GENETIC DISSECTION OF THYROID-HORMONE RECEPTOR-BETA - IDENTIFICATION OF MUTATIONS THAT SEPARATE HORMONE-BINDING AND TRANSCRIPTIONAL ACTIVATION

The thyroid hormone receptors (TR) are members of the nuclear receptor family of ligand-mediated transcription factors. The large region of TR that lies C-terminal to its DNA-binding domain subserves functions of ligand binding, dimerization, and transactivation. Little is known regarding the structural or functional determinants of these processes . We have utilized genetic screening in the yeast Saccharomyces cerevi siae to identify residues involved in these functions. Random mutation s of the rat TR beta 1 isoform between amino acid residues 179 and 456 were screened, and mutants with reduced hormone-dependent activation of reporter gene activity were isolated. In this paper we describe the characterization of a class of mutants that exhibit a dissociation be tween hormone binding and transcriptional activation. These mutants re tained hormone binding (>15% of the wild-type level) yet failed to tra nsactivate a reporter gene. A number of these mutations occurred withi n the D region, which links the DNA-binding and ligand-binding domains of the receptor. One subset of these mutations abrogated DNA binding, supporting a role of the D region in this process. The remainder reta in DNA binding and thus highlight residues critical for receptor activ ation. In addition, an unexpected group of ''superactivator'' mutation s that led to enhanced hormone-dependent activation in S. cerevisiae w ere found. These mutations localized to the carboxy-terminal portion o f the receptor in a region which contains elements conserved across th e superfamily of nuclear receptors. The hormone-dependent phenotype of these superactivator mutations suggests an important role of this seg ment in ligand-mediated transcriptional activation.