Abstract
Orphan receptor Nurr1 participates in the acquisition and maintenance of the dopaminergic cell phenotype, modulation of inflammation, and cytoprotection, but little is known about its regulation. In this study, we report that Nurr1 contains a bipartite nuclear localization signal (NLS) within its DNA binding domain and two leucine-rich nuclear export signals (NES) in its ligand binding domain. Together, these signals regulate Nurr1 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nurr1 is mostly nuclear. A Nurr1 mutant lacking the NLS failed to enter the nucleus. The Nurr1 NLS sequence, when fused to green fluorescent protein, led to nuclear accumulation of this chimeric protein, indicating that this sequence was sufficient to direct nuclear localization of Nurr1. Furthermore, two NES were characterized in the ligand binding domain, whose deletion caused Nurr1 to accumulate predominantly in the nucleus. The Nurr1 NES was sensitive to CRM1 and could function as an independent export signal when fused to green fluorescent protein. Sodium arsenite, an agent that induces oxidative stress, promoted nuclear export of ectopically expressed Nurr1 in HEK293T cells, and the antioxidant N-acetylcysteine rescued from this effect. Similarly, in dopaminergic MN9D cells, arsenite induced the export of endogenous Nurr1, resulting in the loss of expression of Nurr1-dependent genes. This study illustrates that Nurr1 shuttling between the cytosol and nucleus is controlled by specific nuclear import and export signals and that oxidative stress can unbalance the distribution of Nurr1 to favor its cytosolic accumulation.
Highlights
Little is known about the regulation of transcription factor Nurr1
In HEK293T cells transfected with an expression vector for Nurr1-V5, subcellular fractionation assays (Fig. 1C) and immunofluorescence staining (Fig. 1D) indicated that ectopically expressed Nurr1 exhibits a similar distribution as endogenous protein in dopaminergic cells, with a tendency to be located at the nucleus
We report for the first time that Nurr1 is regulated at the level of subcellular localization
Summary
Results: We identified a bipartite NLS and two NES signals implicated in its subcellular trafficking, and we report that oxidative stress induces nuclear export of Nurr. Orphan receptor Nurr participates in the acquisition and maintenance of the dopaminergic cell phenotype, modulation of inflammation, and cytoprotection, but little is known about its regulation. We report that Nurr contains a bipartite nuclear localization signal (NLS) within its DNA binding domain and two leucine-rich nuclear export signals (NES) in its ligand binding domain. Together, these signals regulate Nurr shuttling in and out of the nucleus. These results uncover a new mechanism of regulation of nuclear/cytoplasm shuttling of Nurr that explain how oxidative stress participates in loss of Nurr transcriptional activity
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