Expression and cellular localization of naturally occurring β estrogen receptors in uterine and mammary cell lines

The protein ER-α has been exhaustively characterized in estrogen-sensitive tissues and cell lines. However, little is known regarding the expression and cellular distribution of the newly identified ER-β protein. We first quantified the specific estradiol binding site content in the estrogen-responsive cell lines MCF-7 (mammary) and SHM (myometrial). In the two cell types, these sites were associated to the expression of both ER-α and -β isoforms. Native ER-β was visualized to reside inside the nucleus by means of conventional indirect immunofluorescence. The cells expressed ER-β as a tight ∼50 kDa triplet when resolved by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and blotted using antibodies mapping different domains of the cloned ER-β version. When the cells were subjected to homogenization and differential centrifugation, a substantial proportion of ER-β immunolabeling was localized at membrane subfractions. ER-β expression and partitioning was confirmed by Ligand blotting assays using estrogen derivatives coupled to different macromolecular tags. However, ER-α was expressed as the major estrogen binding protein in both cell lines. Similar localization experiments were performed on HeLa cells (cervix). Though usually considered ER-negative, this cell line displayed basal significant estrogen binding capacity and co-expression of both ER isoforms. Taken as a whole, the results indicate that ER-β could be expressed as functional estrogen binding proteins among a dominant population of ER-α sites in the cell lines under study.