCGAATG-3') primer. The generated PCR products were used as a template for the second PCR using the EQ1 and EQ4 as sense and antisense primers, respectively. The resulting PCR fragment was directly ligated into the pCR-Blunt II-TOPO vector using the Zero Blunt TOPO Cloning kit (Invitrogen). The mutated AR fragment was digested with EcoR1 and exchanged for the corresponding wild-type fragment in pAR0.

The GST-AR.LBD construct was described previously (24). The Q902K mutation was introduced into this construct using QuikChange Site-Directed Mutagenesis (Stratagene, La Jolla, CA) using the oligo’s 5'-GAGATCATCTCTGTGaAAGTGCCCAAGATC-3' and 5'-GATCTTGGGACTTtCACAGAGATGATCTC-3'.

The AR.N1 and AR.C constructs that were used for the N/C interaction and TIF2 activation studies were described previously (17). The Q902K mutation was introduced into the AR.C construct via the EcoR1 restriction fragment of pARQ902K.

Luciferase (LUC) assay

For transcription activation studies, CHO cells were cultured in DMEM/F12 medium, supplemented with 5% dextran-coated charcoal-treated (Invitrogen). For all transcription activation studies, CHO cells were plated in 24-well plates (Nalge Nunc International, Naperville, IL) at a density of 2 x 104 cells per well. After 24 h, cells were transfected using FuGENE reagent (Roche Diagnostics, Basel, Switzerland), according to the instructions of the manufacturer, at a DNA:FuGENE ratio of 1:2. The DNA mixture was composed of 50 ng/well mouse mammary tumor virus (MMTV)-LUC (LUC) reporter plasmid, increasing concentrations of wild-type AR or AR Q902K (0.3–30 ng/well), and carrier plasmid pTZ19 to adjust to a total amount of 250 ng DNA per well. Five hours after transfection, 1 nM synthetic androgen R1881 or vehicle (0.1% ethanol) was added to the cells, or in the case of the dose-response curves, a range of 1 pM to 100 nM R1881 was added. After overnight incubation, cells were lysed in 50 µl lysis buffer [25 mM Tris-phosphate (pH 7.8), 15% glycerol, 1% Triton X-100, and 1 mM dithiothreitol], and 25 µl lysate was used to measure LUC activity using Steady-Glo LUC substrate (Promega, Madison, WI). The data shown are the mean of three independent experiments (average ± SEM).

N/C interaction assay, TIF2 activation assay

The functional N/C interaction assay and TIF2 activation assay were performed in essentially the same way as the trans-activation assay described above, except for the constructs that were used. For N/C interaction, 100 ng/well AR.N1 in combination with increasing concentrations of AR.C or AR.C-Q902K (0.3–30 ng/well) were used. For TIF2 activation, 100 ng/well TIF2 in combination with increasing concentrations of AR.C or AR.C-Q902K (0.3–30 ng/well) were used, in both cases together with 50 ng/well MMTV-LUC reporter and pTZ carrier to adjust to 250 ng/well.

Western blot analysis

For AR Western blot analysis, GSFs containing either the wild-type AR or the Q902K AR were cultured in the presence of FCS for 7 days, as described above. When grown to confluency, medium was replaced by medium containing 10% dextran-coated charcoal-stripped-FCS in the presence of increasing concentrations of R1881 (0.1–100 nM) or vehicle (0.1% ethanol) for 24 h. Additionally, CHO cells were seeded in 80-cm2 culture flasks at a density of 8 x 105 cells per well. The next day, cells were transfected with equivalent amounts of pAR0 or pARQ902K DNA, compared with the LUC assay. AR (120 ng) plasmid and 9880 ng pTZ19 was mixed with 20 µl FuGENE reagent and added to the cells. After 5 h, increasing concentrations of R1881 (0.1–100 nM) or vehicle (0.1% ethanol) were added to the culture medium. The next day, both CHO cells and GSF were washed with PBS, collected in ice-cold PBS, and centrifuged for 5 min at 800 x g. The cell pellet was resuspended in 100 µl ice-cold RIPA buffer [40 mM Tris-HCl (pH 7.4), 5 mM EDTA, 10% glycerol, 10 nM sodium phosphate, 10 mM sodium molybdate, 50 mM NaF, 0.5 mM sodium orthovanadate, 10 mM dithiothreitol, 1% Triton X-100, 0.08% SDS, and 0.5% desoxycholate] containing Complete protease inhibitors (Roche Diagnostics) and centrifuged for 10 min at 400,000 x g. Protein concentration was determined using the RCDC Protein Assay (Bio-Rad, Hercules, CA). From the GSF cell lysate, 50 µg protein was used to load onto a 7% SDS-polyacrylamide gel. The CHO cell lysate was immunoprecipitated with the anti-AR monoclonal antibody F39.4.1 (29) and subjected to SDS-PAGE. Proteins were separated and blotted to nitrocellulose membrane (Schleicher & Schuell, Keene, NH). Western immunoblotting was performed using polyclonal antibody SP197 (30), and proteins were visualized by Western Lightning chemiluminescence detection (Perkin-Elmer, Boston, MA).

