Mutations at the Boundary of the Hinge and Ligand Binding Domain of the Androgen Receptor Confer Increased Transactivation Function

Grant Buchanan, Miao Yang, Jonathan M. Harris, Hyun S. Nahm, Guangzhou Han, Nicole Moore, Jacqueline M. Bentel, Robert J. Matusik, David J. Horsfall, Villis R. Marshall, Norman M. Greenberg and Wayne D. Tilley

Flinders Cancer Centre (G.B., M.Y., N.M., J.B., D.J.H., V.R.M., W.D.T.) Flinders University and Flinders Medical Centre Adelaide SA 5042, Australia Institute for Molecular Biosciences (J.M.H.) University of Queensland Brisbane Qld 4072, Australia Department of Cell Biology and Department of Medicine (H.S.N., G.H., N.M.G.) aylor College of Medicine Houston, Texas 77030 Department of Cell Biology (R.J.M.) Urological Surgery and the Vanderbilt Cancer Center Nashville, Tennessee 37232


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The androgen receptor (AR), a member of the steroid receptor superfamily of nuclear transcription factors, mediates androgen signaling in diverse target tissues. Here we report AR gene mutations identified in human prostate cancer and the autochthonous transgenic adenocarcinoma of the mouse prostate model that colocate to residues 668QPIF671 at the boundary of the hinge and ligand-binding domain, resulting in receptors that exhibit 2- to 4-fold increased activity compared with wild-type AR in response to dihydrotestosterone, estradiol, progesterone, adrenal androgens, and the AR antagonist, hydroxyflutamide, without an apparent effect on receptor levels, ligand binding kinetics, or DNA binding. The expression of these or similar variants could explain the emergence of hormone refractory disease in a subset of patients. Homology modeling indicates that amino acid residues 668QPIF671 form a ridge bordering a potential protein-protein interaction surface. The naturally occurring AR gene mutations reported in this study result in decreased hydrophobicity of this surface, suggesting that altered receptor-protein interaction mediates the precocious activity of the AR variants.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The androgen receptor (AR) is a ligand-activated nuclear transcription factor that mediates the cellular effects of androgens including differentiation, homeostasis, morphogenesis, and growth (1). The AR consists of four broadly defined functional domains: a large amino-terminal domain containing a strong constitutive transactivation function (AF-1); a DNA binding domain; a hinge region; and a carboxy-terminal ligand binding domain (LBD) that contains a highly conserved ligand-dependent transactivation function (AF-2) (2). The activity of both AF-1 and AF-2 is mediated by interaction with accessory proteins termed cofactors, which function to either up-regulate (coactivators) or down-regulate (corepressors) AR activity (3, 4).

In the inherited form of androgen insensitivity, germline mutations in the AR gene result in loss of receptor function and cause abnormal male sexual development (5). In contrast, somatic missense mutations in the AR gene that have been detected in primary and metastatic forms of prostate cancer at frequencies of up to 44% and 50%, respectively (6, 7, 8, 9), as well as in prostate cancer cell lines and xenografts (10, 11), consistently exhibit a gain of function. The majority of these mutations colocate to two discrete regions in the LBD of the AR [i.e. the signature sequence which corresponds to helix 3 (codons 669–728) and a region containing AF-2 (codons 872–908)] that play key roles in regulation of receptor function, and result in receptor variants with reduced specificity for ligand binding and/or activation (7, 9, 11, 12, 13, 14, 15). While mutations identified in the signature sequence and the AF-2 region of the AR gene potentially explain the failure of androgen ablation therapies in a subset of patients with metastatic disease, the genetic and pathological heterogeneity of clinical prostate cancer has hindered a comprehensive analysis of the incidence and nature of AR gene mutations in vivo.

In the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, generated with a probasin-directed SV40 T/t antigen in C57Bl/6 inbred mice, expression of the transgene is initially regulated by androgens and restricted to the prostate epithelial cells of the dorsolateral and ventral lobes (16, 17). Spontaneous prostate tumors that histologically resemble the human disease arise with a short latency period and exhibit progression from prostatic intraepithelial neoplasia to severe hyperplasia and adenocarcinoma. We present here the first report of a spontaneous AR gene mutation in an autochthonous animal model of prostate cancer (i.e. TRAMP) and the colocation of this mutation with somatic AR gene mutations identified in human prostate tumors to amino acids 668QPIF671 at the boundary of the hinge and LBD. This colocation to 668QPIF671 suggests that mutations in this poorly characterized region of the AR, like those in the signature sequence and AF-2, might have functional consequences relevant to tumor progression. In this study, we demonstrate that the naturally occurring mutations identified in prostate cancer in the 668QPIF671 region result in AR variants with a 2- to 4-fold greater transactivation capacity in response to dihydrotestosterone (DHT) and other nonclassical ligands compared with wtAR. Moreover, homology modeling indicates that the 668QPIF671 residues define a motif that forms a major structural component of a protein-protein interaction surface that is disrupted by the mutations reported in this study. Collectively, these findings implicate 668QPIF671 as an important motif for AR function and suggest that mutations in this motif may contribute to progression of prostate cancer in a subset of patients.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PCR single-stranded conformational polymorphism analysis of the entire coding region of the AR in four tumors derived from TRAMP mice (29 weeks of age) identified, in one tumor, a region of exon 4 with altered mobility compared with the wild-type DNA sequence. DNA sequencing of independently amplified fragments of exon 4 of the AR gene in this tumor demonstrated that the mobility shift resulted from a T-A base transition leading to substitution of phenylalanine for isoleucine at codon 653. Phe653 is conserved in the human AR (i.e. Phe671) and is located at the boundary of the hinge region and LBD (Fig. 1Go). This represents the first report of a naturally occurring AR gene mutation in an autochthonous animal model of prostate cancer. Moreover, Phe-Ile671 colocates with a subset of AR gene mutations (i.e. A-G, Gln-Arg668; and T-C, Ile-Thr670) identified in human prostate tumors (8, 9), the latter variant being identified in tumors derived from two individuals (Fig. 1Go). The colocation of these mutations to 668QPIF671 residues suggested that they, like the mutations colocating to the signature sequence and AF-2, might also have functional consequences relevant to tumor progression.



