Date Thesis Awarded
Honors Thesis -- Access Restricted On-Campus Only
Bachelors of Science (BS)
Thyroid hormone receptor α1 (TRα1) is a nuclear receptor that is functionally dependent on its nucleocytoplasmic shuttling for gene regulation. Some sequence motifs responsible for import and export of the receptor have been previously identified, but its full range of nuclear export signal (NES) motifs has not been fully defined. We have previously determined the minimal, transferable sequence for a conserved NES in helix 12 of the receptor but the helix 3/helix 6 (H3/H6) bipartite NES, or monopartite NESs, was not fully characterized. Similarly, prior studies show that TRa1 also possesses a CRM1/calreticulin-dependent export pathway; however, the NES responsible for this mechanism is not yet defined. A candidate sequence located from residues 188-206 of the ligand binding domain has shown a potential role in export regulation. To study the role of these potential NES sequences in TRα1 nuclear shuttling, we performed transient transfection of mCherry-tagged fusion proteins in HeLa cells followed by scoring of the intracellular distribution by fluorescence microscopy. This was accomplished by designing protein constructs containing mCherry-maltose binding protein (MBP) (to create a fusion protein too large to diffuse through the nuclear pore complexes), the TRα1 hinge region NLS (to allow nuclear import), and each NES motif of interest excluding their overlapping residues. These motifs are from helix 3 residues 209-236 and helix 6 residues 239-265 (NES-H3 and NES-H6, respectively). A third fusion protein, H3/H6-219-254, was designed to include central residues from each helix (residues 219-254) to determine if the functional NES lies within the overlapping regions of the helices. Both NES-H3 and NES-H6 motifs show significant individual export function of localizing the mCherry-MBP-Hinge nuclear protein to the cytosol. The combined H3/H6-219-254 fragment and the CRM1-dependent candidate sequence LBD-188-206 did not show significant cytosolic localization when analyzed using quantitative and semi-quantitative scoring. These results suggest that the two helices each contain independently acting NES motifs. Furthermore, the inability of the overlapping residue sequence H3/H6 to shuttle proteins to the cytosol indicates that the functional regions of these sequences may lie towards the peripheral ends of each motif. Further work was done on NES-H3 and NES-H6 interaction with specific exportins. Prior studies show TRα1 localization is influenced by exportins 4, 5, and 7. To better understand the mechanism of NES-H3 and NES-H6-directed shuttling, we performed “RFP-trap” coimmunoprecipitation assays to determine which exportins interact specifically with these signals. However, results of these assays were inconclusive, and indicate an area for future work in analyzing TRα1-exportin interactions within the context of the full-length receptor. Ultimately, these multiple export signals emphasize the meticulous balance between nuclear import and export of TRα1 for its crucial role in gene regulation and metabolism.
Williams, Cheyenne, "Characterization of Novel Export Sequences in the Thyroid Hormone Receptor" (2016). Undergraduate Honors Theses. William & Mary. Paper 913.
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