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 d omain from the human retinoic acid receptor alpha (hRARalphaLBD) in Es cherichia coli. The resulting fusion protein, containing the glutathio ne S-transferase separated from the truncated receptor (hRARalpha 186- 462) by a thrombin cleavage site, was purified with use of affinity ch romatography on immobilized glutathione. A 90% homogeneity was obtaine d, with a specific activity of 100 pmol/mg and an overall 10% yield. F ollowing purification and thrombin cleavage, a predominant monomeric ( stokes radius = 2.3 nm, molecular mass of 32 kDa) [H-3]retinoic acid h RARalpha LBD complex was characterized by high-performance size-exclus ion 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-lengt h hRARalpha expressed in COS cells. Kinetic studies at 0-degrees-C dem onstrate that the association of [H-3]retinoic acid and [3H]CD367, a s ynthetic retinoid, to the overexpressed receptor was extremely rapid ( complete in less than 3 min), whereas their dissociation from the rece ptor was slower, with half-lives of about 40 min at 0-degrees-C. Exper iments performed at various subzero temperatures allowed a more accura te assay of the association rate constant and indicate that the entrop y of activation (DELTAS(a)) is positive, which is characteristic of hy drophobic interactions. The ligand-binding activity was markedly decre ased by pretreatment with various sulfhydryl modifying agents. 5,5'-Di thiobis(2-nitrobenzoic acid) (DTNB) appeared to be the most potent, wh ereas iodoacetamide was the least active. Furthermore, a series of N-a lkylmaleimides was shown to inactivate the recombinant receptor. Compa rison of these agents revealed a striking increase of receptor inactiv ation with increasing chain length of the maleimide derivative. Full p rotection against inactivation was afforded by previous [H-3] retinoid -binding on the receptor. The receptor binding activity was insensitiv e to arsenite, a reagent able to preferentially oxidize vicinal dithio ls. Taken together, these results demonstrate that one or several sulf hydryl groups but probably no vicinal dithiols are involved in the ret inoid-binding activity of hRARalpha, lying most probably in the retino id-binding site itself.