View larger version (13K):
[in this window]
[in a new window]
 
Figure 1. Schematic Showing the DNA Binding Domain (DBD), Hinge Region (H), and LBD of the AR

The T-A base transition identified in the AR gene in a TRAMP tumor results in an amino acid substitution (Phe-Ile653), which is equivalent to Phe-Ile671 in the human AR. Phe-Ile671 colocates to a small peptide 668QPIF671 at the boundary of the hinge and LBD with two AR gene mutations (i.e. A-G, Gln-Arg668; and T-C, Ile-Thr670) identified in human prostate tumors. Additional regions of the AR where AR gene mutations identified in human prostate cancer colocate (i.e. the signature sequence/helix-3, and AF-2) are indicated (stippled boxes). Together, mutations in these three regions account for 73% of the amino acid substitutions identified in the hinge and LBD of the AR in clinically important prostate cancer, and collectively span only 8% of the receptor coding sequence.

 
In vitro analysis using the androgen-responsive probasin reporter gene in PC-3 cells demonstrated that the level of transactivation activity of the transfected AR variants was greater than wtAR for each concentration of AR plasmid (Fig. 2AGo) and all concentrations of DHT (0.01–10 nM) examined (Fig. 2BGo). Furthermore, the increased transactivation capacity of the 668QPIF671 receptor variants in the presence of 1 nM DHT was independent of promoter and cellular context, in that a similar increase in receptor activity was also observed using the AR-responsive mouse mammary tumor virus promoter in both PC-3 and CV-1 cells (data not shown).



View larger version (36K):
[in this window]
[in a new window]
 
Figure 2. Transactivation Capacity of wtAR and 668QPIF671 AR Variants in Transiently Transfected PC-3 Cells

Data are expressed as a percentage of the luciferase activity induced by wtAR in the presence of 1 nM DHT and represents the mean (±SEM) of three to seven independent experiments (B–E), or mean of a representative experiment performed in triplicate (A). A, Optimization of transactivation analysis. Maximal transactivation response was obtained with transfection of 10 ng of either wtAR or variant AR expression vector. B–D, Transactivation of wtAR and 668QPIF671 AR variants in the presence of (B) increasing concentrations (0–10 nM) DHT; (C) nonandrogenic ligands 17ß-estradiol (17ß-E2, 1 nM), progesterone (PROG, 1 nM) and hydroxyflutamide (OHF, 1000 nM); (D) adrenal androgens androstenedione (1 nM) and dehydroepiandrosterone (DHEA, 1 nM). E, Comparison of transactivation activities of wtAR, the naturally occurring 668QPIF671 AR variants identified in prostate tumors (Arg668, Thr670, Ile671), and a receptor variant, Pro-Arg669, generated by in vitro mutagenesis, induced by 1 nM DHT.

 
Consistent with a precocious receptor phenotype, the 668QPIF671 variants displayed up to 10-fold greater transactivation of the androgen-responsive probasin reporter gene induced by 17ß-estradiol, progesterone, and the adrenal androgens, androstenedione and dehydroepiandrosterone, compared with wtAR (Fig. 2Go, C and D). Moreover, agonist activity of the AR antagonist, hydroxyflutamide, was observed for each receptor variant (Fig. 2CGo). Taken together, our findings suggest that the 668QPIF671 or other AR variants with a similar gain of function phenotype could provide a significant growth advantage in the presence of normal physiological levels of DHT before treatment, and have sufficient activity in the presence of 1) alternative ligands (e.g. estrogens, progestins and antiandrogens such as hydroxyflutamide) or 2) low levels of DHT after androgen ablation therapy, to promote the cell survival functions of the AR in cells expressing these variants. In contrast to these naturally occurring AR variants, in vitro mutagenesis of the only amino acid (Pro669) in this region conserved among the closely related receptors for progestins, glucocorticoids, and mineralocorticoids (18) resulted in a receptor that exhibited reduced capacity to activate transcription of the probasin reporter gene compared with wtAR (Fig. 2EGo), presumably due to decreased receptor stability (see below). An Arg669 AR variant is unlikely to be detected in vivo as it would not be expected to provide a growth advantage to prostate tumor cells.

Previous studies have shown that mutations in the AR gene in both the androgen insensitivity syndrome and human prostate cancer can alter AR mRNA or protein levels and/or the kinetics of ligand binding and dissociation (10, 19). Immunoblot analysis demonstrated that AR variants identified in this study exhibited similar steady-state AR protein levels compared with wtAR in transfected PC-3 cells (Fig. 3Go, A–D). Consistent with the immunoblot analysis, similar AR steady state RNA levels of the 668QPIF671 variants and wtAR were observed by RNAse protection assay (RPA) in transfected PC-3 cells (Fig. 3Go, E and F). In addition, there was no appreciable change in the affinity of the 668QPIF671 AR variants for DHT (Table 1Go) or the synthetic nonmetabolizable androgen, methyltrienolone (R1881; data not shown) when compared with wtAR. Furthermore, the naturally occurring 668QPIF671 AR variants exhibited no change in either receptor stability or rate of dissociation of DHT compared with wtAR (Fig. 4Go, A and B). The observation that the amino acid substitutions in 668QPIF671 residues do not alter receptor-ligand binding kinetics suggests that these residues do not contribute to the formation of the ligand-binding pocket, which is consistent with observations from the recently determined AR crystal structure (14). In contrast, the reduced transactivation capacity of the in vitro derived Arg669 receptor variant appears, in part, to be due to reduced stability (Fig. 4AGo).



