Studying the Nuclear Import of Thyroid Hormone Receptor Alpha using a Mammalian Two-Hybrid System

Publication Year:
2010
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Repository URL:
https://scholarworks.wm.edu/honorstheses/741
Author(s):
Giddens, John Patrick
thesis / dissertation description
Thyroid Hormone Receptor α1 (TRα) is a nuclear hormone receptor that plays a vital role in differentiation, development, and maintenance of homeostasis in mammals. TRα functions as a transcriptional repressor when thyroid hormone (TH) is not bound, but upon TH binding TRα undergoes a conformational change which causes TRα to function as a transcriptional activator. TRα shuttles rapidly between the nucleus and the cytoplasm across the nuclear membrane. This thesis research focused on the mechanisms regulating nuclear import of TRα. Prior studies have shown that TRα contains two nuclear localization signals (NLSs) and that TRα can be imported into the nucleus in vitro by the classical nuclear import pathway mediated by importin α and importin β. Here, to determine the specific mechanism of nuclear import and the specific importins that mediate this import in mammalian cells, both in vitro and in vivo binding methods were considered and were optimized for use. First, in order to perform in vitro binding experiments, optimization of protein expression of TRα and importin αs was performed with limited success as expression in E. coli proved extremely difficult even under a vast array of conditions. After it was clear that overexpression in E coli. was not possible with current expression vectors, a different method to study nuclear import of TRα was developed. A mammalian two-hybrid system was developed and optimized for use in both HeLa (human) and NIH3T3 (mouse) cells using positive controls. Genes encoding importin α isoforms were subcloned into the pACT vector and confirmed by sequencing while genes encoding TRα domains: A/B, DBD, Hinge, and LBD were unable to be successfully cloned into the pBIND vector due to time constraints as rapid screening was unable to be performed due to the small size of these inserts. Taken together this thesis research provides a powerful method to study protein-protein interactions among transcription factors and importins, which can be used to understand the mechanisms regulating both the nuclear import and export of TRα.