Nuclear Receptor Interaction Domain Research Articles

Roles of the nuclear receptor corepressor 1 (NCoR1) and the silencing mediator of retinoid and thyroid hormone receptors (SMRT) in the developing brain

The nuclear receptor corepressor 1 (NCoR1) and the silencing mediator of retinoic acid and thyroid hormone (SMRT) are critical coregulators of the nuclear receptor (e.g., thyroid hormone receptors and retinoic acid receptors), mediating transcriptional repression via histone deacetylation. Although they are highly homologous and have similar nuclear receptor interaction domains, they have different roles in different organs. Recently, de novo genetic variants in nuclear corepressors were found in pediatric patients with neurodevelopmental disorders. Thus, we generated the mouse models to understand the role of NCOR1 and SMRT in the central nervous system. We used the mice with conditional NCoR1 or SMRT (NCoR1 lox/lox or SMRT lox/lox ) alleles in combination with the mice that express Cre recombinase in a neuronal specific fashion (Snap25-Ires2-Cre). First, we performed a battery of behavioral tests to screen behavioral phenotype of mice. We found that hypoactivity, social deficits, and mild anxiety in neuronal specific NCoR1 or SMRT KO mice. In addition, NCoR1 KO mice showed high learning abilities in pairwise visual discrimination task. Next, we performed RNA-sequencing analysis with amygdala from postnatal day 21 to investigate gene expression mediated by NCoR1 and SMRT. We found that 449 genes were upregulated by SMRT deletion, whereas only 8 genes were upregulated by NCoR1 deletion. Overall, our data demonstrate for the first time that NCoR1 and SMRT have separate functions in the central nervous system. JSPS Grant-in-Aid for Early-Career Scientists 19K16486, 21K15340 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Structural Insights into the Interaction of the Intrinsically Disordered Co-activator TIF2 with Retinoic Acid Receptor Heterodimer (RXR/RAR)

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) form heterodimers that activate target gene transcription by recruiting co-activator complexes in response to ligand binding. The nuclear receptor (NR) co-activator TIF2 mediates this recruitment by interacting with the ligand-binding domain (LBD) of NRs trough the nuclear receptor interaction domain (TIF2NRID) containing three highly conserved α-helical LxxLL motifs (NR-boxes). The precise binding mode of this domain to RXR/RAR is not clear due to the disordered nature of TIF2. Here we present the structural characterization of TIF2NRID by integrating several experimental (NMR, SAXS, Far-UV CD, SEC-MALS) and computational data. Collectively, the data are in agreement with a largely disordered protein with partially structured regions, including the NR-boxes and their flanking regions, which are evolutionary conserved. NMR and X-ray crystallographic data on TIF2NRID in complex with RXR/RAR reveal a multisite binding of the three NR-boxes as well as an active role of their flanking regions in the interaction.

Pathological Interactions Between Mutant Thyroid Hormone Receptors and Corepressors and Their Modulation by a Thyroid Hormone Analogue with Therapeutic Potential.

Thyroid hormone receptors (TRs) are tightly regulated by the corepressors nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptors. Three conserved corepressor/NR signature box motifs (CoRNR1-3) forming the nuclear receptor interaction domain have been identified in these corepressors. Whereas TRs regulate multiple normal physiological and developmental pathways, mutations in TRs can result in endocrine diseases and be associated with cancers due to impairment of corepressor release. Three mutants that are located in helix H11 of TRs are of special interest: TRα-M388I, a mutant associated with the development of renal clear cell carcinomas (RCCCs), and TRβ-Δ430 and TRβ-Δ432, two deletion mutants causing resistance to thyroid hormone syndrome. Several cell-based and biophysical methods were used to measure the affinity between wild-type and mutant TRα and TRβ and all the CoRNR motifs from corepressors to quantify the effects of different thyroid hormone analogues on these interactions. This study was coupled with the measurement of interactions between wild-type and mutant TRs in the context of a heterodimer with RXR to a NCoR fragment in the presence of the same ligands. Structural insights into the binding mode of corepressors to TRs were assessed in parallel by nuclear magnetic resonance spectroscopy. The study shows that TRs interact more avidly with the silencing mediator of retinoic acid and thyroid hormone receptors than with NCoR peptides, and that TRα binds most avidly to S-CoRNR3, whereas TRβ binds preferentially to S-CoRNR2. In the studied TR mutants, a transfer of the CoRNR-specificity toward CoRNR1 was observed, coupled with a significant increase in the binding strength. In contrast to 3,5,3'-triiodothyronine (T3), the agonist TRIAC and the antagonist NH-3 were very efficient at dissociating the abnormally strong interactions between mutant TRβs and corepressors. A strong impairment of T3-binding for TRβ mutants was shown compared to TRIAC and NH-3 and could explain the different efficiencies of the different ligands in releasing corepressors from the studied TRβ mutants. Consequently, TRIAC was found to be more effective than T3 in facilitating coactivator recruitment and decreasing the dominant activity of TRβ-Δ430. This study helps to clarify the specific interaction surfaces involved in the pathologic phenotype of TR mutants and demonstrates that TRIAC is a potential therapeutic agent for patients suffering from resistance to thyroid hormone syndromes.

