Molecular determinants of the stereoselectivity of agonist activity of estrogen receptors (ER) alpha and beta.

The two known estrogen receptors, ERalpha and ERbeta, are hormone-inducible transcription factors that have distinct roles in regulating cell proliferation and differentiation. Previously, our laboratory demonstrated that ERalpha exhibits stereoselective ligand binding and transactivation for several structural derivatives and metabolites of the synthetic estrogen diethylstilbestrol. We have previously described the properties of indenestrol A (IA) enantiomers on ERalpha. In the study presented here, the estrogenic properties of the S and R enantiomers of IA, IA-S and IA-R, respectively, were expanded to examine the activity in different cell and promoter contexts using ERalpha and ERbeta. Using human cell lines stably expressing either ERalpha or ERbeta, we found that IA-S was a more potent activator of transcription than IA-R through ERalpha in human endometrial Ishikawa and breast MDA-MB 231 (MDA) cells. Interestingly, IA-R was more potent on ERbeta when compared with ERalpha in MDA, but not in Ishikawa cells, and IA-R exhibited equally low binding affinities to ERalpha and ERbeta in vitro in contrast to its cell line-dependent preferential activation of ERbeta. Alignment of the protein structures of the ligand-binding domains of ERalpha and ERbeta revealed one mismatched residue, Leu-384 in ERalpha and Met-283 in ERbeta, which may be responsible for making contact with the methyl substituent at the chiral carbon of IA-S and IA-R. Mutagenesis and exchange of this one residue showed that the binding of IA-R and IA-S was not affected by this mutation in ERalpha and ERbeta. However, in transactivation studies, IA-R showed higher potency in activating L384M-mutated ERalpha and wild-type ERbeta compared with wild-type ERalpha and M283L-mutated ERbeta in all cell and promoter contexts examined. Furthermore, IA-R-bound ERalpha L384M and wild-type ERbeta displayed enhanced interactions with the nuclear receptor interaction domains of the coactivators SRC-1 and GRIP1. These data demonstrate that a single residue in the ligand-binding domain determines the stereoselectivity of ERalpha and ERbeta for indenestrol ligands and that IA-R shows cell type selectivity through ERbeta.