DIVERGENT DIMERIZATION PROPERTIES OF MUTANT BETA-1 THYROID-HORMONE RECEPTORS ARE ASSOCIATED WITH DIFFERENT DOMINANT-NEGATIVE ACTIVITIES

Syndromes of resistance to thyroid hormones are caused by mutations in the T-3-binding domain of the c-erbA beta thyroid hormone receptor ge ne. The S receptor (deletion of THR332) is a potent dominant negative protein cloned from a kindred with generalized resistance to thyroid h ormones. The G-H receptor (ARG311HIS) has compromised dominant negativ e function and was found in both normal individuals and in a patient w ith severe pituitary resistance to thyroid hormones. We have investiga ted the mechanism responsible for the difference in receptor phenotype s by analyzing the binding of S and G-H receptors to thyroid hormone r esponse elements with electrophoretic mobility shift analysis. Wild-ty pe human c-erbA beta 1 (WT), S, and G-H receptors were synthesized in reticulocyte lysate, reacted with a thyroid hormone response element c onsisting of a direct repeat with 4 base pairs (DR+4; AGGTCA CAGG AGGT CA), and the products analyzed by gel shift. G-H receptor homodimeriza tion was greatly impaired; G-H formed predominantly monomeric complex compared with monomeric and homodimeric WT complexes. The G-H receptor was able to form heterodimeric complexes with cellular thyroid hormon e receptor auxiliary protein (TRAP) factors including the human retino id X receptor-alpha. When TRAP was limiting, the levels of G-H heterod imeric complex were 2- to 3-fold reduced compared with Wi receptor. In contrast to the WT and G-H receptors, the S receptor formed almost ex clusively homodimeric complex with DR+4; the approximate ratio of S:WT :G-H homodimeric complexes at equivalent concentrations of receptors w as 60:20:1. A measurable increase (1.2- to 2.6-fold) in heterodimeric complex formation was observed with the S receptor relative to WT when TRAP was at limiting concentration. As reported previously by others, thyroid hormone significantly reduced the WT homodimeric complex with DR+4. There was no effect on the S homodimeric complex. Finally, the WT, S, and G-H receptors formed different complexes with the element c onsisting of an inverted repeat with 5 base pairs (IR+5; AGGTCA ACAGT TGACCT) and the IR element (AGGTCA TGACCT), which were differently reg ulated by thyroid hormone. The S receptor bound as a homodimer with IR +5, whereas the WT receptor bound as a homodimer only with thyroid hor mone. No homodimeric complex formed with IR+5 and the G-H receptor. Qu alitatively similar results were observed with the IR element. We conc lude that the ARG311HIS mutation severely perturbs the homodimerizatio n and, to a much less degree, heterodimerization functions of the c-er bAB1 receptor. Furthermore, the THR332 deletion mutation augments homo dimerization of the c-erbA81 receptor. These results indicate that dif ferent mutations in the c-erbAB1 thyroid hormone receptor have diverge ntly affected dimerization activities which seem to influence the leve l of dominant negative activity in man.