Structure-function analysis of the orphan nuclear hormone receptors NR4A1 and NR4A3

Wansa, K. Disnika Senali Abayratna (2004). Structure-function analysis of the orphan nuclear hormone receptors NR4A1 and NR4A3 PhD Thesis, School of Chemistry & Molecular Bioscience, The University of Queensland.

       
Attached Files (Some files may be inaccessible until you login with your UQ eSpace credentials)
Name Description MIMEType Size Downloads
THE17425.pdf Full text application/pdf 36.13MB 12
Author Wansa, K. Disnika Senali Abayratna
Thesis Title Structure-function analysis of the orphan nuclear hormone receptors NR4A1 and NR4A3
School, Centre or Institute School of Chemistry & Molecular Bioscience
Institution The University of Queensland
Publication date 2004
Thesis type PhD Thesis
Total pages 200
Collection year 2004
Language eng
Subjects 03 Chemical Sciences
0304 Medicinal and Biomolecular Chemistry
030401 Biologically Active Molecules
Formatted abstract

NR4A1-3 (Nur77, NURRl, and NOR1) are 'orphan members' of the nuclear hormone receptor superfamily. These nuclear receptors are classified as immediate early response genes, which are induced through multiple signal transduction pathways. They have been implicated in cell proliferation, differentiation, steroidogenesis, apoptosis, chondrosarcoma's, neurological disorders, inflammation and atherogenesis. However, the mechanism of coactivation and ligand independent and dependent transactivation remains unclear. Using transient transfection assays and the GAL4 hybrid system we examined the molecular basis of Nur77 and NOR-1-mediated transactivation of gene expression in myogenic and nonmyogenic cell lines. We observed that Nur77 and NOR-1 transactivate gene expression in a cell specific manner. Nur77 and NOR-1 operated in an activation function-1 (AF-1) dependent manner. The AB region of Nur77 and NOR-1 encoded an uncommonly potent N-terminal AF-1 domain delimited to between amino acids 50 -160 and 1-112 respectively. The N-terminal AF-1 domain was essential for the ligand independent activation of gene expression. Interestingly, SRC-2 (GRIP-1) modulated the activity of the N-terminal AF-1 domain of Nur77 and NOR-1 and not the LBD of either NR. In vitro GST-pull down assays demonstrated that, the N-terminal AB region of Nur77 and NOR-1 (not the LBD) facilitated coactivator recruitment and directly interacts with SRC-2, p300, PCAF, and TRAP220/DRIP205. Consistent with this, homology modelling indicated that the Nur77 and NOR-1 LBD coactivator binding cleft was substantially different from that of RARƴ and RORβ, a closely related AF-2 dependent receptors. In particular, the characteristic hydrophobic cleft of NRs was replaced with a much more hydrophilic surface with a distinct topology. This observation accounts for the inability of these nuclear receptor LBDs to directly mediate cofactor recruitment. Furthermore, we demonstrated that the AF-1 domain of both Nur77 and NOR-1 physically associates with the C-terminal LBD, and that this intramolecular interaction was potentiated by AF-1 mediated recruitment of SRC-2. 

 

Nur77 (not NOR-1) can heterodimerise with RXR and mediate transactivation in response to RXR-specific agonists from hormone response elements (HREs) with direct repeats (DR5). In a GAL4-hybrid assay in the presence of 9-cis-RA, RXRƴ-LBD increased the activity of Nur77-LBD, and this activity was further potentiated in the presence of SRC-2. We further demonstrated that the Nur77 AF-1 domain synergises with RXRƴ LBD in a retinoid dependent manner and enhanced Nur77 mediated transactivation of gene expression. The Nur77 AF-1 domain could directly interact with RXRƴ in agreement with the GAL4-hybrid assay. Thus, the AF-1 domain plays a major role in Nur77 and NOR-1 mediated transcriptional activation, cofactor recruitment, and intra- and inter- molecular interactions. 

 

In the search for small molecule regulators for the NR4A1-3 subfamily we identified (in collaboration with X-ceptor therapeutics) the purine anti-metabolite 6-Mercaptopurine (6- MP), a widely used anti-neoplastic and anti-inflammatory drug as an activator of the NR4A1- 3 subfamily (Nur77, NURR1 and NOR-1). This suggested that the signalling pathways that inhibit proliferation via inhibition of de novo purine and/or nucleic acid biosynthesis are involved in the regulation NR4A1-3 activity. Interestingly we found that in contrast to other small molecule compounds that target/bind the LBD, 6-MP targeted the AF-1 domain of NOR-1 and potentiated NOR-1 mediated transactivation of gene expression. The AB region of the NR4A1-3 subfamily has been shown to be modulated by kinase signalling pathways that results in a increase or decrease of the transcriptional activity of this subfamily. Therefore a potential mechanism for NR4A1-3 activation by 6-MP was postulated to be posttranslational modification. To determine the accuracy of this hypothesis we mutated several serine and threonine residues implicated in kinase-dependent regulation in the AF-1 domain. Mutation of these residues did not compromise the ability of 6-MP to activate NOR- 1. Furthermore, to determine the specificity and selectivity of 6-MP we investigated whether general trans-activators, myogenic transactivators, primary or secondary coactivators could be activated by 6-MP. We observed that only the secondary coactivator TRAP220 was activated by 6-MP. The region of TRAP220 that was activated by 6-MP was delimited to amino acids 1-800. In this study we showed that NOR-1 interacted with TRAP220 and that the AF-1 domain of NOR-1 supports the recruitment of TRAP220, in the presence of SRC-2 and p300. This indicated that the activation of the AF-1 domain of NOR-1 by 6-MP also involves the activation of TRAP220. In conclusion we hypothesize that the NR4A1-3 subgroup mediates the genotoxic stress response, and suggest that this subgroup may function as sensors, which respond to genotoxicity. 

Keyword Hormone receptors -- Analysis

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
 
Citation counts: Google Scholar Search Google Scholar
Created: Wed, 24 Oct 2012, 17:59:21 EST by Mr Lachlan Wong on behalf of Library Corporate Services