DNA-binding mechanism of the monomeric orphan nuclear receptor NGFI-B.

The 2.7 A X-ray crystal structure of the DNA-binding domain (DBD) of the orphan nuclear receptor, nerve growth factor-induced-B (NGFI-B), complexed to its high-affinity DNA target, represents the first structure analysis of a nuclear receptor DBD bound as a monomer to DNA. The structure of the core DBD and its interactions with the major groove of the DNA are similar to previously crystallographically solved DBD-DNA complexes in this superfamily; however, residues C-terminal to this core form a separate and unique substructure that interacts extensively and in a sequence-specific way with the minor groove of its DNA target, in particular with the characteristic 3 A-T base-pair identity element that extends 5' to the usual nuclear receptor half-site (AGGTCA).     

Constitutive activation of retinoic acid receptor beta2 promoter by orphan nuclear receptor TR2

The orphan nuclear receptor TR2 functions as a constitutive activator for the endogenous retinoic acid receptor beta2 (RAR(beta2)) gene expression in P19 embryonal carcinoma cells and for reporters driven by the RAR(beta2) promoter in COS-1 cells. The activation of RAR(beta2) by TR2 is mediated by the direct repeat-5 (DR5) element located in the RAR(beta2) promoter. Furthermore, cAMP exerts an enhancing effect on the activation of RAR(beta2) by TR2, which is mediated by the cAMP response element located in the 5'-flanking region of the DR5. The constitutive activation function-1 (AF-1) of TR2 is mapped to amino acid residues 10-30 in its N-terminal A segment. A direct molecular interaction occurs between CREMtau and TR2, detected by co-immunoprecipitation, which is mediated by the N-terminal AB segment of TR2. In gel mobility shift assays, TR2 competes with P19 nuclear factor binding to the RAR(beta2) promoter, and TR2 and CREMtau bind simultaneously to this DNA fragment. The role of TR2 in the early events of RA signaling process is discussed.     

Recent advances in the TR2 and TR4 orphan receptors of the nuclear receptor superfamily

The human testicular receptor 2 (TR2) and TR4 orphan receptors are two evolutionarily related proteins belonging to the nuclear receptor superfamily. Numerous TR2 and TR4 variants and homologs have been identified from different species, including vertebrates (e.g. human, murine, rabbit, fish, and amphibian) and invertebrates (e.g. Drosophila, sea urchin, and nematode) since TR2 was initially isolated over a decade ago. Specific tissue distribution, genomic organization, and chromosomal assignment of both orphan receptors have been investigated. In order to reveal the physiological functions played by both TR2 and TR4, upstream modulators of TR2 and TR4 gene expression, their downstream target gene regulation, feedback mechanisms, and differential modulation mediated by the recruitment of other nuclear receptors and coregulators have been investigated. Studies summarized in the present report have provided unexpected insights into the TR2 and TR4 functions in a variety of biological processes. The essential and difficult tasks of identifying orphan receptor ligands, agonist/antagonist assignment, their physiological functions, and mechanisms of action will continue to challenge nuclear receptor researchers in the future.     

Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3

The Bcl-2 family proteins are key regulators of apoptosis in human diseases and cancers. Though known to block apoptosis, Bcl-2 promotes cell death through an undefined mechanism. Here, we show that Bcl-2 interacts with orphan nuclear receptor Nur77 (also known as TR3), which is required for cancer cell apoptosis induced by many antineoplastic agents. The interaction is mediated by the N-terminal loop region of Bcl-2 and is required for Nur77 mitochondrial localization and apoptosis. Nur77 binding induces a Bcl-2 conformational change that exposes its BH3 domain, resulting in conversion of Bcl-2 from a protector to a killer. These findings establish the coupling of Nur77 nuclear receptor with the Bcl-2 apoptotic machinery and demonstrate that Bcl-2 can manifest opposing phenotypes, induced by interactions with proteins such as Nur77, suggesting novel strategies for regulating apoptosis in cancer and other diseases.     

Feedback regulation between orphan nuclear receptor TR2 and human papilloma virus type 16

The human TR2 orphan receptor (TR2), initially isolated from testis and prostate cDNA libraries, is a member of the steroid receptor superfamily. TR2 can regulate several target genes via binding to a consensus response element (AGGTCA) in direct repeat orientation (AGGTCAX((n))AGGTCA, n = 0-6). Here we show that TR2 is able to induce the expression of human papilloma virus type 16 (HPV-16) genes via binding to a DR4 response element in the long control region of HPV-16. Additionally, one of the HPV-16 gene products, the E6 oncogene, regulates TR2 gene expression. A likely mechanism for this regulation involves E6-mediated degradation of the tumor suppressor p53, a protein known to suppress TR2 expression. Together our data provide evidence for feedback regulation between TR2 and HPV-16, which represents a novel regulatory pathway involving a member of the steroid receptor superfamily and the HPV-16 DNA tumor virus.     

