The unusual binding properties of the third distinct teleost estrogen receptor subtype ERβa are accompanied by highly conserved amino acid changes in the ligand binding domain

Three forms of estrogen receptor: ERalpha, ERbeta (ERbetab), and a second ERbeta, ERbetaa (formerly ERgamma) are present in teleost fish. All ERbetaas share amino acid changes in the ligand binding domain that may influence ligand specificity and receptor function. We compared binding specificities of the three ERs of the teleost fish, Atlantic croaker Micropogonias undulatus. Bacterially expressed Atlantic croaker (ac) ERalpha, -betab, and -betaa fusion proteins showed specific, high affinity binding to [H-3]estradiol, with K-d values of 0.61 +/- 0.013, 0.40 +/- 0.006, and 0.38 +/- 0.059 nM, respectively. Rank orders of binding were: diethylstilbestrol much greater than ICI182780 > 4-hydroxytamoxifen > ICI164384 > estradiol zearalenone > moxestrol > tamoxifen > estrone greater than or equal to 17alpha-estradiol > estriol > 2-hydroxyestrone = genistein much greater than RU486 for acERalpha; ICI182780 > diethylstilbestrol > 4-hydroxytamoxifen > estradiol > ICI164384 > genistein > inoxestrol > tamoxifen > zearalenone = estrone > estriol = 17alpha-estradiol > 2-hydroxyestrone much greater than RU486 for acERbetab; and estradiol greater than or equal to diethylstilbestrol > 4-hydroxytamoxifen > ICI182780 > ICI164384 > estriol greater than or equal to genistein > moxestrol > zearalenone > estrone > 17alpha-estradiol > RU486 greater than or equal to tamoxifen >.2-hydroxyestrone for acERbetaa. acERbetaa showed higher relative binding affinities for estradiol, estriol, and RU486 and lower relative binding affinities for synthetic estrogens and antiestrogens than previously characterized ERs. Mutation of the conserved teleost substitutions (acERbetaaPhe(396)) to the ERalpha or ERbetab counterpart shifted diethylstilbestrol and tamoxifen affinities toward those of wild-type acERa and acERbetab, supporting the hypothesis that the positions with conserved residue changes in teleost ERs are important to ER structure and function.