Insight Into Molecular Determinants of T3 vs T4 Recognition From Mutations in Thyroid Hormone Receptor α and β

Abstract

Context: The two major forms of circulating thyroid hormones (THs) are T3 and T4. T3 is regarded as the biologically active hormone because it binds to TH receptors (TRs) with greater affinity than T4. However, it is currently unclear what structural mechanisms underlie this difference in affinity.

Objective: Prompted by the identification of a novel M256T mutation in a resistance to TH (RTH)α patient, we investigated Met256 in TRα1 and the corresponding residue (Met310) in TRβ1, residues previously predicted by crystallographic studies in discrimination of T3 vs T4.

Methods: Clinical characterization of the RTHα patient and molecular studies (in silico protein modeling, radioligand binding, transactivation, and receptor-cofactor studies) were performed.

Results: Structural modeling of the TRα1-M256T mutant showed that distortion of the hydrophobic niche to accommodate the outer ring of ligand was more pronounced for T3 than T4, suggesting that this substitution has little impact on the affinity for T4. In agreement with the model, TRα1-M256T selectively reduced the affinity for T3. Also, unlike other naturally occurring TRα mutations, TRα1-M256T had a differential impact on T3- vs T4-dependent transcriptional activation. TRα1-M256A and TRβ1-M310T mutants exhibited similar discordance for T3 vs T4.

Conclusions: Met256-TRα1/Met310-TRβ1 strongly potentiates the affinity of TRs for T3, thereby largely determining that T3 is the bioactive hormone rather than T4. These observations provide insight into the molecular basis for underlying the different affinity of TRs for T3 vs T4, delineating a fundamental principle of TH signaling.

Figure 1.

(a) Pedigree chart demonstrating that only the index patient (II.1) has the clinical phenotype of RTHα. (b) Sequence analysis of exon 8 of the THRA gene shows a de novo heterozygous missense mutation (c.767T>C) in the index patient, resulting in a Met to Thr substitution at codon 256 (p.M256T).

Figure 2.

Comparison of the architecture of the TRα1 ligand binding pocket in the presence of T3 and T4. (a and b) Close-up view of the ligand-binding pocket of the TRα1 crystal structure in complex with (a) T3 (PDB ID: 2h77) and (b) T4. The residue side-chains lining the niche that accommodates the outer ring of T3 and T4 are highlighted, and their molecular surface is shown except for Phe405 for clarity. The 5′-iodine group of T4 is represented by the green ball in the T4-bound TRα1 model. The hydrophobic contacts between Met256 and the phenolic outer ring are depicted as dashed lines. (c and d) Structural models of the TRα1-M256T mutant in complex with (c) T3 and (d) T4. (e and f) Structural models of the TRα1-M256A mutant in complex with (e) T3 and (f) T4. (g and h) Overlay of the structural orientation of the residue side-chains that face the (g) T3 and (h) T4 ligands at the 5′ position in WT (gray), M256T (blue), and M256A (red) mutant TRα1 models. All figures were created in YASARA Structure using PovRay imaging software.

Figure 3.

(a, c, e) [¹²⁵I]T3 dissociation curves showing that compared with (a) WT, (c) the TRα1-M256T mutation, and (e) TRα1-M256A mutation reduces the affinity for T3 (solid line) more than for T4 (dashed line) (mean ± SEM of three experiments for WT and M256T and two experiments for M256A performed in duplicate). (b, d, f) The TRα1-M256T and TRα1-M256A mutations also had a larger effect on T3- than on T4-dependent transcriptional activation (mean ±SEM of three experiments performed in triplicate). The effect of the Ala substitution on the ligand binding affinity and the transcriptional activity of TRα1 was less than the effect of the Thr substitution. The insert in (b) shows immunoblots confirming an equal expression of WT, M256T, and M256A FLAG-tagged TRα1 and Histone 3 as a loading control in the nuclear fraction of JEG-3 cells.

Figure 4.

The TRα1-M256T mutation had a larger effect on T3- than on T4-dependent (a and b) GAL4-NCoR1 dissociation and (c and d) GAL4-SRC1 association (mean ± SEM of at least three experiments performed in triplicate). The insert in (a) shows immunoblots confirming an equal expression of WT and M256T VP16 TRα1 fusion proteins and Histone 3 as loading control in the nuclear fraction of JEG-3 cells.

Figure 5.

(a–c) The T4-induced transcriptional activity of three TRα1 mutations identified in RTHα patients is lower than that is induced by T3, which is similar to WT [Fig. 2(d)] (mean ± SEM of three experiments performed in triplicate). (d) The EC50 of T4 is ∼30- to 50-fold higher than the EC50 of T3, except for TRα1-M256T. ***P < 0.001 (one-way ANOVA with Tukey post test).

Figure 6.

The T3- and T4-induced transcriptional activity of (a) WT and (b) TRβ1-M310T in JEG-3 cells shows that the TRβ1-M310T mutation affects T3- more than T4-dependent transcriptional activation (mean ± SEM of four experiments performed in triplicate), which is in line with the results of TRα1-M256T [Fig. 3(d)].