Ligand- and Nuclear Factor-Dependent Change in Hydrophobicity of Thyroid Hormone β 1 Receptor

Abstract

An aqueous two-phase partitioning assay was performed using in vitro translated human thyroid hormone β₁ receptor (TRβ₁). Wild-type TRβ₁ was less hydrophobic in the presence of both triiodothyronine (T₃) and nuclear extract. This reflects a conformational change, or change in electrostatic properties, of the TRβ₁-nuclear factor complex as a result of T₃ binding. Mutant TRβ₁s with reduced T₃ binding affinity required a higher concentration of T₃ for the shift of hydrophobicity, and a mutant without T₃ binding activity did not show any shift, even in the presence of 1 mM T₃. The unique mutant receptor, R243Q, has impaired transcriptional function despite virtually normal binding affinity for T₃. When this mutant was examined in this assay, the shift of hydrophobicity was significantly impaired even in the presence of both nuclear extract and a high concentration of T₃. Nuclear extact of COS1 cells did not affect the T₃-binding affinity of R243Q. These results indicate that the R243Q mutant has impaired a ligand-dependent conformational change and interaction with nuclear factor(s). Inability of R243Q to interact normally with nuclear factor(s) may explain, in part, the molecular mechanism of discordance between ligand binding and transactivation function of this mutant.