LACK OF COACTIVATOR INTERACTION CAN BE A MECHANISM FOR DOMINANT-NEGATIVE ACTIVITY BY MUTANT THYROID-HORMONE RECEPTORS

We studied the interactions of two natural thyroid hormone receptor (T R) mutants from patients with resistance to thyroid hormone (RTH) and an artificial TR mutant with a nuclear receptor corepressor, N-CoR, an d a steroid receptor coactivator, SRC-I. In electrophoretic mobility s hift assays, wild-type TR beta-1 interacted with N-CoR in the absence of ligand, whereas T-3 caused dissociation of the TR beta-1/N-CoR comp lex and formation of TR beta-1/SRC-1 complex. In contrast, a natural m utant (G345R) with poor T-3-binding affinity formed TR beta-1/N-CoR co mplex, both in the absence and presence of T-3, but could not form TR beta-1/SRC-1 complex. Another TR mutant, which bound T-3 with normal a ffinity and containing a mutation in the AF-2 region (E457D), had norm al interactions with N-CoR but could not bind SRC-1. Both these mutant s had strong dominant negative activity on wild-type TR transactivatio n. Studies with a TR mutant that had slightly decreased T-3-binding af finity (R320H) showed a T-3-dependent decrease in binding to N-CoR and increase in binding to SRC-1 that reflected its decreased ligand bind ing affinity. Additionally, when N-CoR and SRC-1 were added to these r eceptors at various T-3 concentrations in electrophoretic mobility shi ft assays, TR/N-CoR and TR/SRC-1 complexes, but not intermediate compl exes were observed, suggesting that N-CoR release is necessary before SRC-1 binding to TR. Our data provide new insight on the molecular mec hanisms of dominant negative activity in RTH and suggest that the inab ility of mutant TRs to interact with coactivators such as SRC-1, which results from reduced T-3-binding affinity, is a determinant of domina nt negative activity.