Abstract
Thyroid hormone receptor (TR) is a member of the nuclear receptor superfamily that shuttles between the cytosol and nucleus. The fine balance between nuclear import and export of TR has emerged as a critical control point for modulating thyroid hormone-responsive gene expression; however, sequence motifs of TR that mediate shuttling are not fully defined. Here, we characterized multiple signals that direct TR shuttling. Along with the known nuclear localization signal in the hinge domain, we identified a novel nuclear localization signal in the A/B domain of thyroid hormone receptor α1 that is absent in thyroid hormone receptor β1 and inactive in the oncoprotein v-ErbA. Our prior studies showed that thyroid hormone receptor α1 exits the nucleus through two pathways, one dependent on the export factor CRM1 and the other CRM1-independent. Here, we identified three novel CRM1-independent nuclear export signal (NES) motifs in the ligand-binding domain as follows: a highly conserved NES in helix 12 (NES-H12) and two additional NES sequences spanning helix 3 and helix 6, respectively. Mutations predicted to disrupt the α-helical structure resulted in a significant decrease in NES-H12 activity. The high degree of conservation of helix 12 suggests that this region may function as a key NES in other nuclear receptors. Furthermore, our mutagenesis studies on NES-H12 suggest that altered shuttling of thyroid hormone receptor β1 may be a contributing factor in resistance to thyroid hormone syndrome. Taken together, our findings provide a detailed mechanistic understanding of the multiple signals that work together to regulate TR shuttling and transcriptional activity, and they provide important insights into nuclear receptor function in general.
Highlights
Thyroid hormone receptor (TR) shuttles between the cytosol and nucleus and regulates gene expression in response to hormone
The G3-Hinge fusion protein has a number of important properties; it can be tracked in real time in transiently transfected cells; it is too large for diffusion through the nuclear pore complex, and it serves the purpose of demonstrating that a putative nuclear localization signal (NLS) is sufficient to target G3, a cytosolic protein, to the nucleus
The expression vector for G3-Hinge was transfected into HeLa cells, and the fusion protein was analyzed for its ability to localize to the nucleus or cytosol, in comparison with GFPtagged full-length TR␣1 and G3 alone (Fig. 1B)
Summary
Thyroid hormone receptor (TR) shuttles between the cytosol and nucleus and regulates gene expression in response to hormone. In this report we have identified a novel NLS, designated NLS-2, in the A/B domain of TR␣1 that is absent in TR1, and a novel, conserved CRM1-independent NES in the AF-2 region of helix 12 in the TR␣1 LBD, designated NES-H12 Mutations in this NES markedly reduce both nuclear export and transactivation of thyroid hormone-mediated gene expression. Our mutagenesis studies point to the intriguing possibility that altered shuttling of TR1, due to defective nuclear export, may be a contributing factor in resistance to thyroid hormone syndrome These findings highlight the complexity of the signals that regulate the shuttling of TR and emphasize the importance of detailed molecular characterization for understanding nuclear receptor function
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