Identification of sulfhydryl-modified cysteine residues in the ligand binding pocket of retinoic acid receptor β

The diverse biological functions of retinoic acid (RA) are mediated through retinoic acid receptors (RARs) and retinoid X receptors. HAHs contain a high affinity binding site for RA which is sensitive to treatment with sulfhydryl modification reagents. In an attempt to identify which Cys residues are important for this loss of binding, we created three site- specific RARβ mutants: C228A, C258A, and C267A. The affinity 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) function 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 Cys²²⁸ is necessary to inhibit RA binding when RARβ is modified by reagents which transfer large bulky groups while both Cys²²⁸ and Cys²⁶⁷ must be modified when a small functional group is transferred. These data suggest that both Cys²²⁸ and Cys²⁶⁷ but not Cys²⁵⁸ lie in the ligand binding pocket of RARβ. However, Cys²²⁸ lies closer to the opening of the RARβ ligand binding pocket whereas Cys²⁶⁷ lies more deeply buried.