Hormone-binding Domain Research Articles

Activation of ER-regulated Fusion Proteins In Vivo

Regulatable protein fusions can be made by attaching the hormone binding domain of the estrogen receptor to the N or C-terminus of a protein of interest. Activation of such ER-fusion proteins in vivo can be achieved by daily administration of a synthetic ligand, 4-hydroxytamoxifen (4OHT), via intraperitoneal (i.p.) injection., 可调节的蛋白质融合体可以通过将雌激素受体的激素结合结构域连接到目标蛋白质的N或C末端来制备。 这种ER-融合蛋白在体内的激活可以通过每日通过腹膜内（i.p.）注射施用合成配体4-羟基他莫昔芬（4OHT）来实现。

Epstein–Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines

EBV nuclear antigen 3C (EBNA3C) is an essential transcription factor for EBV transformed lymphoblast cell line (LCL) growth. To identify EBNA3C-regulated genes in LCLs, microarrays were used to measure RNA abundances in each of three different LCLs that conditionally express EBNA3C fused to a 4-OH-Tamoxifen-dependent estrogen receptor hormone binding domain (EBNA3CHT). At least three RNAs were assayed for each EBNA3CHT LCL under nonpermissive conditions, permissive conditions, and nonpermissive conditions with wild-type EBNA3C transcomplementation. Using a two-way ANOVA model of EBNA3C levels, we identified 550 regulated genes that were at least 1.5-fold up- or down-regulated with false discovery rates < 0.01. EBNA3C-regulated genes overlapped significantly with genes regulated by EBNA2 and EBNA3A consistent with coordinated effects on cell gene transcription. Of the 550 EBNA3C-regulated genes, 106 could be placed in protein networks. A seeded Bayesian network analysis of the 80 most significant EBNA3C-regulated genes suggests that RAC1, LYN, and TNF are upstream of other EBNA3C-regulated genes. Gene set enrichment analysis found enrichment for MAP kinase signaling, cytokine-cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecules, implicating these pathways in EBNA3C effects on LCL growth or survival. EBNA3C significantly up-regulated the CXCL12 ligand and its CXCR4 receptor and increased LCL migration. CXCL12 up-regulation depended on EBNA3C's interaction with the cell transcription factor, RBPJ, which is essential for LCL growth. EBNA3C also up-regulated MYC 1.3-fold and down-regulated CDKN2A exons 2 and 3, shared by p16 and p14, 1.4-fold, with false discovery rates < 5 × 10(-4).

Conserved amino acids participate in the structure networks deputed to intramolecular communication in the lutropin receptor.

The luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) particularly susceptible to spontaneous pathogenic gain-of-function mutations. Protein structure network (PSN) analysis on wild-type LHR and two constitutively active mutants, combined with in vitro mutational analysis, served to identify key amino acids that are part of the regulatory network responsible for propagating communication between the extracellular and intracellular poles of the receptor. Highly conserved amino acids in the rhodopsin family GPCRs participate in the protein structural stability as network hubs in both the inactive and active states. Moreover, they behave as the most recurrent nodes in the communication paths between the extracellular and intracellular sides in both functional states with emphasis on the active one. In this respect, non-conservative loss-of-function mutations of these amino acids is expected to impair the most relevant way of communication between activating mutation sites or hormone-binding domain and G protein recognition regions.

Muscleblind-like 1 (Mbnl1) Promotes Insulin Receptor Exon 11 Inclusion via Binding to a Downstream Evolutionarily Conserved Intronic Enhancer

The insulin receptor exists as two isoforms, IR-A and IR-B, which result from alternative splicing of exon 11 in the primary transcript. These two isoforms show a cell-specific distribution, and their relative proportions also vary during development, aging, and in different disease states. We have previously demonstrated that both intron 10 and the alternatively spliced exon 11 contain regulatory sequences that affect insulin receptor splicing both positively and negatively and that these sequences bind the serine/arginine-rich (SR) proteins SRp20 and SF2/ASF and the CELF protein CUG-BP1. In this study, we describe a new intronic splicing element within intron 11 that is highly conserved across species. Using minigenes carrying deletion mutations within intron 11, we demonstrated that this sequence functions as an intronic splicing enhancer. We subsequently used RNA affinity chromatography to identify Mbnl1 as a splicing factor that recognizes this enhancer. By ribonucleoprotein immunoprecipitation, we also established that Mbnl1 binds specifically to the INSR (insulin receptor gene) RNA. Overexpression or knockdown of Mbnl1 in hepatoma and embryonic kidney cells altered the levels of exon 11 inclusion. Finally, we showed that deletion of the intronic enhancer eliminates the ability of Mbnl1 to promote exon inclusion. Collectively, these findings demonstrate a role for Mbnl1 in controlling insulin receptor exon 11 inclusion via binding to a downstream intronic enhancer element.

VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR-RELATED NAC-DOMAIN7 Effectively Induce Transdifferentiation into Xylem Vessel Elements under Control of an Induction System

We previously showed that the VASCULAR-RELATED NAC-DOMAIN6 (VND6) and VND7 genes, which encode NAM/ATAF/CUC domain protein transcription factors, act as key regulators of xylem vessel differentiation. Here, we report a glucocorticoid-mediated posttranslational induction system of VND6 and VND7. In this system, VND6 or VND7 is expressed as a fused protein with the activation domain of the herpes virus VP16 protein and hormone-binding domain of the animal glucocorticoid receptor, and the protein's activity is induced by treatment with dexamethasone (DEX), a glucocorticoid derivative. Upon DEX treatment, transgenic Arabidopsis (Arabidopsis thaliana) plants carrying the chimeric gene exhibited transdifferentiation of various types of cells into xylem vessel elements, and the plants died. Many genes involved in xylem vessel differentiation, such as secondary wall biosynthesis and programmed cell death, were up-regulated in these plants after DEX treatment. Chemical analysis showed that xylan, a major hemicellulose component of the dicot secondary cell wall, was increased in the transgenic plants after DEX treatment. This induction system worked in poplar (Populus tremula x tremuloides) trees and in suspension cultures of cells from Arabidopsis and tobacco (Nicotiana tabacum); more than 90% of the tobacco BY-2 cells expressing VND7-VP16-GR transdifferentiated into xylem vessel elements after DEX treatment. These data demonstrate that the induction systems controlling VND6 and VND7 activities can be used as powerful tools for understanding xylem cell differentiation.

CARM1 mediates the ligand-independent and tamoxifen-resistant activation of the estrogen receptor α by cAMP

The estrogen receptor alpha (ERalpha) is activated as a transcription factor by both estrogen and a large variety of other extracellular signals. The mechanisms of this ligand-independent activation, notably by cAMP signaling, are still largely unknown. We now close the gap in the signaling pathway between cAMP and ERalpha. Whereas the direct phosphorylation of ERalpha by the cAMP-activated protein kinase A (PKA) is dispensable, the phosphorylation of the coactivator-associated arginine methyltransferase 1 (CARM1) by PKA at a single serine is necessary and sufficient for direct binding to the unliganded hormone-binding domain (HBD) of ERalpha, and the interaction is necessary for cAMP activation of ERalpha. Sustained PKA activity promoting a constitutive interaction may contribute to tamoxifen resistance of breast tumors. Binding and activation involve a novel regulatory groove of the ERalpha HBD. As a result, depending on the activating signal, ERalpha recruits different coactivator complexes to regulate alternate sets of target genes.

Estrogen Receptors Recruit SMRT and N-CoR Corepressors through Newly Recognized Contacts between the Corepressor N Terminus and the Receptor DNA Binding Domain

Estrogen receptors (ERs) are hormone-regulated transcription factors that regulate key aspects of reproduction and development. ERs are unusual in that they do not typically repress transcription in the absence of hormone but instead possess otherwise cryptic repressive functions that are revealed upon binding to certain hormone antagonists. The roles of corepressors in the control of these aspects of ER function are complex and incompletely understood. We report here that ERs recruit SMRT through an unusual mode of interaction involving multiple contact surfaces. Two surfaces of SMRT, located at the N- and C-terminal domains, contribute to the recruitment of the corepressor to ERs in vitro and are crucial for the corepressor modulation of ER transcriptional activity in cells. These corepressor surfaces contact the DNA binding domain of the receptor, rather than the hormone binding domain previously elucidated for other corepressor/nuclear receptor interactions, and are modulated by the ER's recognition of cognate DNA binding sites. Several additional nuclear receptors, and at least one other corepressor, N-CoR, share aspects of this novel mode of corepressor recruitment. Our results highlight a molecular mechanism that helps explain several previously paradoxical aspects of ER-mediated transcriptional antagonism, which may have a broader significance for an understanding of target gene repression by other nuclear receptors.

