A novel multifunctional motif in the amino-terminal A/B domain of T sub(3)R alpha modulates DNA binding and receptor dimerization

We reported previously that deletion of the 50-amino acid NH sub(2)-terminal A/B domain of the chicken (c) or rat thyroid hormone (T sub(3)) receptor- alpha (T sub(3)R alpha ) decreased the T sub(3)-dependent stimulation of genes regulated by native thyroid hormone response elements (TREs). This requirement of the NH sub(2)-terminal A/B domain for transcriptional activation was mapped to amino acids 21-30 of cT sub(3)R alpha . Expression of transcription factor IIB (TFIIB) in cells was shown to enhance T sub(3)-dependent transcriptional activation by cT sub(3)R alpha , and this enhancement by TFIIB was dependent on the same 10-amino acid sequence. In vitro binding studies indicated that cT sub(3)R alpha interacts efficiently with TFIIB, and this interaction requires amino acids super(23)KRKRK super(27) in the A/B domain. In this study we document the functional importance of these five basic residues in transcriptional activation by cT sub(3)R alpha , further supporting the biological significance of these residues and their interaction with TFIIB. Interestingly, we also find that the same amino acids also affect DNA binding and dimerization of cT sub(3)R alpha . Gel mobility shift assays reveal that a cT sub(3)R alpha mutant that has all five basic amino acids changed from super(23)KRKRK super(27) to super(23)TITIT super(27) binds to a palindromic TRE predominantly as a homodimer, whereas cT sub(3)R alpha with the wild-type super(23)KRKRK super(27) sequence binds predominantly as a monomer. This results from both a marked decrease in the ability of the cT sub(3)R alpha mutant to bind as a monomer and from an enhanced ability to dimerize as reflected by an increase in DNA-bound T sub(3)R-retinoic X receptor heterodimers. These effects of super(23)KRKRK super(27) on DNA binding, dimerization, transcriptional activation, and the association of T sub(3)R alpha with TFIIB support the notion that this basic amino acid motif may influence the overall structure and function of T sub(3)R alpha and, thus, play a role in determining the distinct context-dependent transactivation potentials of the individual T sub(3)R isoforms.