Hadzic E, Habeos I, Raaka B M, Samuels H H
Division of Molecular Endocrinology, Departments of Medicine and Pharmacology, New York University Medical Center, New York, New York 10016, USA.
J Biol Chem. 1998 Apr 24;273(17):10270-8. doi: 10.1074/jbc.273.17.10270.
We reported previously that deletion of the 50-amino acid NH2-terminal A/B domain of the chicken (c) or rat thyroid hormone (T3) receptor-alpha (T3Ralpha) decreased the T3-dependent stimulation of genes regulated by native thyroid hormone response elements (TREs). This requirement of the NH2-terminal A/B domain for transcriptional activation was mapped to amino acids 21-30 of cT3Ralpha. Expression of transcription factor IIB (TFIIB) in cells was shown to enhance T3-dependent transcriptional activation by cT3Ralpha, and this enhancement by TFIIB was dependent on the same 10-amino acid sequence. In vitro binding studies indicated that cT3Ralpha interacts efficiently with TFIIB, and this interaction requires amino acids 23KRKRK27 in the A/B domain. In this study we document the functional importance of these five basic residues in transcriptional activation by cT3Ralpha, 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 cT3Ralpha. Gel mobility shift assays reveal that a cT3Ralpha mutant that has all five basic amino acids changed from 23KRKRK27 to 23TITIT27 binds to a palindromic TRE predominantly as a homodimer, whereas cT3Ralpha with the wild-type 23KRKRK27 sequence binds predominantly as a monomer. This results from both a marked decrease in the ability of the cT3Ralpha mutant to bind as a monomer and from an enhanced ability to dimerize as reflected by an increase in DNA-bound T3R-retinoic X receptor heterodimers. These effects of 23KRKRK27 on DNA binding, dimerization, transcriptional activation, and the association of T3Ralpha with TFIIB support the notion that this basic amino acid motif may influence the overall structure and function of T3Ralpha and, thus, play a role in determining the distinct context-dependent transactivation potentials of the individual T3R isoforms.