View larger version (25K):
[in this window]
[in a new window]
 
Figure 3. Relative Levels of wtAR and AR Variant Protein and mRNA Determined by Immunoblot and RNAse Protection Analysis A, AR and cytokeratin 8 (CK) immunoblot using 20 µg of total cellular protein derived from PC-3 cells transfected with either wtAR or an 668QPIF671 AR variant. B, Relative protein levels of wtAR and 668QPIF671 AR variants determined by densitometric analysis of immunoblots in panel A, normalized for CK internal control and expressed relative to wtAR. Data represents the mean (±SEM) of three individual transfections. C, Immunoblot analysis of AR and CK using increasing amounts (5–50 µg) of total cellular protein derived from the AR-positive human prostate cancer cell line, LNCaP. D, Densitometric quantitation of AR and CK protein levels in panel C above, demonstrating the linear detection of both proteins over the range of 5–50 µg total cellular protein. Data shown are a representative standard curve. E, RPA of AR and cyclophilin (CY) using total RNA extracted from PC-3 cells transiently transfected with wtAR or 668QPIF671 AR expression vectors. F, Relative wtAR and 668QPIF671 AR variant RNA levels determined by densitometric analysis of RPA in panel E, normalized for CY internal control and expressed relative to wtAR. Data are representative of three independent transfections.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Affinity of wtAR and 668QPIF671 AR Variants for DHT

 


View larger version (26K):
[in this window]
[in a new window]
 
Figure 4. Stability (A) and Dissociation Rate (B) of AR-DHT Complexes in COS-1 Cells Transiently Transfected with wtAR or 668QPIF671 AR Variants

Dissociation rate was also determined for an AR containing an amino acid substitution at position 778 (Met-Thr778). In contrast to 668QPIF671 AR variants, the Thr778 variant exhibited rapid dissociation compared with wtAR, consistent with previous reports for AIS mutations at this residue (15 ). Results represent the mean of two independent determinations.

 
AR electrophoretic mobility shift assays using different amounts of nuclear protein extracts from transiently transfected PC-3 cells demonstrated that the 668QPIF671 AR variants bind to a similar extent as wtAR to a specific, high-affinity androgen response element derived from the probasin gene [i.e. PB-ARE-2 (20); Fig. 5AGo]. The specificity of the AR/PB-ARE-2 interaction was demonstrated by the addition of an AR antibody (21) that resulted in a supershift of the AR/PB-ARE-2 complex (data not shown). Formation of the AR/PB-ARE-2 complex was specifically inhibited by 100-fold excess of unlabeled PB-ARE-2, but not by a similar 100-fold excess of a nonspecific competitor (Fig. 5AGo). AR/PB-ARE-2 complex formation by wtAR and AR-Ile671 was equally competed by 10-, 50-, or 100-fold excess unlabeled PB-ARE-2 (data not shown). Increasing the salt concentration did not discriminate between binding of wtAR and the 668QPIF671 AR variants to PB-ARE-2. The conclusions from mobility shift assays were confirmed by densitometric analysis (data not shown).



View larger version (58K):
[in this window]
[in a new window]
 
Figure 5. Relative DNA Binding Ability of wtAR and 668QPIF671 AR Variants

Gel shift analysis of AR DNA binding ability in PC-3 cells transiently transfected with wtAR, the naturally occurring 668QPIF671 AR variants, or an in vitro derived mutation (Pro-Arg669). Nuclear extract (5 or 10 µg) from the transfected PC-3 cells and from nontransfected LNCaP control cells were incubated with equal amounts of 32P-labeled double-stranded PB-ARE2 oligonucleotide probe in the presence or absence of 100-fold excess unlabeled probe or 100-fold excess of labeled or unlabeled nonspecific (N/S) probe, as described in Materials and Methods.

 
The transactivation capacity of steroid receptors is dependent upon ligand-induced release of corepressors and the active recruitment of coactivators (22, 23). The best characterized of these are the p160 coactivators glucocorticoid receptor-interacting protein 1 (GRIP1), steroid receptor coactivator 1 (SRC1), and amplified in breast cancer-1 (AIB1) (24, 25, 26), which contain conserved LXXLL motifs (L for leucine and X for any amino acid) termed LX domains (LXDs), that confer high-affinity binding to a specific hydrophobic surface containing AF-2 in the LBD of steroid receptors in a ligand-dependent manner (27, 28). Structural changes in regions of steroid receptors responsible for cofactor binding have been shown to impact on receptor activity. For example, mutations in AF-2 in the AR and the estrogen receptor have been shown to either abolish receptor activity or result in receptors that can be activated by antagonists, without affecting the ability of the receptor to bind ligand (7, 10, 11, 23, 29). In addition, amino acid substitutions in the LBD of the AR have been shown to enhance receptor interaction with the coactivator RFG/ELE1 6-fold compared with the wtAR-RFG/ELE1 interaction (30). This evidence suggests that one mechanism whereby an AR variant could exhibit a gain of function is altered interaction with receptor cofactors. However, transactivation analysis demonstrated that the increased activity of the 668QPIF671 AR variants is not due to an altered response to the well-characterized p160 coactivator GRIP1 (Fig. 6Go), consistent with a requirement for regions of the AR distinct from the 668QPIF671 residues for p160 binding. While GRIP1 remains an effective coactivator of the naturally occurring 668QPIF671 AR variants, the fold coactivation was marginally reduced compared with wtAR, possibly reflecting either a reduced requirement for p160 coactivators, or an altered interaction with other cofactors in PC-3 cells.



View larger version (27K):
[in this window]
[in a new window]
 
Figure 6. Effect of GRIP1 on the Transactivation Capacity of wtAR and the 668QPIF671 AR Variants in Transiently Transfected PC-3 Cells

Transactivation was assessed as in Fig. 2Go using a 20:1 ratio of transfected GRIP1:AR expression vectors, and in the presence of 1 nM DHT. Data are expressed as mean (±SEM) of four independent determinations.

