Date Thesis Awarded
Honors Thesis -- Access Restricted On-Campus Only
Bachelors of Science (BS)
The thyroid hormone receptors, TRα1, TRβ1, and TRβ2, are nuclear receptors that regulate key physiological and developmental processes in a thyroid hormone-dependent manner. Although primarily nuclear localized, TRs rapidly shuttle between the nucleus and cytoplasm through nuclear pore complexes via nuclear localization signals (NLS) and nuclear export signals (NES) that interact with specific importins and exportins. We hypothesized that shuttling may be altered in mutant isoforms of TR found in Resistance to Thyroid Hormone (RTH) syndrome. Previous research in our lab showed that the overexpression of Myc-tagged exportin 5 (XPO5) or Myc-tagged exportin 7 (XPO7) produced a significant cytosolic shift of wild-type (WT) GFP-TRα1. Because RTHα-A382PfsX7 is missing the NES on Helix 12 (NES-H12), we hypothesized that the overexpression of either XPO5 or 7 would not cause a significant cytosolic shift if NES-H12 is required for interaction with exportins. Co-transfection of HeLa cells with GFP-tagged exportin and either WT mCherry-TRα1 or mCherry-RTHα-A382PfsX7, followed by fluorescence-based nucleocytoplasmic ratio (N/C) scoring revealed that overexpression of GFP-XPO5 has no significant effect on the localization of WT TRα1 or RTHα-A382PfsX7. This suggests that, in contrast to the Myc tag, the GFP tag may alter the functional activity of XPO5. In contrast, we found that the overexpression of GFP-XPO7 causes a significant cytosolic shift in both wild-type TRα1 and RTHα-A382PfsX7, indicating that NES-H12 is not essential for interaction with XPO7.
Intracellular localization can also be influenced by post-translational modifications of TRs. A prior study in our lab revealed that the acetylation of three lysines within the NLS of TRβ1 produced a significant cytosolic shift in comparison to wild-type TR. Here, we investigated whether acetylation of all three sites is required for altered localization, by transfecting HeLa cells with GFP-TRβ1 single or triple site acetylation mimics (K184Q, K188Q, K190Q), GFP-TRβ1 single or triple site non-acetylation mimics (K184R, K188R, K190R), and WT TRβ1. There was a significant decrease of N/C in each single site acetylation mimic, relative to WT. Average relative N/C’s of the single site acetylation mimics were not significantly different from the triple site (Q) acetylation mimic, indicating that the acetylation of one site alters intracellular localization to the same extent as the acetylation of all three sites. The average relative N/C’s of the single site non-acetylation mimics also were not significantly different from the triple site non-acetylation mimic or from wild-type TR, suggesting that the inability to acetylate either a single site or all three sites of TR does not impact intracellular localization. Finally, we validated the localization of TR subtypes tagged with the human influenza hemagglutinin (HA) tag. Since GFP itself can be acetylated, new expression plasmids have been made using the HA tag. We transfected HeLa cells with either wild-type, or triple site acetylation or non-acetylation mimics, of HA-TRα1, HA-TRβ1, or HA-TRβ2. All subtypes displayed similar localization patterns to GFP-tagged TRs, validating their efficacy for use in investigating the intracellular acetylation levels of TR. This thesis further characterizes the complex mechanisms regulating the intracellular shuttling of TR and provides new areas for future research into the pathogenic role of dysregulated TR intracellular shuttling.
White, Olivia, "Characterizing the Complex Mechanisms of Thyroid Hormone Receptor Intracellular Shuttling" (2023). Undergraduate Honors Theses. William & Mary. Paper 1989.
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