Abstract: Characterizing the Subcellular Localization of Mutant TRa1 (Ala382ProfsX7)

Wild-type GFP-TR1

Thyroid hormone (TH) is received by thyroid hormone receptors (TRs), resulting in ligand-dependent interactions with TH-target genes. There are multiple forms of TRs which are expressed in different tissues; the TRa1 subtype is found predominantly in the central nervous system, bone, the gastrointestinal tract, and cardiac/skeletal muscle (Moran et al., 2013). Ala382ProfsX7 is a novel heterozygous mutation found in TRa1, implicated in Resistance to Thyroid Hormone Syndrome (RTH).

As a critical step in regulating gene expression, TRa1 shuttles between the nucleus and cytoplasm (Bunn et al., 2001). The Ala382ProfsX7 mutation deletes the second nuclear export domain in TRa1 (Mavinakere, Powers, Subramanian, Roggero, & Allison, 2012). This potential disruption of standard TRa1 shuttling may contribute to the phenotype of RTH. Characterizing the subcellular localization of Ala382ProfsX7 expands upon the knowledge of RTH pathology and may offer targets for future treatment.

Using a liposome-based protocol, I will introduce DNA into cells via expression plasmids for wild-type GFP-TRa1 and mutant mCherry-Ala382ProfsX7. The cell cultures will be stained for visibility, fixed to slides, and viewed under an epifluorescent microscope. I will rank the relative intensity of fluorescence between the nucleus and cytoplasm and to generate a nuclear-to-cytoplasmic ratio of fluorescence which will indicate the relative TR localization for each treatment. These results will be analyzed to determine whether the expression of Ala382ProfX7 or its coexpression with wild-type TRa1 has a statistically significant effect on the localization of thyroid hormone receptor. If there is no difference in the nuclear-to-cytoplasmic ratio of localization, I will use heterokaryon shuttling assays and fluorescence recovery after photobleaching (FRAP) to analyze the ability of the mutant to be exported.