 
To examine whether mutations in 668QPIF671 residues resulted in structural alterations in the receptor that could potentially influence ligand binding and/or cofactor interactions, we applied a homology modeling protocol to generate a model of the AR-LBD (14). This approach was recently used to produce a model of the AR-LBD that accurately predicted the AR crystal structure (14). Our analyses indicate that residues 668QPIF671 do not contribute directly to ligand binding or to binding of the p160 coactivators (Fig. 7AGo; Refs. 14, 31, 32). Moreover, from this modeling we have identified that AR residues 668QPIF671 and the carboxy-terminal residues of helix 9 form complete opposing ridges that frame a small highly hydrophobic cleft (Fig. 7Go, B and Ca). A salt bridge between residues Glu835 and Arg838 forms the hydrophobic floor of this pocket (Fig. 7CGo) which, together with the ridges, is conserved in the closely related progesterone receptor (PR), but not other nuclear receptors (data not shown). Crystal structure analysis of other unliganded nuclear receptors reveals minimal repositioning of helix 9 after ligand binding (33), strongly suggesting that the cleft bordered by the 668QPIF671 ridge is a ligand-independent structure. Significantly, each of the naturally occurring AR gene mutations identified in prostate tumors reduced the hydrophobicity of the cleft while having no discernable effect on residues required for ligand or p160 binding (Fig. 7CGo, b,c,e,f). A more pronounced reduction in hydrophobicity was predicted for the in vitro derived mutation in the conserved proline residue (Pro-Arg669) which, in addition to perturbation of the 668QPIF671 ridge, appears to decrease the structural stability of the receptor due to a loss of backbone cyclization (Fig. 7CGod), possibly accounting for the reduced receptor stability and transactivation capacity observed with this mutant (Figs. 2EGo and 4AGo). Ligand-independent formation of the cleft framed by 668QPIF671 suggests that mutations that disrupt this motif would alter AR activity independent of the nature of the bound ligand, supporting the transactivation data (Fig. 2Go). The hydrophobic cleft bordered by the 668QPIF671 motif exhibits a number of features in common with the region of the AR formed by helices 3, 4, and 12 that binds the LXDs of p160 coactivators (Fig. 7AGo). This suggests that it may accommodate related motifs such as the two LXD-like peptides in the amino-terminal transactivation domain (NTD) of the AR, which were recently shown to mediate the N-C interaction between the AR NTD and the LBD resulting in stabilization of the receptor-ligand complex (34). In that report, the FXXLF peptide was shown to interact with high affinity to the hydrophobic p160 binding site after ligand binding. While the interaction surface for the second motif (i.e. WXXLF) is not known, it is possible that it could bind to the cleft bordered by 668QPIF671 residues. However, we did not observe altered dissociation of the ligand-receptor complex during characterization of the 668QPIF671 variants (Fig. 4BGo), as would be predicted if the WXXLF motif bound to this region (34).



View larger version (82K):
[in this window]
[in a new window]
 
Figure 7. Homology Modeling of the AR LBD

A, MOLSCRIPT/Raster3D ribbon diagram (60 61 ) of the AR LBD bound to testosterone showing {alpha}-helix (H1–12) and ß-sheet (S1–4) structures. Ligand (green) is shown docked in the ligand binding pocket; the NR box LXXLL peptide from the p160 coactivator GRIP1 (purple) docked in the hydrophobic coactivator binding cleft formed by helices 3, 4, and 12; and the 668QPIF671 region (red). Leucines in the GRIP1 motif and the side chains of the 668QPIF671 residues are depicted in stick form. There are no direct interactions between 668QPIF671 residues and those in either the ligand binding pocket or the hydrophobic coactivator binding cleft. B, Electrostatic potential surface of the AR LBD. Regions of the surface with negative electrostatic potential are shown in red, and positive electrostatic potential in blue; hydrophobic surfaces appear as white regions. The GRIP1 peptide and leucine side chains of the LXXLL motif are shown in purple. Side chains from the 668QPIF671 residues form a short ridge delineating a small hydrophobic cleft (indicated by an arrow) bordering a pronounced hydrophobic area. C, Disruption of a small hydrophobic cleft by mutations in 668QPIF671 residues. a, Semitransparent surface diagram of the AR 668QPIF671 region showing a small pocket with a hydrophobic floor bordered on one side by a ridge consisting of 668QPIF671 residues and on the other by a ridge formed by the carboxy terminus of helix 9. 668QPIF671 residue side chains are depicted as sticks and colored according to the standard CPK scheme. A hydrophobic floor is formed between these ridges by a salt bridge between Glu835 and Arg838 in helix 9. b–f, Electrostatic potential surface diagrams showing that each of the naturally occurring mutations (c, e, f) in the 668QPIF671 region alter the hydrophobicity of the 668QPIF671 ridge around the cleft, and that this is more pronounced for the in vitro derived Pro-Arg669 substitution (d). Surface coloring is as for panel B.

 
The region of the AR containing the 668QPIF671 residues has previously been shown to contain a repressor function that impairs ligand-dependent activity directed by AF-2 (35, 36). In addition, a four-amino acid deletion in the hinge region has been shown to relieve the dependence of the AR on the hinge-binding coactivator, Ubc9, for maximal activity (37), presumably by disrupting the binding of a corepressor normally displaced by Ubc9. Therefore, one possible explanation for increased activity of the 668QPIF671 AR variants is a reduced interaction with a nuclear corepressor. The best characterized nuclear receptor corepressors, N-CoR (nuclear receptor corepressor) and SMRT (38, 39), contain extended nuclear receptor interaction motifs (LXXI/HIXXXI/L) termed the CoRNR box (40, 41, 42). CoRNR boxes appear to mediate N-CoR and SMRT binding to the hydrophobic cleft of nuclear receptors normally occupied by the LXDs of coactivators on the addition of ligand. Moreover, ligand-induced repositioning of AF-2 results in dissociation of N-CoR and SMRT, consistent with their apparent inability to interact with nuclear receptors in the presence of ligand (42, 43, 44). Collectively, these data suggest N-CoR or SMRT are not likely to interact with the cleft bordering the 668QPIF671 motif, and therefore unlikely to mediate the enhanced ligand-dependent activity of the 668QPIF671 AR variants. The observations presented in this study suggest that perturbation of the interaction between the AR and an undetermined receptor cofactor mediates the precocious activity of the naturally occurring 668QPIF671 AR variants. Identification of the critical protein factor(s) interacting with the hydrophobic cleft adjacent to the 668QPIF671 motif may provide new insights into AR signaling.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
COS-1, CV-1, and PC-3 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and maintained in RPMI 1640 medium (Life Technologies, Inc., Melbourne, Australia) supplemented with 5% FBS and penicillin (100 U/ml) and streptomycin (100 U/ml).

