Mutations in the Conserved C-Terminal Sequence in Thyroid Hormone Receptor Dissociate Hormone-Dependent Activation from Interference with AP-1 Activity

A short C-terminal sequence that is deleted in the v-ErbA oncoprotein and conserved in members of the nuclear receptor superfamily is required for normal biological function of its normal cellular counterpart, the thyroid hormone receptor alpha (T3R alpha). We carried out an extensive mutational analysis of this region based on the crystal structure of the hormone-bound ligand binding domain of T3R alpha. Mutagenesis of Leu398 or Glu401, which are surface exposed according to the crystal structure, completely blocks or significantly impairs T3-dependent transcriptional activation but does not affect or only partially diminishes interference with AP-1 activity. These are the first mutations that clearly dissociate these activities for T3R alpha. Substitution of Leu400, which is also surface exposed, does not affect interference with AP-1 activity and only partially diminishes T3-dependent transactivation. None of the mutations affect ligand-independent transactivation, consistent with previous findings that this activity is mediated by the N-terminal domain of T3R alpha. The loss of ligand-dependent transactivation for some mutants can largely be reversed in the presence of GRIP1, which acts as a strong ligand-dependent coactivator for wild-type T3R alpha. There is excellent correlation between T3-dependent in vitro association of GRIP1 with T3R alpha mutants and their ability to support T3-dependent transcriptional activation. Therefore, GRIP1, previously found to interact with the glucocorticoid, estrogen, and androgen receptors, may also have a role in T3R alpha-mediated ligand-dependent transcriptional activation. When fused to a heterologous DNA binding domain, that of the yeast transactivator GAL4, the conserved C terminus of T3R alpha functions as a strong ligand-independent activator in both mammalian and yeast cells. However, point mutations within this region have drastically different effects on these activities compared to their effect on the full-length T3R alpha. We conclude that the C-terminal conserved region contains a recognition surface for GRIP1 or a similar coactivator that facilitates its interaction with the basal transcriptional apparatus. While important for ligand-dependent transactivation, this interaction surface is not directly involved in transrepression of AP-1 activity.