Regulation of corepressor alternative mRNA splicing by hormonal and metabolic signaling

Alternative mRNA splicing diversifies the products encoded by the NCoR and SMRT corepressor loci. There is a programmed alteration in NCoR mRNA splicing during adipocyte differentiation from an NCoRω isoform, which contains three nuclear receptor interaction domains, to an NCoRδ isoform that contains two nuclear receptor interaction domains. This alternative mRNA splicing of NCoR has profound effects on adiposity and on diabetes in mouse models. We report here that dexamethasone, a powerful regulator of metabolism and of adipocyte differentiation, confers this change in NCoR mRNA splicing in cultured adipocytes. We also demonstrate that changes in dietary components can consistently, if moderately, modulate the total transcript levels and the mRNA splicing of NCoR and SMRT in both cultured cells and intact mice. This ability of alternative corepressor mRNA splicing to respond to nutritional changes confirms its importance in regulating glucose and lipid metabolism, and its promise as a therapeutic candidate for metabolic disorders such as type 2 diabetes.

Development of novel Vitamin D Receptor-Coactivator Inhibitors.

Nuclear receptor coregulators are master regulators of transcription and selectively interact with the vitamin D receptor (VDR) to modulate cell differentiation, cell proliferation and calcium homeostasis. Herein, we report the syntheses and evaluation of highly potent and selective VDR-coactivator inhibitors based on a recently identified 3-indolylmethanamine scaffold. The most active compound, PS121912, selectively inhibited VDR-mediated transcription among eight other nuclear receptors tested. PS121912 is also selectively disrupting the binding between VDR and the third nuclear receptor interaction domain of the coactivator SRC2. Genetic studies revealed that PS121912 behaves like a VDR antagonist by repressing 1,25-(OH)2D3 activated gene transcription. In addition, PS121912 induced apoptosis in HL-60.

The Orphan Receptor/Corepressor DAX‐1 Modulates Human CAR Transcriptional Activity

The human constitutive androstane receptor (CAR) is a nuclear receptor (NR) primarily expressed in liver that regulates genes involved in xenobiotic metabolism, as well as lipid and energy homeostasis. Screening a panel of phage display peptides identified DAX‐1, a NR with corepressor properties, as a potential CAR coregulator. We next determined that DAX‐1 dose‐dependently inhibited CAR‐mediated transactivation. Directed mutation studies of DAX‐1 NR interaction domains showed that mutation of two N terminal domain ‘LXXM/LL’ motifs had no impact on DAX‐1 repression of CAR activity, whereas mutation of downstream ‘LXXLL’ or ‘PCFQVLP’ motifs restored CAR activity to 50% or 65% of control, respectively. Further, deletion of a transcription silencing domain (TSD) located in the DAX‐1 carboxy‐terminal region restored CAR transactivation to 90% of control levels. Two‐hybrid data demonstrated that full length DAX‐1 inhibited CAR‐SRC1 interaction by 50%, while the interaction was once again restored to 90% of control upon deletion of the TSD. Results of an alpha screen assay corroborated a direct interaction between CAR and DAX‐1 that was enhanced by CAR ligands. To examine ex vivo effects of DAX‐1, primary human hepatocytes were transfected with DAX‐1 and subsequent RT‐PCR analyses established that DAX‐1 inhibited CAR‐mediated induction of the CYP2B6 target gene by CITCO. In summary, these studies show that DAX‐1 is a functional corepressor of CAR transcriptional activity. Supported by NIH GM066411