TR3 orphan nuclear receptor mediates apoptosis through up-regulating E2F1 in human prostate cancer LNCaP cells

Early studies suggested both TR3 orphan receptor (TR3) and apoptosis mediator E2F1 might play an important role in mediating prostate cancer cell apoptosis. Their linkage and relationship, however, remain unclear. Here we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) could induce cell apoptosis via induction of TR3 and E2F1 expression in LNCaP prostate cancer cells. Addition of antisense E2F1 could partially rescue the TR3-mediated cell apoptosis, and transfection of the TR3 dominant-negative plasmid could block the TR3-induced E2F1 expression. These data suggest that TPA is able to induce LNCaP cell apoptosis via induction of TR3 resulting in the induction of E2F1. Promoter reporter assays show that TR3 can induce E2F1 expression via binding to the TR3 response element (TR3RE) in the E2F1 promoter -316 to -324 bp region. TR3 can bind specifically to this TR3RE with a Kd of 6.29 nm, and mutations of this E2F1-TR3RE can partially block the TR3-mediated E2F1 expression. Taken together, these data suggest that TPA is able to induce cell apoptosis via a TPA --> TR3 --> E2F1 --> apoptosis pathway in LNCaP cells. Further studies of how to modulate this pathway may allow us to better understand how to control the prostate cancer growth.     

The nuclear orphan receptor TR4 promotes proliferation of myeloid progenitor cells.

Nuclear receptors represent key regulators in cell proliferation, differentiation, and development. Here we demonstrate that the nuclear orphan receptor TR4 is highly expressed in hematopoietic cells and tissues and have analyzed the impact of TR4 in this cell compartment. We show that TR4, when ectopically expressed in bone marrow cells via retrovirus vector, promotes proliferation of myeloid progenitor cells. Cells represent promyelocytes as judged by morphological features, expression of cell surface molecules, and specific markers like Mim-1 and CAAT/enhancer binding protein beta. We also demonstrate that the growth promoting activity of TR4 is not exclusively dependent on its association with DNA, because expression of a mutated TR4 version devoid of its DNA binding domain exhibits a similar proliferative potential as wild-type TR4. In conclusion, these data position the orphan receptor TR4 as an important regulator of myeloid progenitor cell proliferation and development.     

孤儿受体TR3在小鼠睾丸中的定位和表达

本文采用原位杂交和免疫组织化学技术,观察孤儿受体ＴＲ３及其ｍＲＮＡ在小鼠睾丸中的表达及细胞定位。结果表明,在小鼠睾丸中有显著量的孤儿受体ＴＲ３ ｍＲＮＡ和蛋白表达,其表达量在不同曲细精管有明显的差异;孤儿受体ＴＲ３蛋白主要定位于生精细胞,其ｍＲＮＡ在生精细胞特异表达,主要在精原细胞和发育早期的初级精母细胞表达,提示孤儿受体ＴＲ３在小鼠曲细精管精子发生的早期阶段中起着调控作用。     

Probing conformational changes in orphan nuclear receptor: the NGFI-B intermediate is a partially unfolded dimer

Human nerve growth factor-induced B (NGFI-B) is a member of the NR4A subfamily of orphan nuclear receptors (NRs). Lacking identified ligands, orphan NRs show particular co-regulator proteins binding properties, different from other NRs, and they might have a non-classical quaternary organization. A body of evidence suggests that NRs recognition of and binding to ligands, DNA, homo- and heterodimerization partners and co-regulator proteins involve significant conformational changes of the NR ligand-binding domains (LBDs). To shed light on largely unknown biophysical properties of NGFI-B, here we studied structural organization and unfolding properties of NGFI-B ligand (like)-binding domain induced by chemical perturbation. Our results show that NGFI-B LBD undergoes a two-state guanidine hydrochloride (GndHCl) induced denaturation, as judged by changes in the alpha-helical content of the protein monitored by circular dichroism spectroscopy (CD). In contrast, changes in the tertiary structure of NGFI-B LBD, reported by intrinsic fluorescence, reveal a clear intermediate state. Additionally, SAXS results demonstrate that the intermediate observed by intrinsic fluorescence is a partially folded homodimeric structure, which further unfolds without dissociation at higher GndHCl concentrations. This partially unfolded dimeric assembly of NGFI-B LBD might resemble an intermediate that this domain access momentarily in the native state upon interactions with functional partners.     

Mxi2 promotes stimulus-independent ERK nuclear translocation

Spatial regulation of ERK1/2 MAP kinases is an essential yet largely unveiled mechanism for ensuring the fidelity and specificity of their signals. Mxi2 is a p38alpha isoform with the ability to bind ERK1/2. Herein we show that Mxi2 has profound effects on ERK1/2 nucleocytoplasmic distribution, promoting their accumulation in the nucleus. Downregulation of endogenous Mxi2 by RNAi causes a marked reduction of ERK1/2 in the nucleus, accompanied by a pronounced decline in cellular proliferation. We demonstrate that Mxi2 functions in nuclear shuttling of ERK1/2 by enhancing the nuclear accumulation of both phosphorylated and unphosphorylated forms in the absence of stimulation. This process requires the direct interaction of both proteins and a high-affinity binding of Mxi2 to ERK-binding sites in nucleoporins, In this respect, Mxi2 acts antagonistically to PEA15, displacing it from ERK1/2 complexes. These results point to Mxi2 as a key spatial regulator for ERK1/2 and disclose an unprecedented stimulus-independent mechanism for ERK nuclear import.