Single-Stranded Conformational Polymorphism Analysis
Total RNA was isolated from four TRAMP mice at >= 24 weeks of age as previously described (45). RT-PCR single-stranded conformational polymorphism analysis was performed on the entire open reading frame of the AR gene and analyzed on a nondenaturing acrylamide gel (6% polyacrylamide, 5% glycerol gel; either 10 C or 28 C for 16 h) according to previously published methods (46, 47) using 10 sets of overlapping primers spanning the full length of the mouse AR cDNA.

DNA Sequencing
Sequencing was performed using a SEQUENASE 7-deaza-dGTP DNA sequencing kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) and a 373A automated DNA sequencer (PE Applied Biosystems, Foster City, CA).

Construction of AR Variant Expression Plasmids
The required base substitutions were introduced into an expression vector containing the complete human AR gene coding sequence under the control of the cytomegalovirus promoter (48) by PCR-based megaprimer in vitro mutagenesis (49) using pCMV-AR as the template. Megaprimers were created using a common downstream primer (5'-CCTCTAGAGTCGACCTGCAGG-3'), which spans an XbaI restriction site unique to pCMV-AR (underlined), and one of the following upstream primers:

AR-Arg668, 5'-GCTATGAATGTCGGCCCATCTT-3';

AR-Arg669, 5'-GAATGTCAGCGCATCTTTCTG-3';

AR-Thr670, 5'-GTCAGCCCACCTTTCTGAATGTC-3';

AR-Ile671 5'-CAGCCCATCATTCTGAATGTC-3'.

Megaprimers were purified and used in a second PCR with a common upstream primer (5'-TGGAGATGAAGCTTCTGGGTGT-3') spanning a HindIII site (underlined) unique to pCMV-AR. Products were digested with HindIII and XbaI and cloned into the pCMV-AR digested with the same enzymes using normal techniques. The presence of the required base substitution (underlined in the primers above) and the integrity of each AR variant plasmid construct were confirmed by DNA sequencing of both strands of the recreated expression plasmid including the entire region amplified in the PCR-based mutagenesis procedure.

In Vitro Transactivation Assay
The AR-negative human prostate cancer cell line, PC-3 (20,000 cells per well in 96-well plates) was cotransfected with wtAR or AR variant expression vectors (0–40 ng), an androgen-responsive minimal probasin luciferase reporter construct (100 ng) (50), and a control Renilla luciferase plasmid (pRL-tk, Promega Corp., Sydney, Australia; 1 ng) using LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s protocol. Cofactor analysis was performed in an analogous manner using 2.5 ng AR expression vector and cotransfecting 50 ng of GRIP1 (51). Five hours after transfection, the reaction mixture was replaced with DMEM (Life Technologies, Inc.) containing 5% charcoal-stripped FBS and supplemented with 0 or 0.01–10 nM steroids. After 36 h, cells were lysed using Passive Lysis Buffer (Promega Corp.) according to the manufacturer’s specifications and assayed immediately for reporter and control gene activities with the Dual-Luciferase Reporter Gene Assay (Promega Corp.) using a plate reading luminometer (Packard Top Count, Canberra Packard, Mount Waverley, Australia). The luciferase activity induced by 1 nM DHT on wtAR was determined in each experiment and assigned as 100%. All other data are expressed as a percentage of this value.

Dissociation Rate, Stability, and Affinity (kDa) of DHT-AR Complexes
Dissociation of [1,2,4,5,6,7-3H]-5{alpha}-dihydrotestosterone ([3H]-DHT; Amersham Pharmacia Biotech) from wtAR and 668QPIF671 AR variants was examined in monolayer cultures of COS-1 cells (106 cells per 10-cm culture dish) as previously described (52). Cells were transiently transfected with 20 µg of wtAR or 668QPIF671 AR variant expression vectors using diethylaminoethyl (DEAE) Dextran (0.5 mg/ml; Sigma, Castle Hill, Australia; 30 min at 37 C), and subsequently cultured in DMEM (Life Technologies, Inc.) containing 5% charcoal-stripped FBS for 48 h. Parallel cultures were preincubated in DMEM (Life Technologies, Inc.) containing 5% charcoal-stripped FBS and 3 nM [3H]-DHT in the presence or absence of a 200-fold excess of unlabeled DHT for 1 h at 37 C. Media were replaced with DMEM (Life Technologies, Inc.) containing 5% charcoal-stripped FBS and 0.5 µmol/liter unlabeled DHT. Cyclohexamide (500 µmol/liter) was included to minimize synthesis of new AR. Assays for AR stability and affinity for DHT were performed as previously described (19, 48). COS-1 cells transfected as above with wtAR or 668QPIF671 AR variants were harvested into 1.2 ml ice-cold cytosol buffer [10 mM Tris·HCl, 1.0 mM EDTA, 10% (wt/vol) glycerol, 10 mM sodium molybdate, 0.2 mM dithiothreitol, 1.0 mM phenylmethylsulfonylfluoride; pH 7.2]. Soluble cytosol fractions were prepared by centrifugation at 100,000 x g for 30 min. For analysis of receptor stability, cytosol fractions were prelabeled with 3 nM [3H]-DHT for 4 h at 4 C in the presence and absence of a 200-fold excess of unlabeled DHT and incubated at 37 C for 0, 0.5, 1, and 3 h. Unbound steroid was removed by addition of dextran-coated charcoal, and the amount of [3H]-DHT specifically bound was measured by scintillation counting. The affinity of AR variants for DHT was determined in cytosol fractions incubated with 0.1–3.0 nM [3H]-DHT for 16 h at 4 C. Specific binding was determined as above, and the binding data were analyzed by Scatchard plot analysis and linear regression.

