DNA binding affinity of hTRbeta1 mutants as heterodimers with traps from different tissues

Patients with generalized resistance to thyroid hormone (GRTH) show various organ-specific features, for example mental retardation, growth abnormalities, liver damage, delayed bone age or cardiac disorders. Could this reflect aberrant mutant thyroid hormone receptor beta1 (TRbeta1) heterodimerization with specific TR auxiliary proteins (TRAPs) from different tissues, altering the mutant's ability to transactivate tissue-specific genes? To answer this question, we examined the heterodimerization of TRbeta1 mutants and TRAPs of several rat tissues (cerebrum, cerebellum, liver, heart, lung, spleen, and kidney), and in vitro translated RXRalpha, beta and gamma by electrophoretic gel mobility shift assay (EMSA). Mutant TRbeta1 proteins, synthesized in reticulocyte lysate, were incubated with 32P rat malic enzyme (rME) thyroid hormone response elements (TRE) and nuclear extracts of rat tissues. The TRbeta1 mutants used were Mf (G345R), and GH (R316H). Both have non-detectable T3 binding affinity. GH has weak dominant negative effect and Mf has strong dominant negative effect. Two major bands were observed in EMSA. Cerebrum, cerebellum, lung and liver extracts formed a slower migrating band than a TR homodimer, while kidney extracts formed a faster migrating band, and heart and spleen extracts had both bands. There were no qualitative differences in heterodimerization between TRbeta1wt, and TRbeta1 mutants, when using tissue extracts and DNA in excess ratio to TR. We found that RXRalpha, beta, and gamma were differentially expressed in each rat tissue and formed heterodimer complexes with wild type (WT) TRbeta1. Scatchard analysis of affinity and capacity of the binding of TR-TRAP heterodimers to response elements was performed by competing with 2.5-, 5-, 10-, 25-, and 250-fold excess non-radiolabeled rME-TRE. When using kidney extract, the DNA binding affinity of heterodimers was significantly decreased both in wild type and mutant TRs, suggesting that the DNA binding affinity of the faster migrating band was lower than that of the slower migrating band. Mutant GH, which causes 'pituitary RTH' and shows weak dominant negative effect, tended to form heterodimers with lower DNA binding affinity than TRbeta1wt with all extracts. Mutant Mf, which has strong dominant negative effect, tended to show higher DNA binding affinity than TRbeta1WT. When the data were pooled for all tissues, GH and Mf were found to form heterodimers with significantly lower, or higher, affinity for TREs than TRbeta1wt. These results indicate that: 1) differences of DNA binding affinity of mutant TR-TRAP heterodimers to response elements in DNA play a part in its reduced or strong dominant negative effect; and 2) differences in formation of heterodimers with TRAPs present in tissues do not appear to explain the apparent tissue-specific and mutant-specific variations seen in RTH.