Negative regulation by thyroid hormone receptor requires an intact
coactivator-binding surface
J. Clin. Invest. Tania M. Ortiga-Carvalho, et al. 115:2517 doi:10.1172/JCI24109 [
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Figure 1In vitro characterization of TR-β (E457A). (
A) Schematic representation of the TR-β isoforms and the location of the E457A mutation. Amino acid numbers are given for the human mutation in the mouse TR-β locus. DBD, DNA-binding domain; LBD, ligand-binding domain. (
B and
C) Gel mobility shift assay showing that the E457A receptor mutant binds to CoR (NCoR) but not to CoA (SRC-1) protein. RXR was added to all the lanes shown in
C. A positive TRE (DR+4 probe) was radiolabeled, and T
3 was added at a concentration of 10 nM. HD, heterodimer; UP, unprogrammed or non-specific. (
D) Gel mobility shift assay showing that the E457A receptor mutant does not bind to RIP140 in the presence of T
3 on the same radiolabeled DR+4 element. RXR and T
3 were added to all lanes. (
E) Effect of mutant TR-β (E457A) on positive and negative TREs. Ligand-dependent transcriptional activity of positively regulated reporter gene DR+4 LUC and negatively regulated reporter genes TRH LUC (human TRH –900 to +55 bp) and TSH-β LUC (human TSH –1192 to +37 bp). TK 109, a minimal thymidine kinase promoter in pA3Luc, was used as a control. Equal amounts of WT or mutant TR-β2–expressing plasmids or empty vector (SG5) were cotransfected into HEK 293 T cells with a luciferase reporter gene. The data are presented as fold change in activity compared with the level in the absence of T
3. Data represent 5 separate experiments performed in triplicate. *
P < 0.001 vs. no T
3; **
P < 0.001; #
P < 0.01.
