Novel roles of retinoid X receptor (RXR) and RXR ligand in dynamically modulating the activity of the thyroid hormone receptor/RXR heterodimer

Many members of the type II nuclear receptor subfamily function as heterodimers with the retinoid X receptor (RXR). A permissive heterodimer (e.g. peroxisome proliferator-activated receptor/RXR) allows for ligand binding by both partners of the receptor complex. In contrast, RXR has been thought to be incapable of ligand binding in a nonpermissive heterodimer, such as that of thyroid hormone receptor (TR)/RXR, where it has been referred to as a silent partner. However, we recently presented functional evidence suggesting that RXR in the TR/RXR heterodimer can bind its natural ligand 9-cis-RA in cells. Here we extended our study of the interrelationship of TR and RXR. We examined the potential modulatory effect of RXR and its ligand on the activity of TR, primarily using a Gal4-TR chimera. This study led to several novel and unexpected findings: 1) heterodimerization of apo-RXRalpha (in the absence of 9-cis-RA) with Gal4-TR inhibits T3-mediated transactivation; 2) the inhibition of Gal4-TR activity by RXRalpha is further enhanced by 9-cis-RA; 3) two different RXR subtypes (alpha and beta) differentially modulate the activity of Gal4-TR; 4) the N-terminal A/B domains of RXR alpha and beta are largely responsible for their differential modulation of TR activity; and 5) the RXR ligand 9-cis-RA appears to differentially affect T3-mediated transactivation from the Gal4-TR/RXRalpha (which is inhibited by 9-cis-RA) and TRE-bound TR/RXRalpha (which is further activated by 9-cis-RA) heterodimers. Taken together, these results further support our recent proposal that the RXR component in a TR/RXR heterodimer is not silent and, more importantly, reveal novel aspects of regulation of the activity of the TR/RXR heterodimer by RXR and RXR ligand.     

Photoaffinity labeling of human retinoid X receptor beta (RXRbeta) with 9-cis-retinoic acid: identification of phytanic acid, docosahexaenoic acid, and lithocholic acid as ligands for RXRbeta

We utilized [20-methyl-(3)H]-9-cis-retinoic acid ([(3)H]9-cis-RA) as a direct photoaffinity probe for the characterization of human recombinant retinoid X receptor beta protein (RXRbeta). The photoaffinity labeling was light- and concentration-dependent, saturable, and protected by unlabeled 9-cis-RA in a concentration-dependent manner, indicating that binding occurred in the RXR retinoid binding site. all-trans-Retinoic acid (atRA) did not affect labeling with the 9-cis derivative, confirming that atRA does not compete for the 9-cis-RA binding site. Several retinoid, fatty acid, and bile acid ligands were evaluated for their ability to recognize the 9-cis-RA binding site. Retinol, atRA glucuronide, 13-cis-RA, dolichol, 5,6-epoxy-RA, and vitamin D(3) did not compete for the 9-cis-RA binding site. However, the saturated diterpenoid phytanic acid (PA) and docosahexaenoic acid, which have been recently shown to activate the nuclear receptor, RXR, competed with 9-cis-RA labeling, showing high affinity for the 9-cis-RA binding site. Oleic acid, arachidonic acid, and butyric acid did not interact. However, the bile acid lithocholic acid competed efficiently with 9-cis-RA for the binding site. These data validated the photoaffinity assay as an excellent system for the identification and evaluation of ligands for RXR.     

Ligand--protein interactions in the glutamate receptor

Fourier transform infrared spectroscopy was used to investigate ligand-protein interactions in the ligand-binding domain of the GluR4 glutamate receptor subunit. Glutamate binding induces more extensive secondary structural changes in the ligand-binding domain than does kainate binding. Glutamate also alters the hydrogen bonding strength of the single free cysteine side chain in the domain, while kainate does not. On the other hand, the interaction of a binding site arginine residue with kainate appears to be stronger than that with glutamate. These results identify chemical and structural differences that may explain the different functional characteristics of the two agonists acting on ionotropic glutamate receptors. In doing so, they complement and extend recent crystallographic structures of the ligand-binding domain.     

Analysis of the ligand-binding domain of human retinoic acid receptor alpha by site-directed mutagenesis.

Three subtypes of retinoic acid receptors (RAR), termed RAR alpha, RAR beta, and RAR gamma, have been described. They are composed of different structural domains, including distinct domains for DNA and ligand binding. RARs specifically bind all-trans-retinoic acid (RA), 9-cis-RA, and retinoid analogs. In this study, we examined the functional role of cysteine and arginine residues in the ligand-binding domain of hRAR alpha (hRAR alpha-LBD, amino acids 154 to 462). All conserved cysteine and arginine residues in this domain were mutated by site-directed mutagenesis, and the mutant proteins were characterized by blocking reactions, ligand-binding experiments, transactivation assays, and protease mapping. Changes of any cysteine residue of the hRAR alpha-LBD had no significant influence on the binding of all-trans RA or 9-cis RA. Interestingly, residue C-235 is specifically important in antagonist binding. With respect to arginine residues, only the two single mutations of R-276 and R-394 to alanine showed a dramatic decrease of agonist and antagonist binding whereas the R272A mutation showed only a slight effect. For all other arginine mutations, no differences in affinity were detectable. The two mutations R217A and R294A caused an increased binding efficiency for antagonists but no change in agonist binding. From these results, we can conclude that electrostatic interactions of retinoids with the RAR alpha-LBD play a significant role in ligand binding. In addition, antagonists show distinctly different requirements for efficient binding, which may contribute to their interference in the ligand-inducible transactivation function of RAR alpha.