Human Androgen Receptor Mutation Disrupts Ternary Interactions between Ligand, Receptor Domains, and the Coactivator TIF2 (Transcription Intermediary Factor 2)

Abstract

The androgen receptor (AR) is a ligand-dependent X-linked nuclear transcription factor regulating male sexual development and spermatogenesis. The receptor is activated when androgen binds to the C-terminal ligand-binding domain (LBD), triggering a cascade of molecular events, including interactions between the LBD and the N-terminal transactivation domain (TAD), and the recruitment of transcriptional coactivators. A nonconservative asparagine to lysine substitution in AR residue 727 was encountered in a phenotypically normal man with subfertility and depressed spermatogenesis. This N727K mutation, although located in the LBD, did not alter any ligand-binding characteristic of the AR in the patient's fibroblasts or when expressed in heterologous cells. Nonetheless, the mutant AR displayed only half of wild-type transactivation capacity when exposed to physiological or synthetic androgens. This transactivation defect was consistently present when examined with two different reporter systems in three cell lines, using three androgen-driven promoters (including the complex human prostate-specific antigen promoter), confirming the pathogenicity of the mutation. In mammalian two-hybrid assays, N727K disrupted LBD interactions with the AR TAD and with the coactivator, transcription intermediary factor 2 (TIF2). Strikingly, the transactivation defect of the mutant AR can be rectified in vitro with mesterolone, consistent with the ability of this androgen analog to restore sperm production in vivo. Mesterolone, but not the physiological androgen dihydrotestosterone, restored mutant LBD interactions with the TAD and with TIF2, when expressed as fusion proteins in the two-hybrid assay. Our data support an emerging paradigm with respect to AR mutations in the LBD and male infertility: pathogenicity is transmitted through reduced interdomain and coactivator interactions, and androgen analogs that are corrective in vitro may indicate hormonal therapy.

Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions

“…To understand the role of FLNa in a physiologically relevant context, transactivation studies were repeated by using the naturally occurring MMTV-long terminal repeat (LTR) and human PSA androgen-responsive promoters driving the luciferase gene. The MMTV-LTR promoter contains several copies of the consensus ARE, and human PSA gene 1.6-kb promoter (with two putative AREs) controls expression of PSA, an androgen-regulated protein that is widely used as a marker for prostate cancer progression (5). A dose-dependent inhibitory effect of FLNa was observed on AR-mediated transcription from both the MMTV-LTR (data not shown) and PSA (Fig.…”

“…ARE 2 -Luc, containing two tandem copies of the androgen response element (ARE) from the aminotransferase gene driving the luciferase reporter gene, was obtained from G. Jenster (Erasmus University, Rotterdam, The Netherlands). Mouse mammary tumor virus-luciferase (MMTV-Luc) and the 1.6-kb prostate-specific antigen-luciferase (PSA-LUC) were described previously (5). Construction of GAL-ARLBD(628-919), GAL-ARLBD(647-919), VP16-ARTAD(1-565), and pG5Luc has been described (17).…”

“…Transactivation functions reside mainly in the ARTAD, and very minimal activity can be demonstrated in the ARLBD (2,3). Unlike other steroid receptors, transactivity depends on ligand-induced interactions between the ARTAD and ARLBD, and mutations that reduce TAD-LBD interactions affect AR activity, causing androgen insensitivity syndromes (4)(5)(6). Like other transcription regulators, the promoter-bound AR serves as a nidus to recruit cofactors that up-regulate (coactivators) or down-regulate (corepressors) AR activity.…”

The Domestic Coalition: The Command and Control Relationship Between Active Component and National Guard Forces in Defense Support of Civil Authorities Operations

“…5C). BF-3 is a target for androgen insensitivity syndrome mutations at Ile-672, Leu-830, Arg-840, and Asn-727, with mutations in the latter two diminishing SRC2 binding in vitro although neither contacts coregulator (41). Finally, mutations in BF-3 of other NRs are implicated in coactivator binding (38,42).…”

Distinct Interaction of Cortivazol with the Ligand Binding Domain Confers Glucocorticoid Receptor Specificity

“…The reason for this discrepancy is ascribed to deficiency in ligand-dependent recruitment of TIF2 by this mutant AR. Interestingly, a synthetic androgen analog mesterolone restores TIF2 recruitment and transactivation of ARN727K (61). As described in the Introduction, ligand-dependent recruitment of coactivators is an essential step for the nuclear receptor to regulate target gene transcription.…”