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. 2002 May;22(10):3358-72.
doi: 10.1128/MCB.22.10.3358-3372.2002.

ERAP140, a conserved tissue-specific nuclear receptor coactivator

Wenlin Shao  1 , Shlomit Halachmi, Myles Brown
Affiliations

ERAP140, a conserved tissue-specific nuclear receptor coactivator

Wenlin Shao et al. Mol Cell Biol. 2002 May.

Abstract

We report here the identification and characterization of a novel nuclear receptor coactivator, ERAP140. ERAP140 was isolated in a screen for ER alpha-interacting proteins using the ER alpha ligand binding domain as a probe. The ERAP140 protein shares no sequence and has little structural homology with other nuclear receptor cofactors. However, homologues of ERAP140 have been identified in mouse, Drosophila, and Caenorhabditis elegans. The expression of ERAP140 is cell and tissue type specific and is most abundant in the brain, where its expression is restricted to neurons. In addition to interacting with ER alpha, ERAP140 also binds ER beta, TR beta, PPAR gamma, and RAR alpha. ERAP140 interacts with ER alpha via a noncanonical interaction motif. The ER alpha-ERAP140 association can be competed by coactivator NR boxes, indicating ERAP140 binds ER alpha on a surface similar to that of other coactivators. ERAP140 can enhance the transcriptional activities of nuclear receptors with which it interacts. In vivo, ERAP140 is recruited by estrogen-bound ER alpha to the promoter region of endogenous ER alpha target genes. Furthermore, the E(2)-induced recruitment of ERAP140 to the promoter follows a cyclic pattern similar to that of other coactivators. Our results suggest that ERAP140 represents a distinct class of nuclear receptor coactivators that mediates receptor signaling in specific target tissues.

