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First published online 4 March 2008
doi: 10.1242/jcs.022103

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Structural basis for the nuclear import of the human androgen receptor

¹ Uro-Oncology Research Group, Cancer Research UK Cambridge Research Institute, Robinson Way, Cambridge, CB2 0RE, UK
² MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK

View larger version (21K): [in this window] [in a new window]   Fig. 1. The major NLS binding motif is conserved in the steroid receptor hinge region. (A) The amino acid sequences from the hinge regions of the androgen receptor (AR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), progesterone receptor (PR), nucleoplasmin and the SV40 large T antigen were aligned using Multalin (http://bioinfo.genopole-toulouse.prd.fr/multalin/). Conserved basic residues are in bold. The classical bipartite NLS is from nucleoplasmin, and binds to importin- via its minor and major NLS motif, boxed in grey. SV40 is a classical monopartite NLS and binds to the major NLS-binding pockets of importin- (area shaded grey). The sequence alignment of several nuclear hormone receptors reveals significant homology with these classical NLSs, with conserved basic residues in bold. (B) Scheme of the human AR, showing the N-terminal transactivation domain (TAD), DNA-binding domain (DBD), hinge domain (H) and ligand-binding domain (LBD). (C) The second zinc finger of the DBD and the hinge region. The ordered terminal -helix of the DBD is boxed, and the unstructured hinge region is underlined. The conserved NLS motif is in bold. Only the stretch of positively charged residues (629RKLKK633; bold) lies within the unstructured hinge domain, whereas the charged residues of the C-terminal lie within the DBD -helix. Adapted from Shaffer et al. (Shaffer et al., 2004). (D) Pathological substitutional mutations within this region that lead to AIS or prostate cancer (CaP).

View larger version (35K): [in this window] [in a new window]   Fig. 2. AR binds to importin- through its hinge region in vitro and in mammalian cell extracts. (A) AR constructs coding for the GST-tagged N-terminal transactivation domain (TAD), GST-tagged DNA-binding domain (DBD), GST-tagged hinge (H) domain, GST-tagged ligand-binding domain (LBD) or GST-tagged LBD and H domain together (H-LBD), were overexpressed and immobilised on glutathione-S-sepharose (GSH) resin. wtAR, wild-type AR. (B) After incubation with purified importin-, beads were washed, proteins eluted and separated, and probed for importin- by western analysis (1:2000). Equal loading of GST-AR domains was verified by Coomassie staining of an identical gel, with GST-AR domains marked by an asterisk. (C) AR was immuno-precipitated from COS-7 cells and probed for bound importin- by western blotting. Immunoprecipitation was carried out with and without androgen (10 nM R1881).

View larger version (46K): [in this window] [in a new window]   Fig. 3. The structure of the AR hinge region bound to importin-. (A) 2.6 Å resolution Fo-Fc difference electron-density map (contoured at 3) showing the density resulting from the AR hinge region after subtraction of density due to importin- in crystals of the complex. The structure of the underlying importin- is shown in yellow. Electron density resulting from the AR hinge region was only seen over the primary NLS-binding site of importin-. (B) Relationship between the binding sites on importin- for the AR hinge region (blue) and the bipartite NLS from nucleoplasmin (red). Whereas the nucleoplasmin NLS binds to both the major and minor binding sites on importin-, the AR hinge region binds to the major site together with an adjacent region of the importin- surface that is not involved in the interaction with other NLSs. (C) Schematic illustration of the positions of the Armadillo (ARM) repeats of importin- corresponding to the models shown in A and B. (D) Schematic representation of the interacting residues for different molecules (AR, SV40 large-T antigen, nucleoplasmin and the importin- IBB domain) bound to importin-. In all cases, binding at the major site involves insertion of side chains between a series of Trp residues (W142, W184, W273) on importin-, complemented by H-bonds between key Asn (N146, N188, N235) and the NLS main-chain peptides, and also neutralization of negative charges by strategically placed acidic residues on importin-. However, details of the interactions at the major site are different and, significantly, a series of residues (621EAGMTLGA628) immediately upstream of the negative cluster (629RKLKK634) in the AR hinge region bind to importin- in a manner not seen in other NLSs. Only the bipartite nucleoplasmin NLS binds to the secondary site.

View larger version (82K): [in this window] [in a new window]   Fig. 4. The AR hinge region has an intrinsic nuclear import function in live cells. COS-7 cells were transfected with GFP-AR constructs corresponding to wtAR, or disease-related mutations (R617P, C619Y, R629W, R629Q, K630T) or mutations predicted to alter nuclear import from the crystal structure (M624D, K630A, K632A, K633A, KKK630/2/3AAA). Cells were treated with 10 nM non-hyrolysible androgen (R1881, + androgen) or vehicle (ethanol,–androgen) for 30 minutes or 48 hours, fixed with methanol and mounted in Vectorshield. Cells were imaged by confocal microscopy. GFP is shown in green. Images of the two top rows show the cytoplasmic location of wtAR and the different AR mutants in the absence of androgen. The middle and bottom rows show the nuclear translocation of the different AR constructs in the presence of androgen after 30 minutes and 48 hours, respectively. Scale bars, 10 µM.

View larger version (14K): [in this window] [in a new window]   Fig. 5. The effect of hinge-region mutations on AR transactivation. COS-7 cells were transfected with GFP-tagged AR constructs, BOS β-galactosidase expression construct, and the androgen-responsive ARE2-TATA-EIB-luc reporter gene. After treatment with 10 nM R1881 (black bars) or vehicle (grey bars) for 48 hours, cells were harvested and assayed for luciferase activity. Transfection efficiency was normalised to β-galactosidase activity and is shown relative to wtAR (*). Mutations of the 630KLKK633 motif alter transactivation. P<0.001 compared with wtAR. P-values for all other samples were between 0.002 and 0.018.