Structural and Biochemical Investigations of the Thyroid Hormone Receptor and v-erbA Oncoprotein
- Author(s): Slivka, Eric John
- Advisor(s): Fletterick, Robert J
- et al.
The thyroid hormone receptor is a ligand-dependent transcription factor crucial for the regulation of a wide variety of processes involved in metabolism, differentiation, and development. While much has been learned about the structure of the thyroid hormone receptor and the molecular determinants of hormone binding, a complete understanding of these determinants has not yet been obtained.
In order to better understand the molecular determinants of hormone binding in the thyroid hormone receptor, v-erbA, a naturally occurring form of the receptor found in a retrovirus, was used as a platform for mutational studies. The use of v-erbA, which is severely compromised in its ability to bind thyroid hormone and activate transcription, provided a convenient and natural template upon which targeted mutations could be made.
Mutational analysis on ligand binding domain constructs determined that the addition of a missing helix (Helix 12) near the C-terminus of v-erbA was insufficient to restore ligand binding ability to the receptor, but an additional single point mutation, S395F, near the same location was capable of restoring hormone affinity to within ten-fold of that of wild-type thyroid hormone receptor. Protein stability assays based on protease degradation and thermal denaturation also demonstrated this constructs improved stability over other v-erbA constructs.
Transcriptional data obtained using full-length constructs in transient transfection assays demonstrated that the S395F mutation significantly enhanced the ability of v-erbA to activate transcription in the presence of hormone. While both v-erbA and v-erbA + Helix 12 constructs not only failed to activate transcription in the presence of hormone but maintained repression seen in the absence of hormone, the v-erbA + Helix 12 S395F mutant activated transcription even beyond the level seen with wild-type thryroid hormone receptor.