A Novel Binding Site for hERalpha and hARNT1 AF2 Domains in the C-terminus of SRC1e Up-regulates Estrogen Receptor Alpha and AhR/ARNT1 Related Responses

Steroid receptor co-activator 1 (SRC1) is a transcriptional co-activator of numerous transcription factors involving nuclear receptors. Aryl hydrocarbon receptor nuclear translocator 1 (ARNT1) is an obligatory transcriptional partner of the aryl hydrocarbon receptor (AhR) and hypoxia inducible factor-1α (HIF-1α), as well as a co-activator of estrogen receptors (ERs). To initiate transcription, the activation function 2 (AF2) domains of estrogen-activated ERs interact with LxxLL motifs in the nuclear receptor interaction domain (NID) of SRC1. Here we describe an estrogen and LxxLL domain-independent ERα AF2 binding to SRC1e exon 21. In addition, we found an AF2 domain in exon 16 of ARNT1 that also binds to SRC1e exon 21. Surprisingly, the interaction between SRC1e exon 21 and the AF2 domain of ERα functions as a crucial enhancer of estrogen-induced transcription. The binding of ARNT1 AF2 to SRC1e exon 21 enhances the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but the upregulation essentially depends on two cyclin destruction boxes (D-boxes), which are also located on exon 16 of ARNT1. Our findings reveal that a binding site for ERα and ARNT1 AF2 domains in the C-terminus of SRC1e upregulates estrogen- and TCDD-related responses in mammalian cells.

Alternative mRNA Splicing Produces a Novel Biologically Active Short Isoform of PGC-1α

The transcriptional co-activator PGC-1alpha regulates functional plasticity in adipose tissue by linking sympathetic input to the transcriptional program of adaptive thermogenesis. We report here a novel truncated form of PGC-1alpha (NT-PGC-1alpha) produced by alternative 3' splicing that introduces an in-frame stop codon into PGC-1alpha mRNA. The expressed protein includes the first 267 amino acids of PGC-1alpha and 3 additional amino acids from the splicing insert. NT-PGC-1alpha contains the transactivation and nuclear receptor interaction domains but is missing key domains involved in nuclear localization, interaction with other transcription factors, and protein degradation. Expression and subcellular localization of NT-PGC-1alpha are dynamically regulated in the context of physiological signals that regulate full-length PGC-1alpha, but the truncated domain structure conveys unique properties with respect to protein-protein interactions, protein stability, and recruitment to target gene promoters. Therefore, NT-PGC-1alpha is a co-expressed, previously unrecognized form of PGC-1alpha with functions that are both unique from and complementary to PGC-1alpha.

Involvement of SMRT Corepressor in Transcriptional Repression by the Vitamin D Receptor

To repress the expression of target genes, the unliganded nuclear receptor generally recruits the silencing mediator of retinoid and thyroid hormone receptor (SMRT)/nuclear receptor corepressor via its direct association with the conserved motif within bipartite nuclear receptor-interaction domains (IDs) of the corepressor. Here, we investigated the involvement of the SMRT corepressor in transcriptional repression by the unliganded vitamin D receptor (VDR). Using small interference RNA against SMRT in human embryonic kidney 293 cells, we demonstrated that SMRT is involved in the repression of the VDR-target genes, osteocalcin and vitamin D(3) 24-hydroxylase in vivo. Consistent with this, VDR and SMRT are recruited to the vitamin D response element of the endogenous osteocalcin promoter in the absence of 1alpha,25-(OH)(2)D(3) in chromatin immunoprecipitation assays. To address the involvement of the VDR-specific interaction of SMRT in this repression, we identified the molecular determinants of the interaction between VDR and SMRT. Interestingly, VDR specifically interacts with ID1 of the SMRT/nuclear receptor corepressor and that ID1 is required for their stable interaction. We also identified specific residues in the SMRT-ID1 that are required for VDR binding, using the one- plus two-hybrid system, a novel genetic selection method for specific missense mutations that disrupt protein-protein interactions. These mutational studies revealed that VDR interaction requires a wide range of the residues within and outside the extended helix motif of SMRT-ID1. Notably, SMRT mutants defective in the VDR interaction were also defective in the repression of endogenous VDR-target genes, indicating that the SMRT corepressor is directly involved in the VDR-mediated repression in vivo via an ID1-specific interaction with the VDR.

