Master of Science (M.Sc.)
Lizabeth A Allison
Diane C Shakes
The thyroid hormone receptor α1 (TRα1) and the thyroid hormone receptor β1 (TRβ1) are transcription factors that modulate the expression of target genes that are important in metabolism and development in response to thyroid hormone. Although primarily localized to the nucleus, prior studies have shown that TRα1 and TRβ1 shuttle rapidly between the nucleus and cytoplasm, and that nuclear import of TRα1 is directed by two nuclear localization signal (NLS) motifs: NLS-1 in the hinge domain, and NLS-2 in the N-terminal A/B domain. In contrast, TRβ1 lacks NLS-2. Previous studies also characterized two nuclear export signal (NES) motifs, NES-H3/H6 and NES-H12, that reside in the ligand-binding domain and mediate TR nuclear export. Here, we investigated which importins mediate nuclear import of TRa1 using a combined approach of shRNA-mediated knockdown and coimmunoprecipitation assays in HeLa (human) cells. Among all the importins we tested in transient transfection assays (importins 4, 5, 6, 7, 8, 9, 13, importin β1, and adaptor importin α variants), only importin 7, importin β1, and adaptor importin α1 knockdown experiments resulted in a significant localization pattern change from primarily nuclear to a more cytosolic distribution of TRα1. To demonstrate direct interaction between TRα1 or TRβ1 and these importins, “GFP-trap” co-immunoprecipitation assays were performed. Importin 7, importin β1, and adaptor importin α1 were shown to interact with TRα1, while importin 4 as a negative control, did not. Our data show that nuclear entry of TRα1 in HeLa cells is facilitated by both importin 7, likely through interactions with NLS-2, and importin β1 and the adapter importin α1 interacting with NLS-1 and NLS-2. In contrast, TRα1 nuclear import is facilitated only by importin α1/β1 interacting with NLS-1. Prior results from knockdown and overexpression studies provided evidence that multiple exportins influence TR localization. Here, we investigated which exportins serve as a direct carrier for each of the multiple NES motifs in TRα1, using “GFP-trap” coimmunoprecipitation assays. The information would be provided in Chapter 5. Consistent with our prior studies, results show protein-protein interactions between TRα1 and XPO4, XPO5, and XPO7, but not with XPO6. Taken together, our findings highlight a fine balance of nuclear import, retention, and export that modulates TR function.
© The Author
Jibo, Zhang, "Thyroid Hormone Receptor Nucleocytoplasmic Transport" (2016). Dissertations, Theses, and Masters Projects. Paper 1477068484.