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Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor alpha.

ABSTRACT: In this study, we demonstrate that receptor-associated protein 80 (RAP80) interacts with estrogen receptor alpha (ERalpha) in an agonist-dependent manner. The interaction is specific for ERalpha as ERbeta and several other nuclear receptors tested did not interact with RAP80. Interaction between RAP80 and ERalpha was supported by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation analyses. The hinge/ligand-binding domain of ERalpha is sufficient for interaction with RAP80. RAP80 overexpression reduces ERalpha polyubiquitination, increases the level of ERalpha protein, and enhances ERalpha-mediated transactivation. Knockdown of endogenous RAP80 expression by small-interfering RNA (siRNA) reduced ERalpha protein level and the E2-dependent induction of pS2. In this study, we also demonstrate that RAP80 contains two functional ubiquitin-interaction motifs (UIMs) that are able to bind ubiquitin and to direct monoubiquitination of RAP80. Deletion of these UIMs does not affect the ability of RAP80 to interact with ERalpha, but eliminates the effects of RAP80 on ERalpha polyubiquitination, the level of ERalpha protein, and ERalpha-mediated transcription. These data indicate that the UIMs in RAP80 are critical for the function of RAP80. Our study identifies ERalpha as a new RAP80-interacting protein and suggests that RAP80 may be an important modulator of ERalpha activity.

SUBMITTER: Yan J

PROVIDER: S-EPMC1865050 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor alpha.

Yan Jun J Kim Yong-Sik YS Yang Xiao-Ping XP Albers Michael M Koegl Manfred M Jetten Anton M AM

Nucleic acids research 20070220 5

In this study, we demonstrate that receptor-associated protein 80 (RAP80) interacts with estrogen receptor alpha (ERalpha) in an agonist-dependent manner. The interaction is specific for ERalpha as ERbeta and several other nuclear receptors tested did not interact with RAP80. Interaction between RAP80 and ERalpha was supported by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation analyses. The hinge/ligand-binding domain of ERalpha is sufficient for interaction with RAP80. RAP80 ove ...[more]

PMID: 17311814

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Project description:Estrogen receptor-? (ESR1) is an important transcriptional regulator in the mammalian oviduct, however ESR1-dependent regulation of the transcriptome of this organ is not well defined, especially at the genomic level. The objective of this study was therefore to investigate estradiol- and ESR1-dependent regulation of the transcriptome of the oviduct using transgenic mice, both with (ESR1KO) and without (wild-type, WT) a global deletion of ESR1. Oviducts were collected from ESR1KO and WT littermates at 23 days of age, or ESR1KO and WT mice were treated with 5 IU PMSG to stimulate follicular development and the production of ovarian estradiol, and the oviducts collected 48 h later. RNA extracted from whole oviducts was hybridized to Affymetrix Genechip Mouse Genome 430-2.0 arrays (n = 3 arrays per genotype and treatment) or reverse transcribed to cDNA for analysis of the expression of selected mRNAs by real-time PCR. Following microarray analysis, a statistical two-way ANOVA and pairwise comparison (LSD test) revealed 2428 differentially expressed transcripts (DEG's, P < 0.01). Genotype affected the expression of 2215 genes, treatment (PMSG) affected the expression of 465 genes, and genotype x treatment affected the expression of 438 genes. With the goal of determining estradiol/ESR1-regulated function, gene ontology (GO) and bioinformatic pathway analyses were performed on DEG's in the oviducts of PMSG-treated ESR1KO versus PMSG-treated WT mice. Significantly enriched GO molecular function categories included binding and catalytic activity. Significantly enriched GO cellular component categories indicated the extracellular region. Significantly enriched GO biological process categories involved a single organism, modulation of a measurable attribute and developmental processes. Bioinformatic analysis revealed ESR1-regulation of the immune response within the oviduct as the primary canonical pathway. In summary, a transcriptomal profile of estradiol- and ESR1-regulated gene expression and related bioinformatic analysis is presented to increase our understanding of how estradiol/ESR1 affects function of the oviduct, and to identify genes that may be proven as important regulators of fertility in the future.

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Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor alpha.

Publications

Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor alpha.

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