Immunoblot Analysis
Lysates of PC-3 cells transfected with wtAR or 668QPIF671 AR variant expression vectors or untransfected LNCaP cells were prepared as for transfected COS-1 cells above, and the protein concentration was determined using the Bradford protein assay kit (Bio-Rad Laboratories, Inc. Regents Park, NSW, Australia). Total cellular protein (20 µg) was electrophoresed on a 6% SDS-polyacrylamide gel, transferred to Hybond-C membrane (Amersham Pharmacia Biotech), and immunostained using an affinity-purified rabbit polyclonal antibody (U402; 1:200 dilution) specific for the N-terminal 21 amino acids of the AR (21) and a mouse monoclonal cytokeratin 8 antibody (Sigma; 1:1,000 dilution). Immunoreactivity was detected using horseradish peroxidase-conjugated sheep antirabbit IgG (Silenus; 1:2,000) and horseradish peroxidase-conjugated goat antimouse IgG (DAKO Corp., Carpinteria, CA; 1:4,000), respectively, and visualized using ECL Western blotting reagents (Amersham Pharmacia Biotech). Relative AR and cytokeratin 8 protein levels were determined from immunoblots using an Imaging Densitometer (Bio-Rad Laboratories, Inc.).

RPA
PC-3 cells (5 x 106 cells per 10-cm dish) were transiently transfected with wtAR or 668QPIF671 AR variant expression vectors (2 µg) using DEAE-Dextran as described above and incubated for 24 h in the presence or absence of DHT. Total RNA was isolated from cells using guanidinium isothiocyanate as previously described (45). [32P]-UTP-labeled cRNA probes specific for AR nucleotides 1,682–1,918 (48) and cyclophilin nucleotides 38 to 140 (53) were synthesized using a T7 RNA polymerase labeling kit (Ambion, Inc., Austin, TX) and hybridized with 10 µg total RNA. The protection assay was performed using the Ambion, Inc. RNAse Protection Assay Kit according to the manufacturer’s protocol. Products were separated on a 6% denaturing polyacrylamide gel and visualized using a PhosphorImager (Molecular Dynamics, Inc. Sunnyvale, CA). AR and cyclophilin- protected fragments were quantified using Image Quant software.

Electrophoretic Mobility Shift Assays (EMSAs)
Nuclear extracts of untransfected LNCaP and PC-3 cells and of PC-3 cells transfected with wtAR or 668QPIF671 AR variant expression vectors were prepared as previously described (54). EMSAs were performed using labeled specific double-stranded oligonucleotide probe [PB-ARE-2; 5'-AGCTTAATAGGTTCTTGGAGTACTTTACGTCGA-3'; (20); 0.2 ng] incubated with 5–10 µg crude nuclear extracts in 20 µl binding buffer (10 mM HEPES, pH 7.9, 50 mM KCl, 1 mM MgCl2 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 10% glycerol) for 15 min at room temperature (55). For competition studies, nuclear extracts were preincubated with 10- to 100-fold excess of unlabeled PB-ARE-2 or a nonspecific double-stranded oligonucleotide [C/EBP; 5'-GATCCGACCTTACCACTTTCACAATCTGCCAG-3'; (56)]. For EMSAs with increasing salt concentrations, the binding buffer was supplemented with 0–450 mM KCl. Unbound probe was separated from protein-DNA complexes by electrophoresis on a 4% nondenaturing polyacrylamide gel for 3 h at 4 C. Products were visualized using a PhosphorImager, and shifted DNA-protein complexes were quantified using Image Quant software.

Homology Modeling
A model of the human AR-LBD was constructed based on the crystal structure coordinates of the human PR LBD in a complex with progesterone (18), as described elsewhere (14). Testosterone and the GRIP1 peptide were docked into the AR model by superimposing the AR-LBD model onto crystal structures of the PR-LBD/progesterone (18) and estrogen receptor-LBD/estrogen/GRIP1 peptide (32) complexes. An energy-minimized structure for testosterone was constructed using ISIS/Draw and Sculpt 3 (57) and aligned with progesterone by a Maximal Common Substructure Search (58). Electrostatic and van der Waals interactions of testosterone and the GRIP1 peptide with the AR-LBD were minimized locally using Monte Carlo Minimization (59); Van der Waals interactions were modeled using a modified Lennard-Jones function between atoms within 6 Å of each other, and electrostatic interactions were modeled with a distance-dependent dielectric between atoms within 10 Å. Molecular surfaces were calculated using a 1.4-Å probe, and transparencies were rendered with the Ray Tracing program POV-RAY 3 (http://ww.povray.org).


    ACKNOWLEDGMENTS
 
The authors thank Dr. Petra Neufing for critical review of this manuscript and Ms. Marie Pickering for technical assistance. Also Dr. Mike R. Stallcup (Department of Pathology, University of Southern California, Los Angeles, CA) for providing the GRIP1 expression vector.


    FOOTNOTES
 
Address requests for reprints to: Wayne D. Tilley, Flinders Cancer Centre, Flinders Medical Centre, Adelaide SA 5042, Australia. E-mail: Wayne.Tilley{at}flinders.edu.au

G.B. and M.Y. contributed equally to this work.

This work was supported by grants from the Anti-Cancer Foundation of South Australia, the Prostate Cancer Foundation of Australia, and the National Health and Medical Research Council of Australia (W.D.T., D.J.H., V.R.M.), the T. J. Martell Foundation (R.J.M.), the National Cancer Institute Grant CA-74737 (N.M.G.), Prostate Cancer Specialist Program of Research Excellence Grant CA-58204 (N.M.G.), and CaP CURE (N.M.G.).