Pull-down assay

In vitro interaction assays (pull-down assays) were performed as described previously (24). In short, CHO cells were transfected with AR.N1 and either GST-AR.LBD-wt or GST-AR.LBD-Q902K. After overnight incubation with 100 nM R1881 or vehicle, cells were lysed and rotated for 5 h at 4 C with glutathione-agarose beads. Next, agarose beads were washed, subsequently boiled in Laemmli sample buffer, and subjected to SDS-PAGE. After Western blotting, visualization of input and precipitated AR.N1 was carried out as described above. Input of GST-AR.LBD was determined using a monoclonal anti-GST antibody (Zymed Laboratories, San Francisco, CA).

Protein structure

The three-dimensional (3D) crystal structure of the AR ligand binding pocket (LBP) complexed with R1881 was obtained from the National Center for Biotechnology Information (NCBI) structure data bank (accession no. 1E3G) deposited in the data bank by Matias et al. 2000 (13). The stereo diagram showing the LBP and selected residues that were subject to mutation was created using the DeepView/Swiss-PDB Viewer 3.7 program.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
The AR Q902K mutation displays decreased transcription activation

The Q902K mutation is located in the LBD of the AR, within helix 12 and next to the reported AF-2 AD core domain (13, 14). From its position in the LBD, it can be predicted that residue Q902 is not part of the LBP but may play a role in coactivator mediated activation of transcription and in the interaction of the LBD with the NTD. Therefore, functional studies were performed to address this question.

First of all, it was determined whether the mutated AR is functionally expressed in GSFs obtained from the index patient. The mutated AR Q902K protein was detectable in GSF cells, although the expression level may be somewhat lower compared with the expression of wild-type AR protein in normal GSF cells (Fig. 2AGo, lanes 1 and 2). In the presence of 1 nM R1881, however, the expression levels are rather similar (Fig. 2AGo, lanes 3 and 4). It was previously described that the AR protein shows a hormone-induced phosphorylated isoform of 114 kDa (31). However, although GSFs expressing wild-type AR respond to hormone by expressing the hyperphosphorylated 114-kDa protein band (Fig. 2AGo, lanes 3, 5, and 7), Q902K AR did not show the 114-kDa band upon addition of 1, 10, or 100 nM R1881 (Fig. 2AGo, lanes 4, 6, and 8). Thus, although the mutated residue is not a direct target for phosphorylation, it seems that the substitution by a lysine residue at position 902 affects the hormone-induced phosphorylation status of the AR.



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  		FIG. 2. Expression and ligand binding properties of AR Q902K in GSFs. A, Protein expression pattern of wild-type AR (wt) and AR Q902K (m) in GSFs in the absence or presence of 1, 10, and 100 nM R1881. Note the absence of the 114-kDa protein band in the mutated AR. B and C, Scatchard analysis of whole cell binding data in the presence of 3H-R1881 (B) or 3H-DHT (C) obtained from GSFs harboring wild-type AR or AR Q902K.

 
It was then tested whether the Q902K mutation in the AR had an effect on ligand binding. Scatchard plot analysis performed on GSFs in the presence of 3H-R1881 revealed that AR Q902K had a Kd value of 0.26 nM vs. a Kd value of 0.075 nM of wild-type AR (normal range is 0.03–0.13 nM), suggesting that hormone binding was slightly but not severely impaired (Fig. 2BGo). In the presence of 3H-DHT, a similar increase in the Kd value was observed, 0.37 vs. 0.14 nM for wild-type AR (Fig. 2CGo). Thus, also in the presence of natural ligand, no severe defect in hormone binding could be observed. In addition, the hormone dissociation rate of 3H-R1881-bound AR Q902K increased 3-fold, from t1/2= 63 min for wild-type AR to t1/2= 22 min for the mutant (Fig. 3Go). This suggests that the slight increase in Kd value of the Q902K mutant is mainly caused by a faster dissociation of the ligand.



