Identification of thiol group at the estrogen-binding site on the cytoplasmic receptor from rabbit uterus by affinity...

The interaction of estrogen with its receptor from rabbit uterus was studied using the affinity labeling technique. An affinity labeling reagent, 3-hydroxy-17 beta-(p-nitrophenyldithio)-1,3,5(10)-estratriene (Reagent A) was synthesized. The compound was designed to meet two requirements: (1) it binds specifically to the steroid-binding site of the receptor; (2) it has functional group capable of forming a covalent bond with an amino acid residue at or near the binding site. The first requirement was demonstrated by competitive binding assay and sedimentation analysis. If the binding affinity of 17 beta-estradiol is defined as 100, that of Reagent A is found to be 0.05. It was shown that the binding was specific for the estradiol receptor. The second requirement was examined by reaction of Reagent A with simple model compounds. 10 microM Reagent A gave rise to approx. 1.3 microM p-nitrothiophenol in the presence of a large excess of L-cysteine or reduced L-glutathione under the experimental conditions employed, indicating that a covalent bond was formed between Reagent A and the model compounds. Furthermore, I have presented evidence by using polyacrylamide gel electrophoresis that 3H-labeled Reagent A is linked covalently to the estrogen-binding site on the receptor of rabbit uterus. The experiment also fulfilled the first requirement. These results indicate that the thiol group present at the binding site is directly involved in the estradiol-receptor binding.