Structural and Biochemical Basis for the Binding Selectivity of Peroxisome Proliferator-activated Receptor γ to PGC-1α

The functional interaction between the peroxisome proliferator-activated receptor gamma (PPARgamma) and its coactivator PGC-1alpha is crucial for the normal physiology of PPARgamma and its pharmacological response to antidiabetic treatment with rosiglitazone. Here we report the crystal structure of the PPARgamma ligand-binding domain bound to rosiglitazone and to a large PGC-1alpha fragment that contains two LXXLL-related motifs. The structure reveals critical contacts mediated through the first LXXLL motif of PGC-1alpha and the PPARgamma coactivator binding site. Through a combination of biochemical and structural studies, we demonstrate that the first LXXLL motif is the most potent among all nuclear receptor coactivator motifs tested, and only this motif of the two LXXLL-related motifs in PGC-1alpha is capable of binding to PPARgamma. Our studies reveal that the strong interaction of PGC-1alpha and PPARgamma is mediated through both hydrophobic and specific polar interactions. Mutations within the context of the full-length PGC-1alpha indicate that the first PGC-1alpha motif is necessary and sufficient for PGC-1alpha to coactivate PPARgamma in the presence or absence of rosiglitazone. These results provide a molecular basis for specific recruitment and functional interplay between PPARgamma and PGC-1alpha in glucose homeostasis and adipocyte differentiation.

Cloning of SmNCoA-62, a novel nuclear receptor co-activator from Schistosoma mansoni: Assembly of a complex with a SmRXR1/SmNR1 heterodimer, SmGCN5 and SmCBP1

The Schistosoma mansoni nuclear receptors (NR) SmRXR1 and SmNR1 have recently been shown to form a heterodimer and to bind to canonic hormone response DNA elements. Recruitment of co-regulatory proteins to NRs is required for their transcriptional and biological activities. Here, we cloned a novel S. mansoni NR co-activator, SmNCoA-62. SmNCoA-62 is highly homologous to the human Vitamin D receptor co-activator NCoA62/SKIP. SmNCoA-62 contains the SNW nuclear receptor interaction domain and a putative C-terminus transactivation domain. By using in vitro pull-down assays, we fully mapped the interaction domains of S. mansoni NR co-activators, SmNCoA-62, SmGCN5 and SmCBP1 with SmRXR1 and SmNR1, as well as the domains that mediate interactions amongst the co-activators themselves. By mutagenesis analysis, we showed that SmCBP1 LxxLL motif 2 and LxxLL motif 3, but not LxxLL motif 1, were essential to mediate the interactions of SmCBP1 with the EF domains of SmRXR1 and SmNR1. Histone acetyltransferases SmGCN5 and SmCBP1 specifically acetylated the C/D domains of SmRXR1 and SmNR1. In addition, two acetylation sites of SmNR1 were identified. SmGCN5 and SmCBP1 also acetylated SmNCoA-62 but with significant differences in their acetylation activities. Using gel shift analysis, we were able to demonstrate, in vitro, the assembly of the co-activators on the SmRXR1/SmNR1 heterodimer bound to DNA. LxxLL motifs 2 and 3 of SmCBP1 seemed to play a crucial role for the assembly of the co-activators to the DNA-bound SmRXR1/SmNR1 heterodimer.

Generation and Validation of a Mouse Line with a Floxed SRC-3/AIB1 Allele for Conditional Knockout