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Figures

FIG. 1.
FIG. 1.
Identification of a novel ER-associated protein, ERAP140. (A) Amino acid sequence of ERAP140. Full-length ERAP140 cDNA encodes a protein of 942 amino acids with an approximate mass of 140 kDa. The bipartite nuclear localization signal located in the extreme N terminus is indicated by the box. Two inserted sequences (underlined) likely represent splice variants. (B) Schematic diagram of the ERAP140 protein structure. Regions rich in basic, acidic, or cysteine and histidine residues are labeled, and the positions of two splice variants are indicated (Δ). NLS, nuclear localization signal. (C) ERAP140 genomic DNA consists of 15 exons and spans 150 kb; its coding region is represented by the solid boxes, and the noncoding region is represented by the stippled boxes. The asterisks indicate the locations of splice variants. (D) ERAP140 has sequence homology with proteins identified in various species, ranging from human to yeast. The three most conserved domains in ERAP140 are shown, and the percentage of identity in each protein relative to ERAP140 is indicated above the homologous domain.
FIG. 2.
FIG. 2.
ERAP140 interacts with ERα LBD in the presence of estradiol in vivo, and the interaction requires the ERα LBD/AF-2 domain. (A) Whole-cell extracts were prepared from MCF-7 cells and incubated with GST-ER LBD in the absence (−) or presence (+) of 1 μM E2. The bound proteins were eluted, electrophoresed on SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed by Western assay with a polyclonal antibody raised against the λ7 region of ERAP140 (amino acids 181 to 637). GST alone was included as a negative control, and 25 μg of MCF-7 whole-cell extracts was included in the gel as a positive control. (B) GST-λ7 fusion protein was incubated with [35S]methionine-labeled in vitro-translated ERα or ERα ΔAF-2 in the absence (−) or presence of 1 μM E2. Twenty percent of the receptor used in the binding reaction is shown as input. The bound protein was eluted, resolved on SDS-PAGE, and visualized by autoradiography.
FIG. 3.
FIG. 3.
ERAP140 mRNA and protein are expressed at different levels in various tumor cell lines and human tissues. (A) ERAP140 mRNA levels vary among different tumor lines. Cells were cultured under estrogen-free conditions for 3 days prior to being harvested. Total RNA was prepared from each cell type and analyzed by Northern blotting; 18S was included as a loading control. (B) ERAP140 mRNA level is not regulated by estradiol. Cells were cultured under estrogen-free conditions for 3 days, followed by treatment with (+) or without (−) 1 μM E2 for 6 h. Total RNA was then collected and analyzed by Northern blotting. (C) ERAP140 mRNA is expressed at low levels in most of the human tissues examined, with the exception of the brain, where it is highly expressed. Northern analysis was performed on a blot containing 1 μg of poly(A)+ RNA per lane from 12 different human tissues (Clonetech). (D) ERAP140 protein is expressed at different levels in a variety of tumor lines. Twenty micrograms of nuclear extract from each cell line was resolved on SDS-PAGE and analyzed by Western blotting using the polyclonal antibody raised against the λ7 fragment.
FIG. 4.
FIG. 4.
ERAP140 is specifically expressed in the mouse renal cortex and in neurons in the brain by in situ hybridization. The areas indicated within the boxes in panels B, E, H, and K were enlarged for better visualization in panels C, F, I, and L. DIG-labeled antisense RNA was used to detect ERAP140 mRNA in a sagittal section of an adult mouse brain; the neuron-specific localization of ERAP140 mRNA is demonstrated in cerebellum (B and C), hippocampus (E and F), and cerebral cortex (H and I). In the cerebellum, ERAP140 mRNA was found in all three layers, including the molecular layer, the Purkinje cell layer, and the granular layer. ERAP140 mRNA in the hippocampus was localized in the pyramidal cell layer, and in the cerebral cortex, it was localized in all neuronal cells. In the kidney, ERAP140 mRNA was found in the cortex but not in the medulla (K and L). Sense RNA was used as a negative control (A, D, G, and J). Bars: A and B, 500 μm; C, 50 μm; D, E, G, and H, 300 μm; F and I, 30 μm; J and K, 120 μm; L, 12 μm.
FIG. 5.
FIG. 5.
ERAP140 interacts with a specific subset of nuclear receptors in vitro. GST-λ7 fusion protein was incubated with [35S]methionine-labeled in vitro-translated receptors in the absence (−) or presence of a 1 μM concentration of their respective ligands. GST alone was included as a negative control.
FIG. 6.
FIG. 6.
The ERα interaction domain maps to a central region in ERAP140 between amino acids 489 and 559 in which the L523 and I524 residues are essential for ERα binding. (A) λ7 and different fragments of ERAP140 are indicated in the schematic diagram. The fragments were fused to GST and incubated with 35S-labeled ERα in the absence (−) or presence of 1 μM E2 or an ER antagonist, tamoxifen (T). Ten percent of the ERα used in the binding reaction was included as input. (B) Specific leucine and isoleucine residues within fragment ERAP 489-559 were mutated to alanines (boldface), and the mutants were examined for their ERα binding ability in the absence (−) or presence of 1 μM E2 or T. The underlined sequence within ERAP 489-559 was predicted to be an α-helical structure by computer analysis.
FIG. 7.
FIG. 7.
ERAP140 interacts with ERα through a binding surface similar to that of the p160 coactivator, mGRIP1. (A) 35S-labeled in vitro translated mGRIP1 was incubated with GST-ER LBD in the absence (−) or presence (+) of 1 μM E2. Increasing concentrations (1, 10, and 100 nM and 1 μM) of purified wild-type ERAP 489-559 fragment or 489-559 Mut B was added to the reaction. (B) The interaction between GST-ER LBD and 35S-ERAP140 was examined in the absence (−) or presence (+) of 1 μM E2, with increasing doses of peptides added. The sequences of the peptides are indicated at the bottom; the NR boxes are in boldface and underlined.
FIG. 8.
FIG. 8.
ERAP140 enhances nuclear receptor transactivation and possesses intrinsic transcription activity when tethered to DNA. (A) HepG2 cells were plated under hormone-free conditions overnight before transfection. The cells were cotransfected with nuclear receptors and their respective reporter genes, and 100 ng of pcDNA3.1-ERAP140 was either included (+) in the transfection or not (−). At 20 h posttransfection, the cells were treated with vehicle (control) or 100 nM E2, 100 nM T3, 1 μM BRL, or 100 nM T-RA as indicated and harvested after 24 h of incubation. Transcriptional activity was measured via luciferase assay. (B) Cos-1 cells were cotransfected with 100 ng of pCMX-βGal, 100 ng of (GAL4 X 5)SV40-Luc reporter, and 400 ng of GAL4 DBD or increasing concentrations (100, 200, 400, and 800 ng and 1 μg) of the GAL4 DBD-ERAP140 fusion gene. The level of transactivation was represented by the relative luciferase activity. The level of coactivation by ERAP140 compared to that of GAL4 DBD alone is statistically significant (P < 0.05) starting from a 200-ng concentration, as indicated by the asterisks. All experiments were performed multiple times with duplicate samples, and the results shown are representative of those studies. The error bars indicate standard deviations.
FIG. 9.
FIG. 9.
In vivo, ERAP140 is recruited by ERα to the promoter of an endogenous ER target gene, pS2. (A and C) The in vivo binding of ERα, AIB1, and ERAP140 to the pS2 promoter was examined by the ChIP assay. Vector alone, pcDNA/T7-ERAP140 (A), or either of the two T7-tagged ERAP140 mutants (C), mutant A or mutant B, was transiently expressed in MCF-7 cells. Soluble chromatin was prepared from cells treated with 100 nM E2 for 45 min (+) or untreated (−) and immunoprecipitated with antibodies against ERα, AIB1, and the T7 tag. Coprecipitated DNA was amplified using primers that flank the ERE in the pS2 promoter region; the 5′ end of the primers relative to the transcriptional initiation site is indicated in the diagram in panel A. The presence of total pS2 promoter DNA in the soluble chromatin prior to immunoprecipitation was included as input. (B and D) Expression of T7-tagged wild-type or mutant ERAP140 protein in MCF-7 cells transfected with respective plasmids. Whole-cell extracts were prepared 2 days after transfection and subjected to Western blot analysis with an anti-T7 antibody.
FIG. 10.
FIG. 10.
ERAP140 is recruited to the pS2 promoter in a dynamic fashion. MCF-7 cells were transiently transfected with pcDNA/T7-ERAP140, and 2 days after transfection, the cells were treated with 100 nM E2 for the times indicated. Soluble chromatin was then prepared from the cells, followed by immunoprecipitation using either anti-ERα, anti-T7, or anti-RNA Pol II antibody. The coprecipitated DNA was amplified by PCR.
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