Date Thesis Awarded
Honors Thesis -- Open Access
Bachelors of Science (BS)
Mark H. Forsyth
Shantá D. Hinton
Thyroid hormone receptor (TR) is critical in many aspects of metabolic control and development. TR acts as a transcription factor when bound to thyroid hormone (T3 and T4) to change gene expression of thyroid hormone response elements (TREs). There are two main isoforms of TR: TRα and TRβ. TR can shuttle into and out of the nucleus via an importin or exportin-mediated pathway through the nuclear pore complex. The importin or exportin binds to TR at a nuclear localization sequence (NLS) or a nuclear export sequence (NES) to transport TR into or out of the nucleus, respectively. A well characterized nuclear export pathway is the cooperative CRM1/calreticulin-mediated export pathway. However, TRα and TRβ also can follow a CRM1-independent pathway for nuclear export. The main objective of this research was to determine if exportin 5 (XPO5) or RanBP17 mediate the export of TR via a CRM1- independent nuclear export pathway. XPO5 has been characterized as an export factor for microRNAs, as well as for the androgen receptor. RanBP17 has only been characterized as a potential exportin based on CRM1 homology. To determine if XPO5 mediates TR's export by the latter pathway, XPO5 was over-expressed in HeLa cells. The distribution and transcriptional activity of GFP-tagged TRα or TRβ was analyzed by fluorescence microscopy and CAT ELISA, respectively. TR is primarily nuclear at steady-state, but TR had a more cytoplasmic distribution when XPO5 was overexpressed. In addition, CAT reporter gene expression under control of a thyroid hormone response element was markedly decreased when XPO5 was over-expressed, indicating less TR was present in the nucleus. TR has multiple nuclear export sequences (NESs) in the ligand-binding domain. We tested the helix 12 NES (NES-H12) and the NES located in the 3rd and 6th helices (NES-H3/H6) on their interactions with XPO5. The NES-H12 construct had a more cytoplasmic distribution when XPO5 was over-expressed, suggesting that XPO5 utilizes NES-H12 during export. We also over-expressed RanBP17 in HeLa cells and analyzed any changes in distribution of GFP-TRα or TRβ, when analyzed by fluorescence microscopy and CAT ELISA, respectively. Our microscopy results suggest thatRanBP17 facilitates nuclear export of TRβ1 but not TRα, while the CAT ELISA results were inconclusive. In conclusion, our results suggest that XPO5 can mediate TR nuclear export via the NES located in helix 12 of the ligand binding domain. Further, RanBP17 may play a role in nuclear export of TRβ; however, replicate experiments are required to confirm this finding. Investigation of the role of other exportins, such as RanBP16/XPO7, is needed to fully characterize the multiple export pathways followed by TR. Further insight gained from the regulation of nuclear export of TR will ultimately enhance understanding of regulation of thyroid hormone-responsive gene expression and the role of TR in growth and development.
Nelson, Hallie N., "Characterization of Thyroid Hormone Receptor Export Pathways" (2013). Undergraduate Honors Theses. William & Mary. Paper 630.
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