IDENTIFICATION OF SULFHYDRYL-MODIFIED CYSTEINE RESIDUES IN THE LIGAND-BINDING POCKET OF RETINOIC ACID RECEPTOR-BETA

The diverse biological functions of retinoic acid (RA) are mediated th rough retinoic acid receptors (RARs) and retinoid X receptors. RARs co ntain a high affinity binding site for RA which is sensitive to treatm ent with sulfhydryl modification reagents. In an attempt to identify w hich Cys residues are important for this loss of binding, we created t hree site-specific RAR beta mutants: C228A, C258A, and C267A. The affi nity for RA of all three mutant receptors was in the range of that of the wild type protein, suggesting that none of these Cys residues are critical for RA binding, Rather, these modified Cys residue(s) functio n to sterically hinder RA binding; however, the modified Cys residues critical for the inhibition of binding differ depending on the reagent employed. Only modification of Cys(228) is necessary to inhibit RA bi nding when RAR beta is modified by reagents which transfer large bulky groups while both Cys(228) and Cys(267) must be modified when a small functional group is transferred. These data suggest that both Cys(228 ) and Cys(267) but not Cys(258) lie in the ligand binding pocket of RA R beta. However, Cys(228) lies closer to the opening of the RAR beta l igand binding pocket whereas Cys(267) lies more deeply buried.