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Homology-Modeled Ligand-Binding Domains of Zebrafish Estrogen Receptors , ß₁, and ß₂: From in Silico to in Vivo Studies of Estrogen Interactions in Danio rerio as a Model System

Chemical Proteomics Facility, Department of Chemistry, Marquette University (A.D.C., P.K.P., P.K., D.S.S.),Milwaukee, Wisconsin 53201; and Marine and Freshwater Biomedical Sciences Center, University of Milwaukee (H.T.), Milwaukee, Wisconsin 53204

Address all correspondence and requests for reprints to: Dr. Daniel S. Sem, Chemical Proteomics Facility at Marquette, Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, Wisconsin 53201. E-mail: daniel.sem{at}marquette.edu.

Homology models were constructed for the ligand-binding domains of zebrafish estrogen receptors (zfERs) , ß₁, and ß₂. Estradiol-binding sites are nearly identical in zfERs and their human homologs, suggesting that zebrafish will serve as a good model system for studying human ER-binding drugs. Conversely, studies of endocrine disruptor effects on zebrafish will benefit from the wealth of data available on xenoestrogen interactions with human ERs. Compounds flagged by the Interagency Coordinating Committee on the Validation of Alternative Methods for endocrine disruptor screening were docked into our zfER homology models. Ideally, these in silico docking studies would be complemented with in vivo binding studies. To this end, fluorescently tagged estradiol was docked into zfER and found to bind in the same manner as in human ER, with fluorescein preferentially occupying a region between helices 11 and 12. Fluorescently tagged estradiol was synthesized and was found to localize along the path of primordial germ cell migration in the developing zebrafish embryo 3 d after fertilization, consistent with previous reports of 1) a role for estradiol in sex determination, and 2) the first appearance of ERs 2 d after fertilization. These data provide a foundation for future in silico and in vivo binding studies of estrogen agonists and antagonists with zebrafish ERs.