conformation change ͉ FQNLF ͉ FRET ͉ nuclear receptor ͉ estrogen receptor T he nuclear receptor (NR) superfamily consists of a large group of ligand-regulated transcription factors. Several NRs are implicated in human physiology and disease (1, 2) and activation of the estrogen receptors (ER) and androgen receptors (AR) are predisposing factors for breast (3) and prostate cancer (4). Indeed, pharmacologic antagonists of AR and ER are used as antineoplastic agents in these diseases (4-7). It is commonly believed that understanding NR structure and function will facilitate development of specific drugs that can replace or supplement current therapies (2). Ligand binding alters NR structure, cofactor interactions, and transcriptional activity (8). Transcriptional activation functions are present in the aminoterminal domain (NTD; AF-1) and the ligand binding domain (LBD; AF-2) of many NRs, including AR (9) and ER (10). AF-1 is not conserved at the primary sequence level and is poorly characterized functionally (11). In contrast, AF-2 is highly conserved (12) and consists of amino acids that form a coactivator binding pocket on the surface of most NR LBDs (13-16).In many NRs, both AF-1 and AF-2 activities are suppressed in the absence of ligand and enabled after ligand binding (9, 10), which implies that ligand binding to the LBD somehow unmasks AF-1 activities in the NTD. The molecular͞structural basis for LBD communication with AF-1 in full-length molecules remains uncertain. However, an intermolecular interaction between NTD peptides and the agonist-bound LBD has been extensively characterized in vitro and with intracellular two-hybrid assays for the AR (14, 17-21) and ER (22). In the AR NTD, deletion or mutation of a sequence ( 23 FQNLF 27 ) that can bind the AF-2 coactivator pocket of the LBD (14, 19) diminishes activity of the AR at certain promoter elements (21). This finding suggests that an NTD-LBD interaction is functionally important, but it remains unknown whether the NTD interacts with the LBD within one molecule or whether it participates in an intermolecular interaction with the LBD of a second AR molecule.Of the currently available experimental approaches, FRET (23) uniquely can resolve conformation changes and protein interactions of the intact NR molecule in living cells. FRET allows real-time detection of protein conformation changes based on energy transfer between fluorophores attached to domains of interest. Here, we used FRET to determine the time and subcellular location of ligand-induced conformational changes in AR that underlie its activity as a transcription factor. We contrasted these studies with other members of the NR family, ER␣ and peroxisome proliferator-activated receptor-␥2 (PPAR␥2), and have determined a role for the AR-specific 23 FQNLF 27 motif in coordinating intramolecular AR conformational changes that precede AR self-association, most likely as a dimer. Materials and MethodsPlasmid Construction. Plasmids that express AR, ER␣, or PPAR␥2 as enhanced cyan f luorescent protein ...

Many neurodegenerative diseases, including tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and the polyglutamine diseases, are characterized by intracellular aggregation of pathogenic proteins. It is difficult to study modifiers of this process in intact cells in a high-throughput and quantitative manner, although this could facilitate molecular insights into disease pathogenesis. Here we introduce a high-throughput assay to measure intracellular polyglutamine protein aggregation using fluorescence resonance energy transfer (FRET). We screened over 2800 biologically active small molecules for inhibitory activity and have characterized one lead compound in detail. Y-27632, an inhibitor of the Rho-associated kinase p160ROCK, diminished polyglutamine protein aggregation (EC(50) congruent with 5 microM) and reduced neurodegeneration in a Drosophila model of polyglutamine disease. This establishes a novel high-throughput approach to study protein misfolding and aggregation associated with neurodegenerative diseases and implicates a signaling pathway of previously unrecognized importance in polyglutamine protein processing.

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Polyglutamine expansion in certain proteins causes neurodegeneration in inherited disorders such as Huntington disease and X-linked spinobulbar muscular atrophy. Polyglutamine tracts promote protein aggregation in vitro and in vivo with a strict length-dependence that strongly implicates alternative protein folding and/or aggregation as a proximal cause of cellular toxicity and neurodegeneration. We used an intracellular polyglutamine protein aggregation assay based on fluorescence resonance energy transfer (FRET) to identify inhibitors of androgen receptor (AR) aggregation in three libraries of biologically active small molecules: the Annotated Compound Library, the NINDS Custom Collection and a kinase inhibitor collection. In the primary screen 10 compounds reduced AR aggregation. While 10/10 also reduced huntingtin (Htt) exon 1 aggregation, only 2/10 reduced aggregation of pure polyglutamine peptides. In a PC-12 model 9/10 compounds reduced aggregation. Five out of nine compounds tested in an Htt exon 1 assay of neurodegeneration in Drosophila partially rescued the phenotype. Three of the five compounds effective in flies are FDA-approved drugs. These compounds provide new leads for therapeutic development for the polyglutamine diseases based on their efficacy in mammalian cells and a Drosophila model. The high predictive value of the primary screen suggests that the FRET-based screening assay may be useful for further primary and secondary screens for genes or small molecules that inhibit polyglutamine protein aggregation.