A NOVEL MULTIFUNCTIONAL MOTIF IN THE AMINO-TERMINAL A B DOMAIN OF T3R-ALPHA MODULATES DNA-BINDING AND RECEPTOR DIMERIZATION/

We reported previously that deletion of the 50-amino acid NH2-terminal A/B domain of the chicken (c) or rat thyroid hormone (T-3) receptor-a lpha (T3R alpha) decreased the T-3-dependent stimulation of genes regu lated by native thyroid hormone response elements (TREs). This require ment of the NH2-terminal A/B domain for transcriptional activation was mapped to amino acids 21-30 of cT(3)R alpha. Expression of transcript ion factor IIB (TFIIB) in cells was shown to enhance T-3-dependent tra nscriptional activation by cT(3)R alpha, and this enhancement by TFIIB was dependent on the same 10-amino acid sequence. In vitro binding st udies indicated that cT(3)R alpha interacts efficiently with TFIIB, an d this interaction requires amino acids (KRKRK27)-K-23 in the A/B doma in. In this study we document the functional importance of these five basic residues in transcriptional activation by cT(3)R alpha, further supporting the biological significance of these residues and their int eraction with TFIIB. Interestingly, we also find that the same amino a cids also affect DNA binding and dimerization of cT(3)R alpha. Gel mob ility shift assays reveal that a cT(3)R alpha mutant that has all five basic amino acids changed from (KRKRK27)-K-23 to (TITIT27)-T-23 binds to a palindromic TRE predominantly as a homodimer, whereas cT(3)R alp ha with the wild-type (KRKRK27)-K-23 sequence binds predominantly as a monomer. This results from both a marked decrease in the ability of t he cT(3)R alpha mutant to bind as a monomer and from an enhanced abili ty to dimerize as reflected by an increase in DNA-bound T3R-retinoic X receptor heterodimers. These effects of (KRKRK27)-K-23 on DNA binding , dimerization, transcriptional activation, and the association of T3R alpha with TFIIB support the notion that this basic amino acid motif may influence the overall structure and function of T3R alpha and, thu s, play a role in determining the distinct context-dependent transacti vation potentials of the individual T3R isoforms.