PURIFICATION AND FUNCTIONAL-CHARACTERIZATION OF THE LIGAND-BINDING DOMAIN FROM THE RETINOIC ACID RECEPTOR-ALPHA - EVIDENCE THAT SULFHYDRYL-GROUPS ARE INVOLVED IN LIGAND-RECEPTOR INTERACTIONS

The pGEX-2T expression vector was used to produce the ligand-binding domain from the human retinoic acid receptor alpha (hRARalphaLBD) in Escherichia coli. The resulting fusion protein, containing the glutathione S-transferase separated from the truncated receptor (hRARalpha 186-462) by a thrombin cleavage site, was purified with use of affinity chromatography on immobilized glutathione. A 90% homogeneity was obtained, with a specific activity of 100 pmol/mg and an overall 10% yield. Following purification and thrombin cleavage, a predominant monomeric (stokes radius = 2.3 nm, molecular mass of 32 kDa) [H-3]retinoic acid hRARalpha LBD complex was characterized by high-performance size-exclusion chromatography.The purified hRARalpha LBD bound retinoic acid with an apparent K(d) of 9 nM, a value close to the K(d) of the full-length hRARalpha expressed in COS cells. Kinetic studies at 0-degrees-C demonstrate that the association of [H-3]retinoic acid and [3H]CD367, a synthetic retinoid, to the overexpressed receptor was extremely rapid (complete in less than 3 min), whereas their dissociation from the receptor was slower, with half-lives of about 40 min at 0-degrees-C. Experiments performed at various subzero temperatures allowed a more accurate assay of the association rate constant and indicate that the entropy of activation (DELTAS(a)) is positive, which is characteristic of hydrophobic interactions. The ligand-binding activity was markedly decreased by pretreatment with various sulfhydryl modifying agents. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) appeared to be the most potent, whereas iodoacetamide was the least active. Furthermore, a series of N-alkylmaleimides was shown to inactivate the recombinant receptor. Comparison of these agents revealed a striking increase of receptor inactivation with increasing chain length of the maleimide derivative. Full protection against inactivation was afforded by previous [H-3] retinoid-binding on the receptor. The receptor binding activity was insensitive to arsenite, a reagent able to preferentially oxidize vicinal dithiols. Taken together, these results demonstrate that one or several sulfhydryl groups but probably no vicinal dithiols are involved in the retinoid-binding activity of hRARalpha, lying most probably in the retinoid-binding site itself.