Martin E. van Royen

Compartmentalization of androgen receptor protein–protein interactions in living cells

Steroid receptors regulate gene expression in a ligand-dependent manner by binding specific DNA sequences. Ligand binding also changes the conformation of the ligand binding domain (LBD), allowing interaction with coregulators via LxxLL motifs. Androgen receptors (ARs) preferentially interact with coregulators containing LxxLL-related FxxLF motifs. The AR is regulated at an extra level by interaction of an FQNLF motif in the N-terminal domain with the C-terminal LBD (N/C interaction). Although it is generally recognized that AR coregulator and N/C interactions are essential for transcription regulation, their spatiotemporal organization is largely unknown. We performed simultaneous fluorescence resonance energy transfer and fluorescence redistribution after photobleaching measurements in living cells expressing ARs double tagged with yellow and cyan fluorescent proteins. We provide evidence that AR N/C interactions occur predominantly when ARs are mobile, possibly to prevent unfavorable or untimely cofactor interactions. N/C interactions are largely lost when AR transiently binds to DNA, predominantly in foci partly overlapping transcription sites. AR coregulator interactions occur preferentially when ARs are bound to DNA.

Stepwise androgen receptor dimerization

Androgen-regulated gene expression is a highly coordinated dynamic process mediated by androgen receptor (AR) ligand binding and DNA binding, and by specific AR protein–protein interactions. The latter include DNA-binding domain (D-box) interactions in AR homodimers, and the interaction of the FQNLF motif in the AR N-terminal domain and the coactivator groove in the ligand-binding domain (N/C interaction). We have studied these interactions in AR homodimerization using quantitative imaging techniques. We found that the initial cytoplasmic intramolecular AR N/C interaction after ligand binding is followed by a D-box-dimerization-dependent transition to intermolecular N/C interaction in a proportion of nuclear ARs. The consecutive steps leading to homodimerization are initiated prior to DNA binding. Our data indicate the presence of nuclear pools of both AR homodimers and monomers. On the basis of AR-regulated reporter assays we propose specificity in regulation of gene expression by AR homodimers and monomers mediated by AR domain interactions. Moreover, our findings elucidate important steps in the spatiotemporal organization of AR intra- and inter-molecular interactions.

Androgen receptor coregulators: Recruitment via the coactivator binding groove

Androgens are key regulators of male sexual differentiation and essential for development and maintenance of male reproductive tissues. The androgens testosterone and dihydrotestosterone mediate their effect by binding to, and activation of the androgen receptor (AR). Upon activation, the AR is able to recognize specific DNA sequences in gene promoters and enhancers from where it recruits coregulators to orchestrate chromatin remodeling and transcription regulation. The number of proteins that bind to the AR has surpassed 200 and many of them enhance (coactivator) or repress (corepressor) its transactivating capacity. For most of these coregulators, their AR binding interface and their exact mode of action still needs to be elucidated, but for some of the more classical coactivators and corepressors, we gained insight in their working mechanisms. Of particular interest are specific sequences (LxxLL and FxxLF-like motifs) in a subset of coactivators that interact with the AR via a coactivator binding groove in the ligand-binding domain. As compared to other steroid receptors, the conformation of the AR coactivator binding pocket is unique and preferentially binds FxxLF-like motifs. This predisposition is expected to contribute to the regulation of specific sets of target genes via recruitment of selected coregulators. This review provides an overview of these (inter)actions with a focus on the unique characteristics of the AR coactivator binding groove.

Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells.

Proteins involved in chromatin-interacting processes, like gene transcription, DNA replication, and DNA repair, bind directly or indirectly to DNA, leading to their immobilisation. However, to reach their target sites in the DNA the proteins have to somehow move through the nucleus. Fluorescence recovery after photobleaching (FRAP) has been shown to be a strong approach to study exactly these properties, i.e. mobility and (transient) immobilisation of the proteins under investigation. Here, we provide and discuss detailed protocols for some of the FRAP procedures that we have used to study protein behaviour in living cell nuclei. In addition, we provide examples of their application in the investigation of the androgen receptor (AR), a hormone-inducible transcription factor, and of two DNA-maintenance factors, the telomere binding proteins TRF1 and TRF2. We also provide protocols for qualitative FRAP analysis and a general scheme for computer modelling of the presented FRAP procedures that can be used to quantitatively analyse experimental FRAP curves.