The steroid receptor coactivator-3 (SRC-3), also known as AIB1, ACTR, p/CIP and NCOA3, is a transcriptional coactivator for nuclear receptors and certain other transcription factors. SRC-3 is widely expressed and plays important physiological functions and pathogenic roles in breast and prostate cancers. SRC-3 knockout (SRC-3(-/-)) mice display genetic background-dependent embryonic lethality and multiple local and systemic abnormalities. Since both the partial lethality and the systemic effects caused by global SRC-3 knockout interfere with downstream investigation of tissue-specific function of SRC-3, we have generated floxed SRC-3 (SRC-3(f/f)) mice with conditional alleles carrying loxP sites in introns 10 and 12 by a gene-targeting strategy. The two SRC-3(f/f) mouse lines (A and B) are indistinguishable from wild type mice. To test if deletion of the floxed exons 11 and 12 for SRC-3 nuclear receptor interaction domains and disruption of its downstream sequence for transcriptional activation domains would inactivate SRC-3 function, SRC-3(f/f) mice were crossbred with EIIa-Cre mice to generate SRC-3(d/d) mice with germ line deletion of the floxed SRC-3 gene. Both lines of SRC-3(d/d) mice exhibited growth retardation and low IGF-I levels, which was similar to that observed in SRC-3(-/-) mice. The line A SRC-3(d/d) mice showed normal viability, while line B SRC-3(d/d) mice showed partial lethality similar to SRC-3-/- mice, probably due to variable distributions of genetic background during breeding. These results demonstrate that the floxed SRC-3 mouse lines have been successfully established. These mice will be useful for investigating the cell type- and developmental stage-specific functions of SRC-3.

A Novel Fusion between ETV6 and NCOA2 Defines a New Entity of Acute Leukemia with Co-Expression of T-Cell and Myeloid Markers.

We have identified a novel recurrent translocation and the genes involved therein, which is associated with both lymphoid and myeloid acute leukemia. Fluorescence in situ hybridization (FISH) and molecular analyses revealed an in-frame fusion of ETV6 (alias TEL) and NCOA2 (alias TIF2) in six cases of childhood acute leukemia. ETV6 encodes an ets transcription repressor and is frequently involved in chromosome rearrangements with a multitude of partners in lymphoid and myeloid leukemia, and to a lesser extent also in solid tumors. NCOA2 is a member of the p160 family of nuclear receptor transcriptional coactivators (NRCoAs), which have a common domain structure with a conserved N-terminal bHLH-PAS domain, a centrally located (nuclear) receptor interaction domain (RID/NID), and a C-terminal transcriptional activation domain (AD2). NRCoAs typically display HAT activity and/or directly interact with CBP. In this context, NCOA2 also contains a C-terminal CBP-interaction domain (CID), and thus is a transcription factor that mediates transcriptional activation in a ligand-dependent fashion by a mechanism involving chromatin remodeling. As a result of the translocation the pointed (PNT) protein interaction domain of ETV6 is fused to the C-terminus of NCOA2 including the CBP and AD2 motifs. The same C-terminal domains of NCOA2 are also involved in the previously identified MOZ-NCOA2 fusion, which is generated by a inv(8)(p11q13). The absence of the reciprocal NCOA2-ETV6 transcript in one of the cases, and the facts that MOZ-NCOA2 is transforming and that the reciprocal NCOA2-MOZ is not expressed, strongly suggest that the ETV6-NCOA2 chimeric protein and not the reciprocal NCOA2-ETV6 is responsible for leukemogenesis. Unlike in cases with a ETV6-RUNX1 fusion, which is frequently associated with a concurrent secondary deletion of the non-rearranged ETV6 allele, in all cases the second ETV6 and also one NCOA2 allele were retained and both genes were readily expressed. With respect to the immunophenotype, according to the European Group for the Immunological Characterization of Leukemias (EGIL), three of the cases were classified as T-ALL (one as T-I My+), two as biphenotypic leukemia (BAL), and one as AML with M1 phenotype. Nevertheless, all leukemias displayed aberrant co-expression of various T-cell and myeloid/NK markers, and were double-negative for CD4/CD8, suggesting transformation of early progenitors. In contrast to the MOZ-NCOA2 fusion, which is exclusively associated with AML, mostly of M4 and M5 subtype, the ETV6-NCOA2 fusion seems to define a new entity of acute leukemia with aberrant co-presence of T lymphoid and myeloid antigens.