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  		FIG. 3. Hormone dissociation rates of AR and AR Q902K in GSFs. GSFs were incubated with 3 nM 3H-R1881 for 2 h at 37 C, followed by a chase with 6 µM nonradioactive R1881 for different time periods (0, 15, 30, 60, 90, and 150 min). Cells were washed, lysed, and 3H-activity in the lysate was measured in a scintillation counter. From the data, the ratio between the radioactivity bound (B) and bound at t = 0 (B0) was calculated. t1/2 values were determined at a natural logarithmic ratio (ln) of B/B0 value of –0.69, so when B/B0 equals 0.5.

 
To determine whether the Q902K mutation affects the transcription activation potential of the AR, the mutation was created in a mammalian expression vector, AR Q902K. Transcription activation was determined in a LUC assay using the MMTV-LUC reporter plasmid. Transfections with increasing amounts (0.3, 1, 3, 10, and 30 ng/well) of wild-type or mutant AR showed that maximum transcription activation was reached with 3 ng/well DNA (Fig. 4AGo). Higher concentrations of DNA resulted in squelching of the signal. Wild-type AR showed a 27-fold induction of activity, whereas the mutated AR induced activity to maximally 23-fold, which is 85% of the wild-type value (Fig. 4AGo). This difference in transactivation was small but significant (P < 0.05). Furthermore, the dose-response curve showed that the half-maximum response of AR Q902K to hormone had shifted to a 5-fold higher hormone concentration compared with wild type, implicating a decreased sensitivity for R1881 (Fig. 4BGo). Furthermore, both receptors showed a maximum activity at 1 nM R1881, with the difference that the mutant only reached 75% of wild-type activity. Thus, the Q to K mutation mildly affects AR transcription activation potential.



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  		FIG. 4. Transcription activation assay in CHO cells. A, Increasing amounts of wild-type AR (wt AR) and mutated AR (AR Q902K) were transfected into CHO cells. LUC activity measured for wild-type AR at 3 ng/well was set at 100%, and all the other points were calculated relative to that. The activities measured in the hormone-treated cells are displayed, together with the fold induction, which represents the ratio between the activities measured in the absence and presence of hormone. Statistical significance was calculated using a Student’s t test. B, Dose-response curves of wild-type AR and AR Q902K in the presence of increasing amounts of R1881. The activity of wild-type AR measured at 1 nM R1881 was set at 100%, and the other points were calculated relative to that. C, Protein expression of wild-type AR and AR Q902K in CHO cells. Equivalent amounts of DNA, compared with the LUC assay, were transfected. Note the presence of the 114-kDa protein band in AR Q902K only when stimulated with 10 or 100 nM R1881.

 
One explanation for the observed decrease in transcription activation potential could be a reduced protein expression or stability of AR Q902K during the LUC assay. We have tested this possibility by transfecting equivalent amounts of DNA into CHO cells, compared with the LUC assay, and monitoring AR protein expression. We did not observe a reduced protein expression of AR Q902K in CHO cells in the absence of R1881 (Fig. 4CGo, lanes 1 and 2), ruling out any severe change in protein stability in the absence of ligand. However, in the presence of ligand (particularly with 100 nM R1881), protein stability seems slightly reduced, which may be explained by the observed increased ligand dissociation rate resulting in reduced receptor occupancy. In addition, it appears that the phosphorylation pattern is slightly different, in comparison with the expression in GSFs (Fig. 2AGo), whereas 1 nM R1881 only partially stimulated the upshift to the 114-kDa phosphoprotein band, 10 and 100 nM R1881 obviously restored the expression pattern (Fig. 4CGo), although this restoration did not result in higher transcription activation in CHO cells (Fig. 4BGo). Thus, the Q902K mutation in the AR results in a 15–25% reduced hormone-induced maximal transcriptional activity, a 5-fold decrease in hormone sensitivity, and a defective ligand-induced phosphorylation at 1 nM R1881.