Akt and c-Myc Differentially Activate Cellular Metabolic Programs and Prime Cells to Bioenergetic Inhibition

The high glucose consumption of tumor cells even in an oxygen-rich environment, referred to as the Warburg effect, has been noted as a nearly universal biochemical characteristic of cancer cells. Targeting the glycolysis pathway has been explored as an anti-cancer therapeutic strategy to eradicate cancer based on this fundamental biochemical property of cancer cells. Oncoproteins such as Akt and c-Myc regulate cell metabolism. Accumulating studies have uncovered various molecular mechanisms by which oncoproteins affect cellular metabolism, raising a concern as to whether targeting glycolysis will be equally effective in treating cancers arising from different oncogenic activities. Here, we established a dual-regulatable FL5.12 pre-B cell line in which myristoylated Akt is expressed under the control of doxycycline, and c-Myc, fused to the hormone-binding domain of the human estrogen receptor, is activated by 4-hydroxytamoxifen. Using this system, we directly compared the effect of these oncoproteins on cell metabolism in an isogenic background. Activation of either Akt or c-Myc leads to the Warburg effect as indicated by increased cellular glucose uptake, glycolysis, and lactate generation. When cells are treated with glycolysis inhibitors, Akt sensitizes cells to apoptosis, whereas c-Myc does not. In contrast, c-Myc but not Akt sensitizes cells to the inhibition of mitochondrial function. This is correlated with enhanced mitochondrial activities in c-Myc cells. Hence, although both Akt and c-Myc promote aerobic glycolysis, they differentially affect mitochondrial functions and render cells susceptible to the perturbation of cellular metabolic programs.

Transgene excision in zebrafish using the phiC31 integrase

Genome engineering strategies employing site-specific recombinases (SSRs) have become invaluable to the study of gene function in model organisms. One such SSR, the integrase encoded by the Streptomyces bacteriophage phiC31, promotes recombination between heterotypic attP and attB sites. In the present study I have examined the feasibility of the use of phiC31 integrase for intramolecular recombination strategies in zebrafish embryos. I report here that (1) phiC31 integrase is functional in zebrafish cells, (2) phiC31 integrase can excise a transgene cassette flanked by an attB and an attP site, analogous to a common use of the Cre/lox SSR system, (3) phiC31 integrase functions in the zebrafish germline, and (4) a phiC31 integrase-estrogen receptor hormone-binding domain variant fusion protein catalyzes attB-attP recombination in zebrafish embryos in a 4-hydroxytamoxifen-dependent manner, albeit less efficiently than phiC31 alone. These features should make this a useful approach for genome manipulations in the zebrafish.

Abstract B96: Akt and c-Myc differentially activate cellular metabolic programs and prime cells to bioenergetic inhibition

Abstract The high glucose consumption of tumor cells even in oxygen rich environments, referred to as the Warburg effect, has been noted as a nearly universal biochemical characteristic of cancer cells. Targeting the glycolysis pathway has been explored as an anti-cancer therapeutic strategy to eradicate cancer based on this fundamental biochemical property of cancer cells. Oncogenes such as Akt and c-Myc regulate cell metabolisms. Accumulating studies have uncovered various molecular mechanisms by which oncoproteins affect cellular metabolism, raising a concern as to whether targeting glycolysis will be equally effective in treating cancers arisen from different oncogenic activities. To address this issue, we establish a dual-regulatable FL5.12 pre-B cell line in which myrAkt is expressed under the control of doxycycline, and c-Myc, fused to the hormone-binding domain of the human estrogen-receptor (ER), is activated by tamoxifen. Using this system we directly compare the effect of these oncoproteins on cell metabolism in an isogenic background. Activation of either Akt or c-Myc leads to the Warburg effect as indicated by increased cellular glucose uptake, glycolysis, and lactate generation. When cells are treated with glycolysis inhibitors, Akt sensitizes cells to apoptosis whereas c-Myc does not. In contrast, c-Myc but not Akt sensitizes cells to the inhibition of mitochondrial function. This is correlated with enhanced mitochondrial activities in c-Myc cells. Hence, while both Akt and c-Myc promote aerobic glycolysis, they differentially affect mitochondrial functions and render cells susceptible to the perturbation of cellular metabolic programs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B96.

