DNA binding affinity of hTR beta 1 mutants as heterodimers with traps fromdifferent tissues

Patients with generalized resistance to thyroid hormone (GRTH) show various organ-specific features, for example mental retardation, growth abnormalit ies, liver damage, delayed bone age or cardiac disorders. Could this reflec t aberrant mutant thyroid hormone receptor beta 1 (TR beta 1) heterodimeriz ation with specific TR auxiliary proteins (TRAPs) from different tissues, a ltering the mutant's ability to transactivate tissue-specific genes? To ans wer this question, we examined the heterodimerization of TR beta 1 mutants and TRAPs of several rat tissues (cerebrum, cerebellum, liver, heart, lung, spleen, and kidney), and in vitro translated RXR alpha, beta and gamma by electrophoretic gel mobility shift assay (EMSA). Mutant TR beta 1 proteins, synthesized in reticulocyte lysate, were incubated with P-32 rat melic enz yme (rME) thyroid hormone response elements (TRE) and nuclear extracts of r at tissues. The TR beta 1 mutants used were Mf (G345R), and GH (R316H). Bot h have non-detectable T3 binding affinity. GH has weak dominant negative ef fect and Mf has strong dominant negative effect. Two major bands were obser ved in EMSA. Cerebrum, cerebellum lung and liver extracts formed a slower m igrating band than a TR homodimer, while kidney extracts formed a faster mi grating band, and heart and spleen extracts had both bands. There were no q ualitative differences in heterodimerization between TR beta 1wt, and TR be ta 1 mutants, when using tissue extracts and DNA in excess ratio to TR. We found that RXR alpha, beta, and gamma were differentially expressed in each rat tissue and formed heterodimer complexes with wild type (WT) TR(31. Sca tchard analysis of affinity and capacity of the binding of TR-TRAP heterodi mers 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 th e faster migrating band was lower than that of the slower migrating band. M utant GH, which causes 'pituitary RTH' and shows weak dominant negative eff ect, tended to form heterodimers with lower DNA binding affinity than TR be ta 1wt with all extracts. Mutant Mf, which has strong dominant negative eff ect, tended to show higher DNA binding affinity than TR beta 1WT. 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 TR beta 1wt. T hese results indicate that: 1) differences of DNA binding affinity of mutan t TR-TRAP heterodimers to response elements in DNA play a part in its reduc ed or strong dominant negative effect; and 2) differences in formation of h eterodimers with TRAPs present in tissues do not appear to explain the appa rent tissue-specific and mutant-specific variations seen in RTH. (C) Societ e francaise de biochimie et biologie moleculaire / Elsevier, Paris.