Decreased N/C interaction due to the Q902K mutation

It was previously determined that the NTD and LBD of the AR interact in a ligand-dependent manner (N/C interaction) (20) and that the AF-2 AD core domain is important for this functional interaction (17). Because the Q902K mutation lies in close proximity of the AF-2 AD core region, we have performed a functional N/C interaction assay to study in more detail the effect of the Q902K mutation on transcription activation. For the N/C interaction, AR.N1, AR.C, and AR.C-Q902K constructs were used (Fig. 5AGo). In the presence of 1 nM R1881, AR.N1 and AR.C coexpression resulted in transactivation of the MMTV-LUC promoter with a 50-fold induction. Maximum activity was reached at 10 ng DNA/well. AR.C-Q902K showed a 50% reduced functional interaction with AR.N at 10 ng DNA/well (Fig. 5BGo). These results were further supported by an in vitro GST-pull-down interaction study. AR.N1 and GST-AR.LBD-wt or GST-AR.LBD-Q902K constructs were transfected into CHO cells for protein production. The interaction was performed in vitro using glutathione agarose and visualized by Western blot (Fig. 5DGo). The pull-down assay clearly shows the reduced interaction of GST-AR.LBD-Q902K with AR.N1 in the presence of R1881.



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  		FIG. 5. Effect of the Q902K mutation on functional and in vitro N/C interaction. A, Constructs used for the functional N/C interactions. AR.C, N-terminally truncated AR; AR.C-Q902K, N-terminally truncated AR with Q902K mutation; AR.N1, C-terminally truncated AR. B, Functional N/C interaction as measured in CHO cells transfected with 100 ng/well AR.N1 and increasing amounts of either AR.C or AR.C-Q902K. Only activities measured in the hormone-treated cells are displayed, with the fold induction indicated above the bars. The activity of AR.C at 10 ng DNA/well was set at 100%, and the other points were calculated relative to that. C, Dose-response curves of the N/C interaction at increasing concentrations of R1881. The activity of AR.C measured at 1 nM R1881 was set at 100%, and the other points were calculated relative to that. D, Interaction of AR.N1 with either GST-AR.LBD-wt or GST-AR.LBD-Q902K as studied by pull-down assay. Proteins were produced in CHO cells by transfection of AR.N1 (1 µg) with the GST-AR.LBD (3 µg) constructs, in the absence and presence of 100 nM R1881 (27 ). Input is 3% of the lysate used in the pull-down experiment.

 
Next, a dose-response curve of the functional N/C-interaction in the presence of R1881 was performed, which showed that functional interaction of AR.C with AR.N1 is first measurable in the presence of 10–10 M R1881 and that maximum interaction is reached at 10–8 M R1881. Interaction between AR.C-Q902K and AR.N1 was shifted to a 5-fold higher R1881 concentration in the dose-response curve, and maximum interaction was reached at 10–8 M but only 50% of wild-type (Fig. 5CGo). These results indicate that the N/C interaction in the mutant AR is severely impaired due to the Q902K mutation. Although 100 nM R1881 can rescue the impaired N/C interaction to some extent, wild-type activities are never reached using supraphysiological doses (Fig. 5CGo).

TIF2 transactivation is partially impaired due the Q902K mutation

The AF-2 AD core domain is not only involved in N/C interaction but is also part of an interaction surface for the binding of nuclear receptor coactivators (14, 17). To further characterize the effect of the Q902K mutation on AR transcription activation, we studied the transactivation potential of TIF2. Because the effect of TIF2 transactivation is more pronounced on the NH2 terminally truncated AR (17), we used the AR.C construct for TIF2 experiments. A constant amount of TIF2 was cotransfected with increasing amounts of either AR.C of AR.C-Q902K (Fig. 6AGo). The maximum transactivation enhanced by TIF2 was reached at 10 ng DNA per well, resulting in a 34-fold induction of transcription with AR.C and a 13-fold induction with AR.C-Q902K. At higher DNA concentrations, autosquelching reduced the transcription activation, but the difference between wild-type and mutant AR.C remained 2- to 3-fold.



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  		FIG. 6. TIF2 activation of wild-type and mutated AR. A, Transactivation of increasing amounts of AR.C and AR.C-Q902K by TIF2 (100 ng/well). Only activities measured in the hormone-treated cells are displayed, with the fold induction indicated above the bars. The activity of AR.C at 10 ng DNA/well was set at 100%, and the other points were calculated relative to that. B, Dose-response curve of TIF2 activation assay in the presence of increasing concentrations of R1881 (1 pM to 100 nM). The activity of AR.C measured at 1 nM R1881 was set at 100%, and the other points were calculated relative to that.