Defining the hair follicle stem cell (Part I)

Defining the hair follicle stem cell (Part I)

Breast cancer-derived M543V mutation in helix 12 of estrogen receptor α inverts response to estrogen and SERMs

We have isolated from human breast cancers several mutations in the Helix 12 component of activation function 2 (AF-2) in the estrogen receptor alpha (ERalpha). We used a novel approach to detect changes in the hormone-binding domain of ERalpha, based on the evidence that antiestrogens, such as 4-hydroxytamoxifen (ZOHT) and ICI 182,780, block the function of ERalpha by binding and folding the AF-2 transcriptional domain in a way that inhibits its association with coactivator proteins. We have identified a Helix 12 mutation, M543V, which leads to greater ERalpha transcription with ZOHT and other antiestrogens (including 1,1-dichloro-2,2,3-triarylcyclopropanes, DTACs) than with 17-beta estradiol (E2). We also found an independent mutation at the same position, M543I, which did not show this inverted ligand phenotype. In comparison to further Helix 12 mutations made in vitro, it appears that relative hydrophobicity of the amino acid side chains on the inner face of Helix 12 is key to maintaining the transcriptionally active, agonist conformation with bound E2. This active conformation can be induced, resulting in increased transcription, by adding excess p160 coactivator AIB1 in transcriptional assays with E2-bound receptors, while the ZOHT-bound receptors were not further activated by AIB1. Other experiments show that the cross talk between ERalpha and AP-1 protein from AP-1-binding sites is not dependent on Helix 12 integrity. We show that two alleles containing a proline substitution in Helix 12 that inactivate AF-2 function of ERalpha at EREs have little negative effect on function through AP-1 elements, supporting a prominent role for the N-terminal AF-1 of ERalpha in AP-1/ERalpha transcriptional cross talk.

Tissue mRNA expression of the glucocorticoid receptor and its splice variants in fatal critical illness

Critical illness results in activation of the hypothalamic-pituitary-adrenal (HPA) axis, which might be accompanied by a peripheral adaptation in glucocorticoid sensitivity. Tissue sensitivity is determined by the active glucocorticoid receptor GRalpha, of which two splice variants involving the hormone-binding domain exist, GRbeta and GR-P. To study tissue mRNA expression of the GR and its splice variants in fatal critical illness. We assessed mRNA expression of the GRalpha, GRbeta and GR-P variants in liver (n = 58) and muscle (n = 65) of patients who had died after intensive care, and had been randomized for insulin treatment. We analysed whether GR mRNA expression was associated with insulin treatment, cortisol levels and glucocorticoid treatment. GRalpha and GR-P mRNA constituted 87 +/- 8% and 13 +/- 2%, respectively, of total GR mRNA in liver. GRbeta mRNA could only be amplified in five liver samples. All variants were present in most muscle samples (alpha = 96 +/- 11%, P = 3.9 +/- 0.4%, beta = 0.010 +/- 0.002%). GR expression was not associated with insulin therapy. A strong positive relationship was observed between the different GR variants in both liver and muscle (P < 0.001 for all). Serum cortisol levels were negatively associated with liver GRalpha and muscle GR-P expression (P < 0.05). mRNA expression of both liver GRalpha and GR-P, but not muscle GR, was substantially lower in patients who had received exogenous glucocorticoids (P < 0.01). We demonstrate the presence of GRalpha and GR-P mRNA in liver and of GRalpha, GRbeta and GR-P mRNA in muscle, with no evidence for altered splicing in critical illness. In contrast to muscle GR, liver GR expression was substantially lower in patients receiving exogenous glucocorticoids.

Delayed sexual maturation through gonadotropin receptor vaccination in the rainbow trout Oncorhynchus mykiss

In fish, gonadotropin hormones FSH-GTH1 and LH-GTH2 are less specific for their cognate receptors than in mammals. The respective reproductive functions of fish LH and FSH are thus difficult to establish. We aimed to study the effect of specific antagonists of the two gonadotropin receptors on trout sexual maturation in both sexes by targeting specific regions of LH and FSH receptors, Lhr and Fshr. Filamentous phages displaying Lhr specific or Fshr specific decapeptides from the extracellular hormone binding domain were engineered. Recombinant phages were used as receptor-specific antagonistic vaccines. Male and female trouts were immunized with anti-LHR, anti-FSHR, anti-FSHR + LHR or adjuvant alone, through multiple injections over 8–24 weeks, starting at different stages of sexual maturation. The consequences of immunization on gonadal development were evaluated by determining gonad growth, by histological analysis of testis and ovaries at the end of the vaccination period and by measuring blood plasma sex steroids using radioimmunoassay. We show for the first time in fish that the anti-receptor vaccinations could have specific antagonistic effects on the development of the reproductive functions; while the anti-FSHR affected the sexual maturation of prepubertal males and delayed sperm production, the anti-LHR blocked vitellogenesis in females. In maturing males, the combined anti-FSHR + LHR vaccine inhibited spermatogenesis and affected steroidogenesis. In that case, the effects of the vaccine on spermatogenesis were transient and reversible when immunization was stopped. Such an immunological strategy to specifically and transiently inhibit a receptor provides a promising approach for discovering their specific functions; it could also lead to a new technology for controlling the onset of puberty in aquaculture species.

