Dominant negative activity of thyroid hormone receptor variant alpha2 and interaction with nuclear corepressors

Abstract

The splicing variant of the thyroid hormone receptor alpha (TRalpha) gene, TR variant alpha2 (TRv alpha2), lacks the second half of the ninth heptad, a domain thought to be important for heterodimerization with retinoid X receptors (RXRs). In transient transfection studies, TRv alpha2 exhibits weak dominant negative inhibition of TRalpha1-mediated transcription. In contrast, a TRv alpha2 mutant in which the ninth heptad was restored (alpha2 + 9H), exhibits very strong dominant negative activity. We have examined the role of nuclear corepressors (CoRs) in the dominant negative activity of TRv alpha2 and alpha2 + 9H. Glutathione S-transferase pull down experiments revealed that TRv alpha2 barely interacts with CoRs, whereas alpha2 + 9H interaction with CoRs is as strong as that of TRalpha1. A P160R CoR box mutation was introduced in the context of TRv alpha2 and alpha2 + 9H, which nearly abolishes the ability of these receptors to interact with CoRs. In transient transfection the dominant negative activity of TRv alpha2 was only marginally impaired by the P160R mutation. In contrast, alpha2 + 9H-P160R had approximately 66% less dominant negative activity than alpha2 + 9H. These results suggest that the weak dominant negative activity of TRv alpha2 is due in part to its lack of interaction with CoRs, and that restoration of the ninth heptad restores CoR interaction and strong dominant negative activity. Further, the data reveal aspects of the dominant negative action that are dependent on the orientation of the TRE.