 
TIF2 activation of AR.C and AR.C-Q902K was tested at increasing hormone concentrations (Fig. 6BGo). Wild-type AR.C was activated by TIF2 initially in the presence of 10–10 M R1881, which was 30% of the maximum activity measured at 10–7 M. In contrast to the dose-response curves of full-length AR and the N/C interaction (Figs. 4BGo and 5CGo, respectively), no maximum activity was reached in the TIF2 activation assay, even in the presence of 100 nM R1881. Activation of AR.C-Q902K by TIF2 was not detectable at hormone concentrations < 10–9 M R1881 and could be maximally induced to 50% of wild-type activity. Furthermore, alike AR.C, AR.C-Q902K response to hormone was not saturated at 10–7 M. In conclusion, TIF2 activation of Q902K AR was impaired by approximately 50%.

Positioning Q902 in the AR LBD 3D model structure

We used the AR LBD model structure deposited in the structure data bank by Matias et al. 2000 (13) to locate the position of Q902 (Fig. 7Go). The 18 amino acid residues described to constitute the LBP are shown in white, whereas residues located in and next to the AF-2 AD core domain in helix 12 that have been reported to be mutated in AIS patients (V889, I898, V903, and P904) are shown in purple. The Q902 residue is shown in green. It is obvious from the 3D model that residue Q902 is not located close to the 18 residues that interact with the bound ligand but may be part of the AF-2 AD core domain-interaction surface.



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  		FIG. 7. Model structure of the AR LBP and the AF-2 AD core domain. Amino acid residues constituting the LBP are represented in white, residues in and close to the AF-2 AD core reported to be mutated in AIS are represented in purple, the Q902 residue is shown in green, and the ligand R1881 is shown in red. Side chains of the amino acid residues constituting the LBP are represented in gray, yellow, blue, and red. The crystal structure was retrieved from the NCBI structure data bank (accession no. 1E3G) and created using the DeepView/Swiss-PDB Viewer 3.7 program.

 

   Discussion
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 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The Q902K mutation, described in this study, has not been associated with AIS before, but a somatic Q902R mutation has been reported in a patient with androgen-independent prostate cancer (32). However, no functional analysis of either two mutations has been described so far. Therefore, we studied the effect of the Q902K mutation on AR transcription activation to see whether the mutation could be correlated with the PAIS phenotype.

It was determined that the Q902K mutation leads to a mild impairment of transcription activation (15–25%) and to defective hormone-induced hyperphosphorylation of the AR protein, leading to the absence of the 114-kDa androgen-induced phosphoprotein band on Western blots. The phosphorylation pattern of the mutant AR in GSFs seems to be more affected than that of the mutant AR in transfected CHO cells. So far, no conclusive role has been ascribed to AR phosphorylation. Some studies performed in vitro report that AR phosphorylation is necessary for full transcription activation and ligand binding (9, 31, 33, 34, 35), whereas a more recent study does not find such a correlation (36). AR Q902K expressed in CHO cells resulted in hyperphosphorylation upon stimulation with a high (10–100 nM) concentration of R1881, but this did not coincide with a rescue of transcription activation. In addition, AR Q902K showed transcriptional activity at lower R1881 concentrations, even in the absence of a 114-kDa protein band. These findings suggest that, for the Q902K mutation, there is no clear correlation between AR phosphorylation and transactivation potential.

Detailed functional analyses revealed that the Q902 residue plays a substantial role in the functional interaction between the LBD and NTD, as well as in coactivator-mediated enhancement of transactivation. These functional interactions are not necessarily linked to ligand binding because the Kd of the AR Q902K receptor is only slightly elevated, but dissociation rate, N/C interaction, and TIF2 transactivation are more severely affected. It has been shown that the N/C interaction prolongs androgen binding by decreasing the ligand dissociation rate, without altering androgen binding affinity (23, 27). Thus, the reduced N/C interaction in AR Q902K increases androgen dissociation from the receptor, whereas the binding affinity is not severely impaired. This explains why even at saturating R1881 concentrations the mutant AR remains less active. In addition, increased dissociation of the ligand may result in the observed slight decrease in protein stability. Because receptor occupancy is a significant determinant of protein stability (37, 38), the decreased transcriptional activity may also partly be due to a reduced protein stability of the mutant receptor.

Similar results were obtained by Thompson et al. (39), who described mutations located between helices 3–11 within the LBD of the AR in PAIS patients that displayed normal or slightly reduced androgen binding activity but severely impaired N/C interaction, decreased coactivator response, and decreased transactivation. Recently, another study described that the decrease in N/C interaction in mutated ARs correlated with the severity of the AIS phenotype, whereas the Kd was not or only slightly affected by the mutation (40). Thus, loss of a functional N/C interaction in the AR may be an important molecular defect in AIS patients, and screening for such a functional interaction, in addition to hormone binding studies, may be a valuable tool to determine the effect of novel mutations in more detail.

