Variable transcriptional activity and ligand binding of mutant β1 3,5,3′-triiodothyronine receptors from four families with generalized resistance to thyroid hormone

Mutations in the gene encoding the human β1 T₃ receptor (hTRβ1) have been associated with generalized resistance to thyroid hormone (GRTH). We measured the T₃-binding affinity and transcriptional regulatory capacity of the mutant hTRβ1 from four unrelated kindreds with GRTH. These mutations are contained in different functional regions of the ligand-binding domain. The T₃ affinity of the mutant receptors correlated well with the degree of impairment of their trans-activating function in a transient cotransfection system in HeLa cells; two mutant receptors with undetectable ligand affinity showed no transcriptional activity, whereas the two other mutants characterized by a 2- and 5-fold reduction in T₃ affinity required 5- and 15-fold higher T₃ concentrations for half-maximal activity in the cotransfection assay, respectively. All of the mutant hTRβ1s were able to inhibit the function of transfected normal hTRβ1 and endogenous retinoic acid receptor in activating a palindromic positive T₃ response element (TRE). In the partially functional mutants this dominant negative effect could be completely reversed by increased T₃ concentrations. The dominant negative potency did not depend on the type of TRE used; mutant hTRβ1s were able to inhibit normal receptor function to the same degree on a dimer-permissive palindromic TRE as on a nondimer-permissive inverted repeat of two identical half-sites separated by five spacer bases. However, the dominant negative potency was dependent on the absolute amount of receptor expression vector transfected. The expression of normal and mutant hTRβ1 was assessed by immunocytochemistry. The hTRβ1 protein levels in HeLa cells paralleled the amount of transfected expression vector. Moreover, all the mutant receptors were properly expressed in the nuclei of the transfected cells. These data suggest that different mutations in the ligand-binding domain of the human hTRβ1 result in a variable degree of functional impairment, which may partially explain the phenotypic differences between kindreds with GRTH. Our findings suggest that competition for binding to the TRE and possibly the binding of limiting accessory factors may be more important in mediating the dominant negative effect than the formation of normal/mutant T₃ receptor dimers.