Ligand-specific allosteric regulation of coactivator functions of androgen receptor in prostate cancer cells

The androgen receptor not only mediates prostate development but also serves as a key regulator of primary prostatic cancer growth. Although initially responsive to selective androgen receptor modulators (SARMs), which cause recruitment of the nuclear receptor-corepressor (N-CoR) complex, resistance invariably occurs, perhaps in response to inflammatory signals. Here we report that dismissal of nuclear receptor-corepressor complexes by specific signals or androgen receptor overexpression results in recruitment of many of the cohorts of coactivator complexes that permits SARMs and natural ligands to function as agonists. SARM-bound androgen receptors appear to exhibit failure to recruit specific components of the coactivators generally bound by liganded nuclear receptors, including cAMP response element-binding protein (CBP)/p300 or coactivator-associated arginine methyltransferase 1 (CARM1) to the SARM-bound androgen receptor, although still causing transcriptional activation of androgen receptor target genes. SARM-bound androgen receptors use distinct LXXLL (L, leucine; X, any amino acid) helices in the p160 nuclear receptor interaction domains that may impose selective allosteric effects, providing a component of the molecular basis of differential responses to different classes of ligands by androgen receptor.

Genotype-Phenotype Associations in Sotos Syndrome: An Analysis of 266 Individuals with NSD1 Aberrations

We identified 266 individuals with intragenic NSD1 mutations or 5q35 microdeletions encompassing NSD1 (referred to as "NSD1-positive individuals"), through analyses of 530 subjects with diverse phenotypes. Truncating NSD1 mutations occurred throughout the gene, but pathogenic missense mutations occurred only in functional domains (P < 2 x 10(-16)). Sotos syndrome was clinically diagnosed in 99% of NSD1-positive individuals, independent of the molecular analyses, indicating that NSD1 aberrations are essentially specific to this condition. Furthermore, our data suggest that 93% of patients who have been clinically diagnosed with Sotos syndrome have identifiable NSD1 abnormalities, of which 83% are intragenic mutations and 10% are 5q35 microdeletions. We reviewed the clinical phenotypes of 239 NSD1-positive individuals. Facial dysmorphism, learning disability, and childhood overgrowth were present in 90% of the individuals. However, both the height and head circumference of 10% of the individuals were within the normal range, indicating that overgrowth is not obligatory for the diagnosis of Sotos syndrome. A broad spectrum of associated clinical features was also present, the occurrence of which was largely independent of genotype, since individuals with identical mutations had different phenotypes. We compared the phenotypes of patients with intragenic NSD1 mutations with those of patients with 5q35 microdeletions. Patients with microdeletions had less-prominent overgrowth (P = .0003) and more-severe learning disability (P = 3 x 10(-9)) than patients with mutations. However, all features present in patients with microdeletions were also observed in patients with mutations, and there was no correlation between deletion size and the clinical phenotype, suggesting that the deletion of additional genes in patients with 5q35 microdeletions has little specific effect on phenotype. We identified only 13 familial cases. The reasons for the low vertical transmission rate are unclear, although familial cases were more likely than nonfamilial cases (P = .005) to carry missense mutations, suggesting that the underlying NSD1 mutational mechanism in Sotos syndrome may influence reproductive fitness.

Coregulator Interactions with the Thyroid Hormone Receptor

The thyroid hormone receptor (TR) directly regulates the transcription of thyroid hormone-responsive genes in response to changing levels of thyroid hormone. Mechanistically TR utilizes a complex set of binding interactions, with hormone, response elements, and coregulatory proteins, to provide specific local control of patterns of transcriptional response that are partially responsible for inducing the tissue-selective responses to the circulating hormone. One of the apparently dominant phenomena in the regulation of thyroid hormone responses is the protein interactions between TR and its coregulators. This review summarizes the current state of knowledge with respect to the identity of these coregulators, their interaction with TR, and the consequences of those interactions.

Characterization of the Interaction between Retinoic Acid Receptor/Retinoid X Receptor (RAR/RXR) Heterodimers and Transcriptional Coactivators through Structural and Fluorescence Anisotropy Studies

