Multiple estrogen binding sites in the uterus: Stereochemistry of receptor and non-receptor binding of diethylstilbestrol and its metabolites

Abstract

Indenestrol A (IA), an oxidative metabolite of the synthetic estrogen diethylstilbestrol (DES), has high binding affinity for estrogen receptor in mouse uterine cytosol but possesses weak biological activity. Racemic mixture of optically active [ 3H]indenestrol A (IA-Rac) was separated and purified into individual enantiomers on a semi-preparative scale by HPLC with a Chiralpak OP(+) column. The structure-activity relationship was investigated among the [ 3H]IA enantiomers (IA- R and IA- S) and [ 3H]DES through direct saturation binding assays using mouse uterine cytosol. Specific binding curves and Scatchard plots were obtained for each [ 3H]ligand; DES, IA-Rac, IA- R and IA- S. IA- S enantiomer ( K d =0.67) binds to the estrogen receptor with the same affinity as DES ( K d =0.71) and four times higher affinity than IA R( K d =2.56). The number of binding sites for IA- S is approximately the same as estradiol, DES and IA-Rac while IA- R binds far fewer sites than the other ligands. Saturation binding assays indicated that [ 3H]DES and [ 3H]IA enantiomers exhibited a higher level of non-specific binding to the cytosol receptor compared to estradiol which has a low level of non-specific binding. These binding studies led to the detection of an additional binding component for the stilbestrol compounds in estrogen target tissue cytosol preparations. Sucrose density gradient separation assays under low salt conditions showed that both [ 3H]DES and [ 3H]IA compounds bound to the 8S form of the receptor, the same as E 2. But, in addition both DES and IA bound to another binding component in 4S region. The binding to the 4S component were partially displayed by the addition of excess unlabeled E 2 and DES. Further characterization of the 4S component is described.