Methodological Approaches to Study the Genetics of Dementia and Cognitive Function
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PEPTIDES AND PROTEINS INTERACTING WITH THE ANDROGEN RECEPTOR Dennis van de Wijngaart Cover: 3-Dimensional representation of the androgen receptor ligand-binding domain (blue) in complex with a peptide containing the FxxLF motif as present in the androgen receptor N-terminal domain (yellow). The studies described in this thesis were performed at the Departments of Urology and Pathology of the Josephine Nefkens Institute, Erasmus MC, Rotterdam, the Netherlands, and were financially supported by the Dutch Cancer Society (KWF). Layout and printing by Optima Grafische Communicatie, Rotterdam The printing of this thesis was financially supported by: Department of Pathology, Erasmus MC Erasmus University Rotterdam (EUR) Dutch Cancer Society (KWF) Schering-Plough Laagland BV Stichting Wetenschappelijk Onderzoek Prostaatkanker (SWOP) ISBN-978-90-8559-501-4 PEPTIDES AND PROTEINS INTERACTING WITH THE ANDROGEN RECEPTOR Peptiden en eiwitten die interacteren met de androgeenreceptor Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof.dr. S.W.J. Lamberts en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op woensdag 1 april 2009 om 11.45 uur door Dennis Johannes van de Wijngaart Geboren te Rotterdam PROMOTIECOMMISSIE Promotor: Prof.dr.ir. J. Trapman Overige leden: Prof.dr. J.A. Grootegoed Prof.dr. J.P.T.M. van Leeuwen Dr. A.B. Houtsmuller Copromotor: Dr.ir. G.W. Jenster Contents LIST OF abbreviations 7 Chapter 1 General introduction 9 1. General introduction 11 1.1 The nuclear receptor family 11 1.2 The androgen receptor 11 1.2.1 The amino-terminal domain 12 1.2.2 The DNA-binding domain 14 1.2.3 The hinge region 14 1.2.4 The ligand-binding domain 14 1.3 AR transcriptional activation 15 1.3.1 Testosterone synthesis and conversion to DHT 15 1.3.2 AR nuclear translocation 16 1.3.3 AR dimerization and N/C interaction 16 1.3.4 AR-DNA binding 18 1.3.5 AR target genes 20 1.3.6 AR transcriptional regulation 20 1.3.7 AR enhancer-promoter dynamics 22 1.4 AR and disease 23 1.4.1 Kennedy’s disease / SBMA 23 1.4.2 Androgen insensitivity syndrome 23 1.4.3 Prostate cancer 24 1.4.3.1 Androgen-dependent prostate cancer 24 1.4.3.2 Androgen-independent prostate cancer 25 2. The AR ligand-binding pocket 27 2.1 Structure of the AR ligand-binding pocket 27 2.2 Structural analysis of agonist-bound wild type AR LBD 28 2.2.1 Binding of R1881, testosterone, and DHT 28 2.2.2 Androgen specificity of the AR ligand-binding 28 pocket 2.3 Binding of antagonists and non-androgenic ligands to 28 mutated AR LBDs 2.3.1 The AR T877A mutation 31 2.3.2 The AR W741C and W741L mutations 32 2.3.3 The AR H874Y mutation 32 2.3.4 The AR L701H/T877A double mutant 33 3. Peptide interactions with the AR coactivator groove 34 3.1 The nuclear receptor coactivator groove and LxxLL motif 34 binding 3.2 The AR LBD preferentially binds FxxLF motifs 35 3.3 Amino acid requirements for peptide binding to the AR 36 LBD 3.3.1 Mutational analysis of the AR FxxLF motif 36 3.3.2 Random screenings for AR-interacting peptides 37 3.4 Structural analysis of peptides binding to the AR LBD 37 3.5 AR mutations and ligands affect peptide recruitment to the 40 AR LBD 3.5.1 AR groove mutations 40 3.5.2 Effects of the ligand-binding pocket on peptide 41 recruitment 3.5.3 Binding function-3 41 4. Scope of this thesis 41 5. References 43 Chapter 2 Differential ligand-responsiveness of androgen receptor L701 57 mutants Chapter 3 Novel FxxFF and FxxMF motifs in androgen receptor cofactors 75 mediate high affinity and specific interactions with the ligand- binding domain Chapter 4 Functional screening of FxxLF-like peptide motifs identifies 97 SMARCD1/BAF60a as an androgen receptor cofactor that selectively modulates TMPRSS2 expression Chapter 5 Blockade of androgen receptor function by peptides targeted to 123 the coactivator-binding groove Chapter 6 General discussion 143 Summary 159 Samenvatting 161 Curriculum vitae 165 List of publications 167 Dankwoord 169 LIST OF ABBREVIATIONS aa amino acid AF activation function AIS androgen insensitivity syndrome AR androgen receptor ARA androgen receptor associated protein ARE androgen response element BAF BRG1-associated factor BF-3 binding function-3 BRG1 brahma-related gene 1 BRM brahma CAIS complete androgen insensitivity syndrome CFP cyan fluorescent protein ChIP chromatin immunoprecipitation CPA cyproterone acetate CTE carboxy-terminal extension DBD DNA-binding domain DHT 5α-dihydrotestosterone DNA deoxyribonucleic acid ER estrogen receptor ERR estrogen-related receptor FRAP fluorescence recovery after photobleaching FRET fluorescence resonance energy transfer GFP green fluorescent protein GR glucocorticoid receptor HAT histone acetyltransferase HDAC histone deacetylase Hsp heat-shock protein LBD ligand-binding domain LH luteinizing hormone LHRH luteinizing hormone-releasing hormone kDa