Quantitative analysis of DNA binding affinity and dimerization properties of wild-type and mutant thyroid hormone...

Thyroid hormone (triiodothyronine [T3]) actions are mediated through binding of thyroid hormone receptors (TRs) to specific DNA sequences (thyroid hormone response elements [TREs]) as monomers, homodimers, and heterodimers with thyroid hormone receptor auxiliary proteins (TRAPs). We quantitatively characterized dimerization of wild-type (WT) and mutant TRbetas by coimmunoprecipitation, and binding to DNA by electrophoretic gel mobility shift assays (EMSA). Binding affinities of TR retinoid X receptor-alpha (RXRalpha) heterodimers to DNA were determined by competing with excess nonradiolabeled TREs in EMSA. TRs in vitro synthesized in reticulocyte lysates (RL), and human RXRalpha expressed in a Sf9 cell-baculovirus system (BAC), were coincubated with 32P-labeled rat malic enzyme (ME), palindromic (PAL), or chicken lysozyme F2 (F2) TREs. The mutant TRbetas tested were R316H and G345R, which have nondetectable T3 binding and have previously been reported to show weak and potent dominant negative effect, respectively. Scatchard analysis showed no significant differences in Kas between WT and mutant TR-RXRalpha heterodimers binding to DNA. We measured affinity of heterodimerization between TRs and RXRalpha in solution in the absence of DNA, and by coimmunoprecipitation using anti-TRbeta1WT specific antibodies. 35S-labeled RL-RXRalpha was incubated with BAC-WT or TRbeta or R316H in the absence or presence of increasing amounts of nonlabeled BAC-RXRalpha. Displacement curves were obtained by counting radioactivity of precipitated 35S-RXRalpha, that was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Kds of WT and TRbeta R316H heterodimerizing with RXRalpha were approximately the same. Binding affinity of TR homodimers for F2-TRE was studied because this TRE binds homodimers strongly. Scatchard analysis clearly showed that DNA binding affinity of BAC-WT homodimers did not differ with or without 100 nM T3, but maximal binding capacity (MBC) was reduced three-fold to fourfold in the presence of 100 nM T3. In contrast, BACTRbeta-R316H homodimers showed a fivefold reduction in DNA binding affinity for F2, both in the presence and absence of T3, and approximately the same MBC as WT in the absence of T3. Mutant RL-G345R homodimers showed approximately the same Ka as RL-WT homodimers for binding to F2 and the same MBC in the presence and absence of T3. These results indicate that (1) T3 reduced TRbeta homodimerization in solution but does not effect DNA binding of formed homodimers; (2) T3 does not influence DNA binding affinity of TR/RxR heterodimers; and (3) TRbeta mutant R316H homodimers have reduced DNA binding affinity but homodimerization and heterodimerization in solution does not differ from WT TRbeta.