Intracellular Localization Patterns of Cancer‐associated Thyroid Hormone Receptor Mutants

Abstract

The thyroid hormone receptor (TR) is a nuclear receptor that plays a critical role in human development and metabolism, regulating thyroid hormone interactions. TR rapidly shuttles between the nucleus and cytoplasm but is primarily localized to the nucleus at steady state, where it acts as a hormone‐dependent transcription factor. This process of nuclear import and export is mediated by two nuclear localization signals (NLSs) and multiple nuclear export signals (NESs). Previous studies have shown that TR may act as a tumor suppressor by regulating key genes in cell growth and proliferation in response to thyroid hormone. However, if TR shuttling is misregulated, it will not be able to perform its transcriptional regulatory functions. In this project, we analyzed the impact of NLS and NES amino acid substitutions on the intracellular localization patterns of TRα1 mutants previously associated with hepatocellular carcinoma (G24E, M256V, E343A, P269L) and thyroid cancer (K29T, C97A). We transfected mammalian cells with expression plasmids for green fluorescent protein (GFP)‐tagged and mCherry‐tagged TRα1 mutants and examined localization patterns through quantitative fluorescence microscopy. Here, we show that both TRα1 mutations exhibit increased cytosolic distributions. Further, mutant TRα1 (K29T, C97A) had an increased tendency to form nuclear and cytosolic protein aggregates. In co‐transfection experiments, TRα1 (K29T, C97A) failed to co‐localize with LC3, a well‐characterized autophagy marker, but showed significant co‐localization with GFP‐170, a marker for aggresome formation. These data suggest that aggregates formed by TRα1 (K29T, C97A) are actively recruited to cellular aggresomes as a cellular stress response. Overall, our study provides evidence that even minor changes in TR’s primary structure can have a significant impact on its intracellular localization and folding patterns.Support or Funding InformationThis project was funded by the National Institutes of Health. Grant information: 2018–2021, P.I., “Thyroid Hormone Receptor Mislocalization and Pathogenesis,” National Institutes of Health (NIH‐NIDDK) Research Grant # 2 R15 DK058028‐05: $405,273