THE ISOLATION AND CHARACTERIZATION OF PURIFIED HETEROCOMPLEXES OF RECOMBINANT RETINOIC ACID RECEPTOR AND RETINOID-X-RECEPTOR LIGAND-BINDINGDOMAINS

Retinoic acid exerts many of its biological effects by interaction wit h heterocomplexes of nuclear retinoic acid receptors (RARs) and retino id X receptors (RXRs). To further examine this interaction, a glutathi one S-transferase (GST) fusion protein containing the ligand binding d omain of human RXR alpha has been used to copurify the ligand binding domain of human RAR gamma by affinity chromatography over glutathione- agarose. Complexes of recombinant RAR-RXR ligand binding domains retai ning full ligand binding capacity were purified, and their interaction s with various retinoids were characterized by fluorometric titration and photoaffinity labeling. Analyses of the distribution of limiting a mounts of [H-3]-all-trans-retinoic acid between cytoplasmic retinoic a cid binding proteins, CRABP-I and CRABP-II, and the purified heterocom plexes indicate that all-trans-retinoic acid binds with comparable aff inity to CRABP-I and the heterocomplexes, but with approximately 10-fo ld less affinity to CRABP-Tl. The aromatic retinoid acitretin, which i s used in the treatment of psoriasis, binds relatively poorly to the p urified heterocomplexes, although it binds with high affinity to the C RABPs. Acitretin displaces [H-3]-all-trans-retinoic acid from the CRAB Ps and increases retinoic acid occupancy of the heterocomplexes. These results suggest that certain retinoids could potentially perturb the distribution of endogenous retinoic acid between the CRABPs and the nu clear receptors and thus affect retinoid signaling. The purified recom binant complexes should provide a useful model system for further stru ctural analysis of the dimerization interface between the RAR and RXR ligand binding domains.