Abstract
Background:
Natural mutant staggerer (sg) mice harbor a mutated retinoic acid receptor–related orphan receptor alpha (RORα). A genetic deletion corresponding to the ligand-binding domain (LBD) of RORα results in aberrant cerebellar development in the sg mice. These mice show similar neurotrophin expression to that seen in perinatal hypothyroid animals. RORα augments thyroid hormone receptor (TR)-mediated transcription, which may be partly responsible for the similar cerebellar abnormalities between sg and hypothyroid animals. The objective of this study is to examine further the mechanisms of augmentation of TR action by RORα. We examined whether TR directly binds to ROR and which regions of TR or ROR are required for the TR-ROR interaction.
Methods:
A transient transfection-based reporter gene assay was performed to measure the activity of TR-mediated transcription in CV-1 cells. To examine TR-RORα binding mammalian two-hybrid and glutathione-S-transferase (GST) pull-down assays were carried out.
Results:
Although full-length RORα augmented TRα1- or β1-mediated transcription, such augmentation was not observed with sg-type mutant RORα (RORsg) that contained the RORα N-terminal and DNA-binding domain (DBD) and a part of the LBD. On the other hand, the transcription of Gal4-DBD–fused TRβ1-LBD was suppressed by RORα, indicating that RORα does not interact with TR-LBD. Full-length TRβ1 bound to RORα or RORsg in GST pull-down assays; however, RORα-LBD did not bind to TRα1 or β1.
Conclusion:
The full-length forms of both RORα and TR are essential for the augmentation of TR-mediated transcription by RORα.