Molecular Determinants of the Stereoselectivity of Agonist Activity of Estrogen Receptors (ER) and *

The two known estrogen receptors, ER and ER , are hormone-inducible transcription factors that have distinct roles in regulating cell proliferation and differentiation. Previously, our laboratory demonstrated that ER 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 ER . 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 ER and ER . Using human cell lines stably expressing either ER or ER , we found that IA-S was a more potent activator of transcription than IA-R through ER in human endometrial Ishikawa and breast MDA-MB 231 (MDA) cells. Interestingly, IA-R was more potent on ER when compared with ER in MDA, but not in Ishikawa cells, and IA-R exhibited equally low binding affinities to ER and ER in vitro in contrast to its cell line-dependent preferential activation of ER . Alignment of the protein structures of the ligand-binding domains of ER and ER revealed one mismatched residue, Leu-384 in ER and Met-283 in ER , 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 ER and ER . However, in transactivation studies, IA-R showed higher potency in activating L384M-mutated ER and wild-type ER compared with wild-type ER and M283L-mutated ER in all cell and promoter contexts examined. Furthermore, IA-R-bound ER L384M and wild-type ER 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 ER and ER for indenestrol ligands and that IA-R shows cell type selectivity through ER . The estrogenic effects of a variety of structurally diverse endogenous and xenobiotic compounds are mediated through the estrogen receptors (ER and ER ), which function as ligand-inducible transcription factors for genes involved in cell growth, proliferation, and differentiation (1). Studies using knock-out mice for the two ER subtypes ( ERKO and ERKO, respectively) have revealed that each receptor plays a unique role in estrogen biology in a wide variety of target tissues (2). Furthermore, in vitro studies indicated that ER and ER display marked differences in binding affinity and activation by natural and synthetic ER ligands (3, 4). Interestingly, although ER shows lower binding affinity for and activation by endogenous estrogens, several xenoestrogens preferentially bind and activate ER (3). These observations have prompted further studies aimed at elucidating the molecular mechanisms of action of ER and ER , and the search for ER subtype-selective agonists that could be used to evaluate the physiological roles of each receptor. Diethylstilbestrol (DES) is a known carcinogen, which is oxidatively metabolized to a variety of metabolites with varying degrees of hormonal activity (34). Indenestrol A (IA) is a metabolite derived from DES that has high binding affinity for ER but weak biological activity (35). IA exists as a racemic mixture of enantiomers, IA-S and IA-R (36), which have a methyl substitution on the chiral carbon (Fig. 1). Previously, our laboratory demonstrated that IA-R displays a lower binding affinity and transactivation potency on ER than IA-S (5), which may help explain the differential biological activity and allow the study of the stereoselectivity of ligand binding and transcriptional responses of ERs. The discovery of ER prompted us to investigate whether both ER subtypes would exhibit the same stereoselectivity for the IA enantiomers. Human ER and ER share high sequence homology (97%) in the central DNA-binding domain, but only moderate conservation (60%) in the C-terminal ligand-binding domain (LBD) and activation function-2 (AF-2). Since AF-2 mediates the ligand-dependent transcriptional activities of both ER subtypes (1), it is possible that structural differences within this region could account for differences in the specificities of ER and ER for several estrogenic chemicals (4, 6). To expand our knowledge about the molecular determinants of the stereoselectivity of ER and ER , we have characterized the ability of IA-S and IA-R to function as agonists/antagonists * This work was supported by a Grant from the Deutsche Forschungsgemeinschaft (Mu 1490/1) (to S. O. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence may be addressed: Merck KGaA, Institute of Toxicology, P.O. Box 64271, Darmstadt, Germany. Fax: 49-6151-7291-8517; E-mail: [email protected]. To whom correspondence may be addressed: NIEHS, MD B3-02, 111 TW Alexander Dr., P.O. Box 12233, Research Triangle Park, NC 27709. Fax: 919-541-0696; E-mail: [email protected]. 1 The abbreviations used are: ER, estrogen receptor; FBS, fetal bovine serum; ERE, estrogen response element; DES, diethylstilbestrol; DCC, dextran-coated charcoal; IA, indenestrol A; THC, tetrahydrocrysene; E2, 17 -estradiol; m, murine; h, human; MEM, minimal essential medium; NR; nuclear receptor-interacting regions; RPA, RNase protection assay; LBD, ligand-binding domain; DBD, DNA-binding domain. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 14, Issue of April 4, pp. 12255–12262, 2003 Printed in U.S.A.