kiloDalton MAIS mild androgen insensitivity syndrome MED mediator MMTV mouse mammary tumour virus MR mineralocorticoid receptor mRNA messenger RNA N/C interaction interaction between NTD and LBD NES nuclear export signal NLS nuclear localization signal NR nuclear receptor NTD N-terminal domain OH-F hydroxyflutamide PAIS partial androgen insensitivity syndrome PCR polymerase chain reaction PPAR peroxisome proliferator-activated receptor PR progesterone receptor PSA prostate-specific antigen RAR retinoic acid receptor RNA ribonucleic acid RXR retinoic X receptor SBMA spinal bulbar muscular atrophy SRC steroid receptor coactivator TAU transcription activation unit TR thyroid hormone receptor TR-FRET time-resolved fluorescence resonance energy transfer VDR vitamin D receptor YFP yellow fluorescent protein CHAPTER 1 General introduction General introduction 1. GENERAL INTRODUCTION Androgens are important sex steroid hormones. The androgens testosterone and dihydrotes- tosterone (DHT) are essential for normal male sexual differentiation and for the development and maintenance of male reproductive tissues, including the prostate. Androgens mediate their effects by binding to, and activation of, the androgen receptor (AR), which is a transcrip- tion factor belonging to the nuclear receptor (NR) family. Upon androgen binding, the AR is able to recognize specific DNA sequences from where it regulates the expression of its target genes. A disregulated androgen-AR pathway is involved in several diseases, such as prostate cancer, androgen insensitivity syndrome (AIS), and Kennedy’s disease or spinal and bulbar muscular atrophy (SBMA). 1.1 THE NUCLEAR RECEPTOR FAMILY NRs are members of a family of transcriptional regulators involved in many diverse cellular processes, including growth, differentiation, apoptosis, and metabolism (1-3). So far, forty- eight receptors have been identified in human, of which 24 require a ligand to be activated (3). NR ligands, such as steroids, retinoids, and fatty acids, are usually small hydrophobic molecules allowing easy crossing of the cell membrane. The other receptors are so-called orphan receptors. For these receptors, regulatory ligands have not yet been identified or they may function without a ligand. NRs have a modular structure composed of an amino-terminal transcription activation domain (NTD), a central DNA-binding domain (DBD), and a carboxyl-terminal ligand-binding domain (LBD), which is connected to the DBD via flexible hinge region (as shown in Figure 1A for AR) (1). Phylogenetic tree reconstruction based on alignment of DBD- and LBD sequences classified the human NR family into six groups (4, 5). Group I contains the receptors for thyroid hormone (TRs), retinoic acids (RARs), and vitamin D (VDR). It also includes the peroxisome proliferator-activated receptors (PPARs) and some orphan receptors such as Rev-erb, RORs, and LXR. Members of Group II are the retinoic X receptors (RXRs) and the orphans COUP-TF, HNF4, TR2, and TR4. The steroid receptors, which include the AR, the progesterone receptor (PR), the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and the estrogen receptors (ERs), as well as the closely related orphan estrogen-related receptors (ERRs) form Group III. Groups IV, V, and VI comprise various orphan receptors, such as NURR1 and ste- roidogenic factor 1 (SF1). A separate group, Group 0, consists of DAX1 and SHP, which lack a DNA-binding domain. 1.2 THE ANDROGEN RECEPTOR The AR is expressed in a variety of tissues, with highest levels present in the male urogenital tract. Lower AR levels are found in many other tissues, including bone, muscle, hair follicles, liver, and brain (6, 7). The AR is encoded by a single copy gene located at chromosome band 11 Chapter 1 Xq11.2-12 and consists of 8 exons (8, 9). Exon 1 encodes the AR NTD, exons 2 and 3 each en- codes a zinc finger of the DBD, whereas exons 4 to 8 encode the hinge region (proximal part of exon 4) and the LBD. The size of the AR protein is 919 amino acid residues, but may vary between individuals because of variations in the lengths of poly-glutamine and poly-glycine stretches in the NTD. Based on a length of 919 amino acid residues, the AR NTD encompasses the first 557 residues. The DBD is formed by residues 558-623, the hinge region by residues 624-670, and the LBD by residues 671-919. 1.2.1 THE AMINO-TERMINAL DOMAIN The NTD harbours the main transcription activation function in AR, termed activation func- tion-1 (AF-1). So far, no crystallographic data are available for any of the NR NTDs, which is probably due to the high flexibility of this domain in solution (10, 11). Although the AR NTD structure is mainly disordered, several regions appear to be in an intermediate folded state (12, 13). This so-called molten globule conformation enables multiple protein-protein inter- actions without the need for high affinities and increases the contact surface for individual interactions (12).