Variable transcriptional activity and ligand binding of mutant beta 1 3,5,3'-triiodothyronine receptors from four families with generalized resistance to thyroid hormone.

Abstract

Mutations in the gene encoding the human beta 1 T3 receptor (hTR beta 1) have been associated with generalized resistance to thyroid hormone (GRTH). We measured the T3-binding affinity and transcriptional regulatory capacity of the mutant hTR beta 1 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 hTR beta 1s were able to inhibit the function of transfected normal hTR beta 1 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 hTR beta 1s 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 hTR beta 1 was assessed by immunocytochemistry. The hTR beta 1 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 hTR beta 1 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.

Interaction of human beta 1 thyroid hormone receptor and its mutants with DNA and retinoid X receptor beta. T3 response element-dependent dominant negative potency.

“…It has been hypothesized that the mutant h-TR,13 inhibits the function of the normal h-TR by a dominant negative mechanism. This was confirmed by in vitro studies demonstrating that h-TR1 I from patients with mutations in the carboxy-terminal part of the LBD have not only reduced T3-binding and transcriptional activity, but are also able to inhibit normal h-TRa1 and h-TR1 I function in transient transfection systems (7)(8)(9).…”

“…After excising the rRXR,8 cDNA from pKS-RXRB with EcoRI/BstXI, the ends were blunt-ended with T4 DNA polymerase. HindIll linkers were added and the fragment was ligated into the HindIII site ofthe pSV2 eucaryotic expression vector (9). The construct was verified by restriction analyses.…”

Mice with a targeted mutation in the thyroid hormone β receptor gene exhibit impaired growth and resistance to thyroid hormone

“…On the basis of the available in vitro biochemical evidence, the mechanism of competition of the wild-type TRs with mutants for binding to TRE is currently favored (mechanism i) (7,25). However, the present in vivo data suggest that the possibility of formation of inactive wild-type͞mutant heterodimers on TRE is also a viable mechanism (mechanism iii).…”

Thyroid hormone receptor β mutants: Dominant negative regulators of peroxisome proliferator-activated receptor γ action

“…PV has a C-insertion at codon 448 that produces a frame-shift in the carboxyl-terminal 14 amino acids of TR␤1 (4). PV has completely lost T3 binding and exhibits potent dominant negative activity (7). Patient PV has only one mutated TR␤ allele and exhibits short stature and delayed bone age, similar to other heterozygous RTH patients (5).…”