Received for publication December 21, 1999. Revision received September 1, 2000. Accepted for publication September 25, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. O’Malley B 1990 The steroid receptor superfamily: more excitement predicted for the future. Mol Endocrinol 4:363–369[Medline]
  2. Kallio PJ, Pakvimo JJ, Janne OA 1996 Genetic regulation of androgen action. Prostate Suppl 6:45–51:45–51[Medline]
  3. Glass CK, Rose DW, Rosenfeld MG 1997 Nuclear receptor coactivators. Curr Opin Cell Biol 9:222–232[CrossRef][Medline]
  4. Torchia J, Glass C, Rosenfeld MG 1998 Co-activators and co-repressors in the integration of transcriptional responses. Curr Opin Cell Biol 10:373–383[CrossRef][Medline]
  5. Quigley CA, De Bellis A, Marschke KB, el-Awady MK, Wilson EM, French FS 1995 Androgen receptor defects: historical, clinical, and molecular perspectives. Endocr Rev 16:271–321[Medline]
  6. Gelmann EP 1996 Androgen receptor mutations in prostate cancer. Cancer Treat Res 1996:285–302
  7. Taplin ME, Bubley GJ, Shuster TD, Frantz ME, Spooner AE, Ogata GK, Keer HN, Balk SP 1995 Mutation of the androgen-receptor gene in metastatic androgen- independent prostate cancer. N Engl J Med 332:1393–1398[Abstract/Free Full Text]
  8. Tilley WD, Buchanan G, Hickey TE, Bentel JM 1996 Mutations in the androgen receptor gene are associated with progression of human prostate cancer to androgen independence. Clin Cancer Res 2:277–285[Abstract]
  9. Bentel JM, Tilley WD 1996 Androgen receptors in prostate cancer. J Endocrinol 151:1–11[Free Full Text]
  10. Veldscholte J, Ris-Stalpers C, Kuiper GG, Jenster G, Berrevoets C, Claassen E, van Rooij HC, Trapman J, Brinkmann AO, Mulder E 1990 A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. Biochem Biophys Res Commun 173:534–540[Medline]
  11. Tan J, Sharief Y, Hamil KG, Gregory CW, Zang DY, Sar M, Gumerlock PH, deVere WR, Pretlow TG, Harris SE, Wilson EM, Mohler JL, French FS 1997 Dehydroepiandrosterone activates mutant androgen receptors expressed in the androgen-dependent human prostate cancer xenograft CWR22 and LNCaP cells. Mol Endocrinol 11:450–459[Abstract/Free Full Text]
  12. Culig Z, Hobisch A, Cronauer MV, Cato AC, Hittmair A, Radmayr C, Eberle J, Bartsch G, Klocker H 1993 Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by adrenal androgens and progesterone. Mol Endocrinol 7:1541–1550[Abstract]
  13. Zhao XY, Malloy PJ, Krishnan AV, Swami S, Navone NM, Peehl DM, Feldman D 2000 Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor. Nat Med 6:703–706[CrossRef][Medline]
  14. Matias PM, Donner P, Coelho R, Thomaz M, Peixoto C, Macedo S, Otto N, Joschko S, Scholz P, Wegg A, Basler S, Schafer M, Egner U, Carrondo MA 2000 Structural evidence for ligand specificity in the binding domain of the human Androgen receptor: implications for pathogenic gene mutations. J Biol Chem 275:26164–26171[Abstract/Free Full Text]
  15. Gottlieb B, Beitel LK, Lumbroso R, Pinsky L, Trifiro M 1999 Update of the androgen receptor gene mutations database. Hum Mutat 14:103–114[CrossRef][Medline]
  16. Gingrich JR, Greenberg NM 1996 A transgenic mouse prostate cancer model. Toxicol Pathol 24:502–504[Medline]
  17. Gingrich JR, Barrios RJ, Kattan MW, Nahm HS, Finegold MJ, Greenberg NM 1997 Androgen-independent prostate cancer progression in the TRAMP model. Cancer Res 57:4687–4691[Abstract]
  18. Williams SP, Sigler PB 1998 Atomic structure of progesterone complexed with its receptor. Nature 393:392–396[CrossRef][Medline]
  19. Marcelli M, Tilley WD, Zoppi S, Griffin JE, Wilson JD, McPhaul MJ 1991 Androgen resistance associated with a mutation of the androgen receptor at amino acid 772 (Arg—-Cys) results from a combination of decreased messenger ribonucleic acid levels and impairment of receptor function. J Clin Endocrinol Metab 73:318–325[Abstract]
  20. Claessens F, Alen P, Devos A, Peeters B, Verhoeven G, Rombauts W 1996 The androgen-specific probasin response element 2 interacts differentially with androgen and glucocorticoid receptors. J Biol Chem 271:19013–19016[Abstract/Free Full Text]
  21. Husmann DA, Wilson CM, McPhaul MJ, Tilley WD, Wilson JD 1990 Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. Endocrinology 126:2359–2368[Abstract]
  22. Freedman LP 1999 Increasing the complexity of coactivation in nuclear receptor signaling. Cell 97:5–8[Medline]
  23. Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:1167–1177[Abstract]
  24. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93:4948–4952[Abstract/Free Full Text]
  25. Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract]
  26. Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan XY, Sauter G, Kallioniemi OP, Trent JM, Meltzer PS 1997 AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277:965–968[Abstract/Free Full Text]
  27. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733–736[CrossRef][Medline]
  28. Ding XF, Anderson CM, Ma H, Hong H, Uht RM, Kushner PJ, Stallcup MR 1998 Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC- 1): multiple motifs with different binding specificities. Mol Endocrinol 12:302–313[Abstract/Free Full Text]
  29. Mahfoudi A, Roulet E, Dauvois S, Parker MG, Wahli W 1995 Specific mutations in the estrogen receptor change the properties of antiestrogens to full agonists. Proc Natl Acad Sci U S A 92:4206–4210[Abstract]
  30. Bonagura TW, Corden JL, Brown TR, Interaction of a transcriptional coactivator with normal and mutant androgen receptors. Proceedings of the 80th Annual Meeting of The Endocrine Society, New Orleans, 1999, LAP1–537 (Abstract)