Decoding the Cryptic Active Conformation of a Protein by Synthetic Photoscanning: INSULIN INSERTS A DETACHABLE ARM BETWEEN RECEPTOR DOMAINS

Proteins evolve in a fitness landscape encompassing a complex network of biological constraints. Because of the interrelation of folding, function, and regulation, the ground-state structure of a protein may be inactive. A model is provided by insulin, a vertebrate hormone central to the control of metabolism. Whereas native assembly mediates storage within pancreatic beta-cells, the active conformation of insulin and its mode of receptor binding remain elusive. Here, functional surfaces of insulin were probed by photocross-linking of an extensive set of azido derivatives constructed by chemical synthesis. Contacts are circumferential, suggesting that insulin is encaged within its receptor. Mapping of photoproducts to the hormone-binding domains of the insulin receptor demonstrated alternating contacts by the B-chain beta-strand (residues B24-B28). Whereas even-numbered probes (at positions B24 and B26) contact the N-terminal L1 domain of the alpha-subunit, odd-numbered probes (at positions B25 and B27) contact its C-terminal insert domain. This alternation corresponds to the canonical structure of abeta-strand (wherein successive residues project in opposite directions) and so suggests that the B-chain inserts between receptor domains. Detachment of a receptor-binding arm enables photo engagement of surfaces otherwise hidden in the free hormone. The arm and associated surfaces contain sites also required for nascent folding and self-assembly of storage hexamers. The marked compression of structural information within a short polypeptide sequence rationalizes the diversity of diabetes-associated mutations in the insulin gene. Our studies demonstrate that photoscanning mutagenesis can decode the active conformation of a protein and so illuminate cryptic constraints underlying its evolution.

Expression, purification, and characterization of the intra-cellular domain of the ANP receptor

The membrane-bound atrial natriuretic peptide receptor (GCA) catalyzes the formation of cGMP from GTP in response to natriuretic peptide hormones. Previous structural studies have focused on the extra-cellular hormone binding domain of this receptor whereas its intra-cellular domain has not yet been amenable to such studies. We report here the baculovirus expression and purification of the GCA intra-cellular domain construct GCA ID comprising the complete intra-cellular region which includes the kinase-homology domain, coiled-coil region, and catalytic cyclase domain. The intra-cellular domain was enzymatically characterized in terms of guanylyl cyclase activity and the effects of ATP, manganese, and Triton X-100. Our results indicate that the activity of the intra-cellular domain of the ANP receptor is about 2 fold less active compared to a truncated cyclase domain construct lacking the kinase-like domain that was also expressed and purified. In addition, unlike the full length receptor, the intra-cellular domain could not be activated by Triton X-100/Mn 2+ or its activity stimulated by ATP. These data therefore indicate that the major part of the transition from the basal state to the fully, ANP/ATP-dependent, activated state as well its stimulation/enhancement by Triton X-100/Mn 2+ requires the full length receptor. These receptor insights could aid in the development of novel therapeutics as the GCA receptor is a key drug target for cardiovascular diseases.