Novel FXXFF and FXXMF Motifs in Androgen Receptor Cofactors Mediate High Affinity and Specific Interactions with the Ligand-binding Domain

Upon hormone binding, a hydrophobic coactivator binding groove is induced in the androgen receptor (AR) ligand-binding domain (LBD). This groove serves as high affinity docking site for α-helical FXXLF motifs present in the AR N-terminal domain and in AR cofactors. Study of the amino acid requirements at position +4 of the AR FXXLF motif revealed that most amino acid substitutions strongly reduced or completely abrogated AR LBD interaction. Strong interactions were still observed following substitution of Leu+4 by Phe or Met residues. Leu+4 to Met or Phe substitutions in the FXXLF motifs of AR cofactors ARA54 and ARA70 were also compatible with strong AR LBD binding. Like the corresponding FXXLF motifs, interactions of FXXFF and FXXMF variants of AR and ARA54 motifs were AR specific, whereas variants of the less AR-selective ARA70 motif displayed increased AR specificity. A survey of currently known AR-binding proteins revealed the presence of an FXXFF motif in gelsolin and an FXXMF motif in PAK6. In vivo fluorescence resonance energy transfer and functional protein-protein interaction assays showed direct, efficient, and specific interactions of both motifs with AR LBD. Mutation of these motifs abrogated interaction of gelsolin and PAK6 proteins with AR. In conclusion, we have demonstrated strong interaction of FXXFF and FXXMF motifs to the AR coactivator binding groove, thereby mediating specific binding of a subgroup of cofactors to the AR LBD.

Quantitation of glucocorticoid receptor DNA-binding dynamics by single-molecule microscopy and FRAP

Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (∼0.7 s) and the other half for longer time periods (∼2.3 s). A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors) show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (≤1 ms) interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.

FRAP and FRET Methods to Study Nuclear Receptors in Living Cells

Quantitative imaging techniques of fluorescently-tagged proteins have been instrumental in the study of the behavior of nuclear receptors (NRs) and coregulators in living cells. Ligand-activated NRs exert their function in transcription regulation by binding to specific response elements in promotor and enhancer sequences of genes. Fluorescence recovery after photobleaching (FRAP) has proven to be a powerful tool to study the mobility of fluorescently-labeled molecules in living cells. Since binding to DNA leads to the immobilization of DNA-interacting proteins like NRs, FRAP is especially useful for determining DNA-binding kinetics of these proteins. The coordinated interaction of NRs with promoters/enhancers and subsequent transcription activation is not only regulated by ligand but also by interactions with sets of cofactors and, at least in the case of the androgen receptor (AR), by dimerization and interdomain interactions. In living cells, these interactions can be studied by fluorescence resonance energy transfer (FRET). Here we provide and discuss detailed protocols for FRAP and FRET procedures to study the behavior of nuclear receptors in living cells. On the basis of our studies of the AR, we provide protocols for two different FRAP methods (strip-FRAP and FLIP-FRAP) to quantitatively investigate DNA-interactions and for two different FRET approaches, ratio imaging, and acceptor photobleaching FRET to study AR domain interactions and interactions with cofactor motifs. Finally, we provide a protocol of a technique where FRAP and acceptor photobleaching FRET are combined to study the dynamics of interacting ARs.

Androgen receptor complexes probe DNA for recognition sequences by short random interactions.

ABSTRACT Owing to the tremendous progress in microscopic imaging of fluorescently labeled proteins in living cells, the insight into the highly dynamic behavior of transcription factors has rapidly increased over the past decade. However, a consistent quantitative scheme of their action is still lacking. Using the androgen receptor (AR) as a model system, we combined three different fluorescence microscopy assays: single-molecule microscopy, photobleaching and correlation spectroscopy, to provide a quantitative model of the action of this transcription factor. This approach enabled us to distinguish two types of AR–DNA binding: very brief interactions, in the order of a few hundred milliseconds, and hormone-induced longer-lasting interactions, with a characteristic binding time of several seconds. In addition, freely mobile ARs were slowed down in the presence of hormone, suggesting the formation of large AR–co-regulator complexes in the nucleoplasm upon hormone activation. Our data suggest a model in which mobile hormone-induced complexes of transcription factors and co-regulators probe DNA by briefly binding at random sites, only forming relatively stable transcription initiation complexes when bound to specific recognition sequences.

A novel mutation F826L in the human androgen receptor in partial androgen insensitivity syndrome; increased NH2-/COOH-terminal domain interaction and TIF2 co-activation

A novel mutation F826L located within the ligand binding domain (LBD) of the human androgen receptor (AR) was investigated. This mutation was found in a boy with severe penoscrotal hypospadias (classified as 46,XY DSD). The AR mutant F826L appeared to be indistinguishable from the wild-type AR, with respect to ligand binding affinity, transcriptional activation of MMTV-luciferase and ARE2-TATA-luciferase reporter genes, protein level in genital skin fibroblasts (GSFs), and sub-cellular distribution in transfected cells. However, an at least two-fold higher NH2-/COOH-terminal domain interaction was found in luciferase and GST pull-down assays. A two-fold increase was also observed for TIF2 (transcription intermediary factor 2) co-activation of the AR F826L COOH-terminal domain. This increase could not be explained by a higher stability of the mutant protein, which was within wild-type range. Repression of transactivation by the nuclear receptor co-repressor (N-CoR) was not affected by the AR F826L mutation. The observed properties of AR F826L would be in agreement with an increased activity rather than with a partial defective AR transcriptional activation. It is concluded that the penoscrotal hypospadias in the present case is caused by an as yet unknown mechanism, which still may involve the mutant AR.

Structure of the homodimeric androgen receptor ligand-binding domain

The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor.