A role for helix 3 of the TRβ ligand-binding domain in coactivator recruitment identified by characterization of a third cluster of mutations in resistance to thyroid hormone

Resistance to thyroid hormone (RTH) has hitherto been associated with thyroid hormone beta receptor (TR beta) mutations which cluster in two regions (alpha alpha 310-353 and alpha alpha 429-461) of the hormone-binding domain and closely approximate the ligand-binding cavity, Here, we describe a third cluster of RTH mutations extending from alpha alpha 234-282 which constitute a third boundary of the ligand pocket. One mutant, T277A, exhibits impaired transactivation which is disproportionate to its mildly reduced ligand affinity (K-a), T3-dependent recruitment of coactivators (SRC-1, ACTR) by mutant receptor-RXR heterodimers was reduced in comparison with wild-type. Cotransfection of SRC-1 restored transactivation by T277A, In the TRP crystal structure this helix 3 residue is surface-exposed and is in close proximity to residues L454 and E457 in helix 12 which are known to be critical for coactivator interaction, suggesting that they all constitute part of a receptorcoactivator interface. The transcriptional function of other mutants (A234T, R243W/Q, A268D, Delta 276I, A279V, R282S) in this cluster correlated with their reduced K-a and they inhibited wild-type TR beta action in a dominant negative manner. DNA binding, heterodimerization and corepressor recruitment were preserved in all mutants, signifying the importance of these attributes for dominant negative activity and correlating with the absence of natural mutations in regions bordering the third cluster which mediate these functions.