The thyroid hormone receptor variant α2 is a weak antagonist because it is deficient in interactions with nuclear receptor corepressors

The thyroid hormone receptor splice variant, α2, is unable to bind thyroid hormone (T₃) and has been proposed to function as an endogenous inhibitor of T₃ action. In this report, we examined further the DNA sequence requirements for α2 binding to thyroid hormone response elements (TREs) in an attempt to identify response elements that mediate potent inhibition by α2. Heterodimers of α2 and retinoid X receptor were found to bind to a subset of TREs (DR4, direct repeats spaced by 4 bp) in which selected flanking and spacer sequences enhanced interactions with the AGGTCA core binding sequence. Despite the optimization of the TRE-binding sites, α2 remained a weak dominant negative inhibitor of TRE-driven transcription. A promoter interference assay was also developed for testing inhibition by α2. In these studies, α2 blocked gene transcription, but it required cotransfected retinoid X receptor, and it was not as potent as unliganded thyroid hormone receptors. These results led to the hypothesis that (α2 might be deficient in interactions with nuclear receptor corepressors. Consistent with this view, α2 did not silence basal transcription in its native form or when linked to Gal4. α2 also failed to interact with corepressors (NCoR and SMRT) in both gel shift assays and mammalian two- hybrid assays. We conclude that α2 is a weak antagonist of thyroid hormone action because it binds weakly to a limited repertoire of response elements, and it does not interact with corepressors. Thus, α2 may be able to compete with thyroid hormone receptors for binding to a limited group of target sites, but it is not able to actively inhibit transcription.