Towards a New SPE Material for EDCs: Fully Automated Synthesis of a Library of Tripodal Receptors Followed by Fast Screening by Affinity LC

AbstractA series of human estrogen receptor (hER) mimics were synthesised. On the basis of the knowledge on the structure of the hormone binding domain of the hER, three different peptide chains were constructed onto a tripodal scaffold. By using a fully automated solid‐phase synthesis protocol, 120 members with known identity were synthesised in only a week. Affinity towards estrogenic compounds was checked by affinity LC. For this purpose, ethinylestradiol was “clicked” onto a modified‐silica phase, and the obtained material was packed into an HPLC column. The results stemming from the affinity LC experiments were confirmed by clicking one library member to silica and by using this solid phase to extract different endocrine‐disrupting chemicals (EDCs) from aqueous media. (© Wiley‐VCH Verlag GmbH &amp; Co. KGaA, 69451 Weinheim, Germany, 2009)

Possible involvement of androgen receptor alterations in hepatocarcinogenesis

Androgen receptors (ARs) act as transcription factors. An increased AR activity could be due either to mutations or to an increased expression of the receptor. AR mutations involving the hormone binding domain could increase AR function and promote carcinogenesis, as suggested for prostate cancer. Herein, we evaluated qualitative (point mutations involving the hormone binding domain) and quantitative AR alterations and their possible correlation with cell proliferation and tumour grading. Carcinomatous and non-cancerous surrounding liver tissue was collected from 14 Caucasian patients with hepatocarcinoma. They were all affected by cirrhosis with different aetiologies. AR missense mutations, AR mRNA and protein levels, AR distribution in the liver, liver cell proliferation, and tumour staging were evaluated by DNA sequencing, quantitative real-time PCR, Western blot analysis, immunofluorescence, PCNA immunostaining, and conventional histological techniques, respectively. AR gene regions encoding the hormone binding domain did not contain any missense mutation. AR mRNA and protein levels were increased in hepatocarcinoma compared to non-cancerous surrounding tissue. Cell proliferation was significantly increased in the tumour compared to non-cancerous surrounding tissue. Mutations of the AR regions studied were not involved in hepatocarcinogenesis. Elevated AR levels in transformed cells could have a tumour promoting effect by stimulating cell growth.

GINS Inactivation Phenotypes Reveal Two Pathways for Chromatin Association of Replicative α and ε DNA Polymerases in Fission Yeast

The tetrameric GINS complex, consisting of Sld5-Psf1-Psf2-Psf3, plays an essential role in the initiation and elongation steps of eukaryotic DNA replication, although its biochemical function is unclear. Here we investigate the function of GINS in fission yeast, using fusion of Psf1 and Psf2 subunits to a steroid hormone-binding domain (HBD) to make GINS function conditional on the presence of beta-estradiol. We show that inactivation of Psf1-HBD causes a tight but rapidly reversible DNA replication arrest phenotype. Inactivation of Psf2-HBD similarly blocks premeiotic DNA replication and leads to loss of nuclear localization of another GINS subunit, Psf3. Inactivation of GINS has distinct effects on the replication origin association and chromatin binding of two of the replicative DNA polymerases. Inactivation of Psf1 leads to loss of chromatin binding of DNA polymerase epsilon, and Cdc45 is similarly affected. In contrast, chromatin association of the catalytic subunit of DNA polymerase alpha is not affected by defective GINS function. We suggest that GINS functions in a pathway that involves Cdc45 and is necessary for DNA polymerase epsilon chromatin binding, but that a separate pathway sets up the chromatin association of DNA polymerase alpha.

N-terminal polyglutamine-containing fragments inhibit androgen receptor transactivation function

Abstract Several neurodegenerative diseases, including Kennedy's disease (KD), are associated with misfolding and aggregation of polyglutamine (polyQ)-expansion proteins. KD is caused by a polyQ-expansion in the androgen receptor (AR), a key player in male sexual differentiation. Interestingly, KD patients often show signs of mild-to-moderate androgen insensitivity syndrome (AIS) resulting from AR dysfunction. Here, we used the yeast Saccharomyces cerevisiae to investigate the molecular mechanism behind AIS in KD. Upon expression in yeast, polyQ-expanded N-terminal fragments of AR lacking the hormone binding domain caused a polyQ length-dependent growth defect. Interestingly, while AR fragments with 67 Q formed large, SDS-resistant inclusions, the most pronounced toxicity was observed upon expression of 102 Q fragments which accumulated exclusively as soluble oligomers in the 100-600 kDa range. Analysis using a hormone-dependent luciferase reporter revealed that full-length polyQ-expanded AR is fully functional in transactivation, but becomes inactivated in the presence of the corresponding polyQ-expanded N-terminal fragment. Furthermore, the greatest impairment of AR activity was observed upon interaction of full-length AR with soluble AR fragments. Taken together, our results suggest that soluble polyQ-containing fragments bind to full-length AR and inactivate it, thus providing insight into the mechanism behind AIS in KD and possibly other polyglutamine diseases, such as Huntington's disease.