Retinoid receptors (RARs and RXRs) are ligand-activated transcription factors that regulate the transcription of target genes by recruiting coregulator complexes at cognate promoters. To understand the effects of heterodimerization and ligand binding on coactivator recruitment, we solved the crystal structure of the complex between the RARbeta/RXRalpha ligand-binding domain heterodimer, its 9-cis retinoic acid ligand, and an LXXLL-containing peptide (termed NR box 2) derived from the nuclear receptor interaction domain (NID) of the TRAP220 coactivator. In parallel, we measured the binding affinities of the isolated NR box 2 peptide or the full-length NID of the coactivator SRC-1 for retinoid receptors in the presence of various types of ligands. Our correlative analysis of three-dimensional structures and fluorescence data reveals that heterodimerization does not significantly alter the structure of individual subunits or their intrinsic capacity to interact with NR box 2. Similarly, we show that the ability of a protomer to recruit NR box 2 does not vary as a function of the ligand binding status of the partner receptor. In contrast, the strength of the overall association between the heterodimer and the full-length SRC-1 NID is dictated by the combinatorial action of RAR and RXR ligands, the simultaneous presence of the two receptor agonists being required for highest binding affinity. We identified an LXXLL peptide-driven mechanism by which the concerted reorientation of three phenylalanine side chains generates an "aromatic clamp" that locks the RXR activation helix H12 in the transcriptionally active conformation. Finally, we show how variations of helix H11-ligand interactions can alter the communication pathway linking helices H11, H12, and the connecting loop L11-12 to the coactivator-binding site. Together, our results reveal molecular and structural features that impact on the ligand-dependent interaction of the RAR/RXR heterodimer with nuclear receptor coactivators.

Purification and crystallization of the heterodimeric complex of RARbeta and RXRalpha ligand-binding domains in the active conformation.

The ligand-binding domains of the retinoid X receptor alpha (RXRalpha) and of the retinoic acid receptor beta (RARbeta) were overexpressed separately and copurified in the heterodimeric form. Using a crystallization solution containing sodium formate and PEG 3350 as precipitant, the heterodimer was cocrystallized with the promiscuous ligand 9-cis-retinoic acid (9C-RA) and a 13-residue fragment of the nuclear receptor interaction domain (NID) of the transcriptional coactivator TRAP220. The crystals grew in the trigonal space group P3(1)21, with unit-cell parameters a = b = 115.7, c = 247.2 angstroms and two heterodimers per asymmetric unit. X-ray diffraction data were collected to 2.9 angstroms resolution. The structure was solved by molecular replacement and is currently being refined.

Interactions between Activating Signal Cointegrator-2 and the Tumor Suppressor Retinoblastoma in Androgen Receptor Transactivation

Activating signal cointegrator-2 (ASC-2), a cancer-amplified transcription coactivator of nuclear receptors and numerous other transcription factors, was previously shown to contain two LXXLL motifs, each of which interacts with a distinct set of nuclear receptors. In this work, we showed that ASC-2 has an indirect, separate binding site for androgen receptor (AR). Interestingly, this region overlapped with the direct interaction interfaces with the tumor suppressor retinoblastoma (Rb). Although ASC-2 alone stimulated AR transactivation in cotransfections of HeLa cells, ectopic expression of Rb effected ASC-2 to act as a transcription coactivator of AR in Rb-null Saos2 cells. These results, along with the previous report in which AR was shown to directly interact with Rb (Yeh, S., Miyamoto, H., Nishimura, K., Kang, H., Ludlow, J., Hsiao, P., Wang, C., Su, C., and Chang C. (1998) Biochem. Biophys. Res. Commun. 248, 361-367), suggest that the AR-ASC-2 interactions in vivo may involve Rb. Thus, ASC-2 appears to contain at least three distinct nuclear receptor interaction domains.

Activating signal cointegrator 2 required for liver lipid metabolism mediated by liver X receptors in mice.

Activating signal cointegrator 2 (ASC-2), a cancer-amplified transcriptional coactivator of nuclear receptors and many other transcription factors, contains two LXXLL-type nuclear receptor interaction domains. Interestingly, the second LXXLL motif is highly specific to the liver X receptors (LXRs). In cotransfection, DN2, an ASC-2 fragment encompassing this motif, exerts a potent dominant-negative effect on transactivation by LXRs, which is rescued by ectopic coexpression of the full-length ASC-2 but not by other LXXLL-type coactivators, such as SRC-1 and TRAP220. In contrast, DN2/m, in which the LXXLL motif is mutated to LXXAA to abolish the interactions with LXRs, is without any effect. Accordingly, expression of DN2, but not DN2/m, in transgenic mice results in phenotypes that are highly homologous to those previously observed with LXRalpha(-/-) mice, including a rapid accumulation of large amounts of cholesterol and down-regulation of the known lipid-metabolizing target genes of LXRalpha in the liver upon being fed a high-cholesterol diet. These results identify ASC-2 as a physiologically important transcriptional coactivator of LXRs and demonstrate its pivotal role in the liver lipid metabolism.