  31. Feng W, Ribeiro RJ, Wagner RL, Nguyen H, Apriletti JW, Fletterick RJ, Baxter JD, Kushner PJ, West BL 1998 Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors. Science 280:1747–1749[Abstract/Free Full Text]
  32. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927–937[Medline]
  33. Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-{alpha}. Nature 375:377–382[CrossRef][Medline]
  34. He B, Kemppainen JA, Wilson EM 2000 FXXLF and WXXLF sequences mediate the NH2-terminal interaction with the ligand binding domain of the androgen receptor. J Biol Chem 275:22986–22994[Abstract/Free Full Text]
  35. Moilanen A, Rouleau N, Ikonen T, Palvimo JJ, Janne OA 1997 The presence of a transcription activation function in the hormone- binding domain of androgen receptor is revealed by studies in yeast cells. FEBS Lett 412:355–358[CrossRef][Medline]
  36. Kuil CW, Berrevoets CA, Mulder E 1995 Ligand-induced conformational alterations of the androgen receptor analyzed by limited trypsinization. Studies on the mechanism of antiandrogen action. J Biol Chem 270:27569–27576[Abstract/Free Full Text]
  37. Poukka H, Aarnisalo P, Karvonen U, Palvimo JJ, Janne OA 1999 Ubc9 interacts with the androgen receptor and activates receptor- dependent transcription. J Biol Chem 274:19441–19446[Abstract/Free Full Text]
  38. Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamei Y, Soderstrom M, Glass CK 1995 Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397–404[CrossRef][Medline]
  39. Chen JD, Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  40. Hu X, Lazar MA 1999 The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors. Nature 402:93–96[CrossRef][Medline]
  41. Nagy L, Kao HY, Love JD, Li C, Banayo E, Gooch JT, Krishna V, Chatterjee K, Evans RM, Schwabe JW 1999 Mechanism of corepressor binding and release from nuclear hormone receptors. Genes Dev 13:3209–3216[Abstract/Free Full Text]
  42. Perissi V, Staszewski LM, McInerney EM, Kurokawa R, Krones A, Rose DW, Lambert MH, Milburn MV, Glass CK, Rosenfeld MG 1999 Molecular determinants of nuclear receptor-corepressor interaction. Genes Dev 13:3198–3208[Abstract/Free Full Text]
  43. Jackson TA, Richer JK, Bain DL, Takimoto GS, Tung L, Horwitz KB 1997 The partial agonist activity of antagonist-occupied steroid receptors is controlled by a novel hinge domain-binding coactivator L7/SPA and the corepressors N-CoR or SMRT. Mol Endocrinol 11:693–705[Abstract/Free Full Text]
  44. Smith CL, Nawaz Z, O’Malley BW 1997 Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. Mol Endocrinol 11:657–666[Abstract/Free Full Text]
  45. Tilley WD, Wilson CM, Marcelli M, McPhaul MJ 1990 Androgen receptor gene expression in human prostate carcinoma cell lines. Cancer Res 50:5382–5386[Abstract]
  46. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T 1989 Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86:2766–2770[Abstract]
  47. Ichikawa A, Hotta T, Takagi N, Tsushita K, Kinoshita T, Nagai H, Murakami Y, Hayashi K, Saito H 1992 Mutations of p53 gene and their relation to disease progression in B-cell lymphoma. Blood 79:2701–2707[Abstract]
  48. Tilley WD, Marcelli M, Wilson JD, McPhaul MJ 1989 Characterization and expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci USA 86:327–331[Abstract]
  49. Sarkar G, Sommer SS 1990 The "megaprimer" method of site-directed mutagenesis. Biotechniques 8:404–407[Medline]
  50. Kasper S, Rennie PS, Bruchovsky N, Lin L, Cheng H, Snoek R, Dahlman-Wright K, Gustafsson JA, Shiu RP, Sheppard PC, Matusik RJ 1999 Selective activation of the probasin androgen-responsive region by steroid hormones. J Mol Endocrinol 22:313–325[Abstract/Free Full Text]
  51. Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol 17:2735–2744[Abstract]
  52. Grino PB, Isidro-Gutierrez RF, Griffin JE, Wilson JD 1989 Androgen resistance associated with a qualitative abnormality of the androgen receptor and responsive to high dose androgen therapy. J Clin Endocrinol Metab 68:578–584[Abstract]
  53. Van Der Kwast TH, Schalken J, Ruizeveld dWJ, van Vroonhoven CC, Mulder E, Boersma W, Trapman J 1991 Androgen receptors in endocrine-therapy-resistant human prostate cancer. Int J Cancer 48:189–193[Medline]
  54. Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489[Abstract]
  55. Ausubel I, Frederick M 2000 Current Protocol in Molecular Biology. John Wiley & Sons, Inc., New York
  56. Cleutjens KB, van Eekelen CC, van der Korput HA, Brinkmann AO, Trapman J 1996 Two androgen response regions cooperate in steroid hormone regulated activity of the prostate-specific antigen promoter. J Biol Chem 271:6379–6388[Abstract/Free Full Text]
  57. Surles MC, Richardson JS, Richardson DC, Brooks FP, Jr 1994 Sculpting proteins interactively: continual energy minimization embedded in a graphical modeling system. Protein Sci 3:198–210[Abstract/Free Full Text]
  58. Kubinyi H, Hamprecht FA, Mietzner T 1998 Three-dimensional quantitative similarity-activity relationships (3D QSiAR) from SEAL similarity matrices. J Med Chem 41:2553–2564[CrossRef][Medline]
  59. Abagyan R, Argos P 1992 Optimal protocol and trajectory visualization for conformational searches of peptides and proteins. J Mol Biol 225:519–532[Medline]
  60. Kraulis PJ 1991 MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr 24:946–950[CrossRef]
  61. Merritt EA, Murray MEP 1994 Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D50:869–873