In addition to interdomain interactions, disrupted coactivator interactions have also been implicated with several forms of AIS. Mild forms of AIS leading to subfertility or oligospermic infertility in men have been associated with mutations in the AR LBD leading to disrupted interdomain interactions (LBD with LBD or LBD with NTD) in combination with defective coactivator TIF2 activation (41, 42). Furthermore, CAIS has been reported in an individual where no mutations in the AR gene were found but where the transmission of the activation signal from AF-1 was disrupted, possibly due to the absence of a functional coactivator (43). Thus, a minor disruption of coactivator binding or a complete loss of coactivator function can result in varying degrees of AIS. Our finding that the Q902K mutation leads to 50% decreased TIF2 transcription activation in a patient with a PAIS phenotype is in line with these reports.

Because the 3D crystal structure of the liganded AR LBD is known (13, 44), it is possible to make assumptions about the role of certain amino acid residues in AR function. From the model structure, it can be predicted that the Q902 residue is in relative close vicinity of the LBP and of the AF-2 AD core motif and that Q902 is surrounded by residues that have been found mutated in individuals with AIS, such as V889, M895, I898, V903, and P904. Except for V903, which is only conserved between different AR species, residues V889, M895, I898, Q902, and P904 are either highly conserved or identical between AR, progesterone receptor, glucocorticoid receptor, and mineralocorticoid receptor, which are other members of the steroid hormone receptor family (45). V889, which is located just before the AF-2 AD core domain, has been reported by several groups to be mutated into a methionine residue, resulting in either PAIS or CAIS (46, 47, 48). Two mutations within the AF-2 AD core domain, M895T and I898T, have been reported in CAIS (49, 50); the V903M substitution was identified in a patient with PAIS (51), and P904S and P904H mutations have been reported to cause CAIS (46, 51). Based on these reports, it can be concluded that the position of residue Q902 in the model structure is informative but cannot be easily correlated to an AIS phenotype.

The V889M and I898T mutations have been characterized at the molecular level, and it was found that both mutations do not affect ligand binding affinity, although dissociation half-times were decreased. Strikingly, the N/C interaction was completely abolished by the I898T mutation (leading to CAIS) and severely hampered by the V889M mutation (leading to either CAIS or PAIS), which could be rescued only at high ligand concentrations (21, 22). The TIF2 interaction seems to be unaffected by these mutations. Detailed functional analysis of all the above-mentioned mutations in the AR would be needed to determine the role of each amino acid residue in inter-/intramolecular domain interaction, coactivator binding, and the relation to AIS. Our results, together with previous reports, indicate that residues outside the AF-2 AD core domain can play an important role in functional N/C interaction, and the severity of this disruption can be correlated with an AIS phenotype. Furthermore, although not every residue in and around the core domain is involved in TIF2-induced activation, amino acid Q902 appears to play an important role in this interaction.

With respect to clinical relevance, the TIF2 activation study showed that high levels of androgen could partially rescue the negative effect induced by the Q902K mutation. Because the dose-response curve with TIF2 did not show a saturation level, it can be speculated that even higher, supraphysiological concentrations of hormone could have a greater effect on activation and thus would be of use as clinical therapy. This study provides further support that detailed functional analyses on mutated ARs not only provide insight in AR functioning but can also show their relevance for clinical therapy.


   Acknowledgments
 
The authors thank Dr. Eniko Teledgy and Karen Roodnat for technical assistance, Drs. Gronemeyer and Chambon for providing the TIF2 construct, Dr. Dijkema for providing MMTV-LUC, and Dr. Katja Wolfenbuttel for the contribution.


   Footnotes
 
First Published Online October 14, 2004

1 A.U. and C.A.B. contributed equally to this work and should both be considered first authors. Back

Abbreviations: AD, Activation domain; AF, activation function; AIS, androgen insensitivity syndrome; AR, androgen receptor; CAIS, complete androgen insensitivity syndrome; 3D, three-dimensional; FCS, fetal calf serum; GSF, genital skin fibroblast; LBD, ligand binding domain; LBP, ligand binding pocket; LUC, luciferase; MMTV, mouse mammary tumor virus; NTD, NH2-terminal domain; PAIS, partial AIS.

Received January 13, 2004.

Accepted October 1, 2004.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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