SAT-559 Intranuclear Dynamics of RTHα Mutant Thyroid Hormone Receptors

Abstract

: Resistance to Thyroid Hormone α (RTHα), a reduced sensitivity to thyroid hormone (T3) in peripheral tissues, is caused by mutations in thyroid hormone receptor α (TRα), a nuclear receptor that regulates gene expression when bound to T3. These mutations often result in receptors incapable of binding T3. All mutations characterized in RTHα, thus far, have been found in the ligand binding domain (LBD), some of which result in truncated proteins lacking all or part of helix 12. Some studies have shown that Nuclear Receptor Corepressor 1 (NCoR1), which interacts with the hinge region of TRα (the region between the DNA-binding domain and LBD) has a higher affinity for RTHα mutants compared to wild-type TRα, supporting the hypothesis that helix 12 of the LBD also functions to disassociate NCoR1 from TRα when it is bound to T3 (1, 2). We proposed that this increased affinity for NCoR1 alters the mobility of the TRα in the nucleus, impacting its function. As a first step in in testing this model, we used Fluorescence Recovery after Photobleaching (FRAP) to examine the effects of select RTHα mutations, Ala382ProfsX7 and F397fs406X, on nuclear localization and intranuclear mobility. After transfecting HeLa cells with expression plasmids for green fluorescent protein (GFP)-tagged wild-type TRα and each of the mutants, we first assessed their intracellular distribution and initial intranuclear mobility. Although wild-type TRα is known to shuttle between the nucleus and cytoplasm, it is primarily localized to the nucleus at a steady state. Here we show that TR-Ala382ProfsX7 and F397fs406X are also primarily localized to the nucleus. FRAP revealed that wild-type TRα is highly dynamic within the nucleus, with recovery half-times ranging from 0.74 to 0.95 seconds and 97.4% (n=3, 20 cells per replicate) of TRα in the mobile fraction, indicating that the receptor rapidly dissociates and reassociates with DNA binding sites and/ or other nuclear binding sites. There was no significant difference in intranuclear dynamics between wild-type TRα and the RTHα mutants, TR-Ala382ProfsX7 and F397fs406X. Next, we wanted to determine the effects of T3 on intranuclear mobility and found that recovery half-times and the percentage of receptors in the mobile fraction were T3 independent for both wild-type TRα and RTHα mutants. Investigation of the effects of overexpression or knock down of NCoR1 will provide further insight into the impact of altered binding affinity for NCoR1 on the intranuclear dynamics of RTHα mutants.