AUTO-SILENCING BY THE RETINOID X RECEPTOR

Abstract Gene transcription is often regulated by small ligands, enabling cells to respond to external and metabolic stimuli. Of particular interest are the mechanisms by which hydrophobic hormones modulate the transcriptional activities of proteins of the nuclear receptor superfamily. It was previously shown that, in the absence of ligand, the retinoid X receptor (RXRα) forms tetramers with a high affinity and a pronounced positive co-operativity such that tetramers become the receptor’s predominant species tat concentrations as low as 60–70 nM. It was shown further that while RXR tetramers are remarkably stable in the absence of ligand, ligand-binding induces their rapid dissociation into smaller species, dimers and monomers. Here, the functional consequences of the self-association properties of RXR were studied by examining two point mutants of RXR that displayed aberrant oligomerization behaviors. One mutant, mRXRα-R321A, was found to form tetramers with a wild-type affinity, but these tetramers failed to dissociate upon ligand-binding. This mutant was found to be impaired in its ability to associate with the nuclear receptor co-activator p/CIP and to activate transcription in response to the RXR ligand 9- cis -retinoic acid. The other mutant, mRXRα-F318A, self-associated into dimers with a wild-type affinity, but was unable to form tetramers. This mutant displayed substantial transcriptional activity even in the absence of ligand. We previously proposed, based on in vitro studies that RXR acts as an auto-silencer by sequestering itself into tetramers, and that an important role for the ligand in activating this receptor is to release active species, dimers and monomers, from the transcriptionally inactive tetrameric pool. The observations reported here provide in-cell evidence in support of this model and indicate that ligand induced dissociation of tetramers is the first step in signalling by RXR.