The transcriptional activity of estrogen receptor-α is dependent on Ca2+/ calmodulin

Abstract Estrogen binds to estrogen receptors in cells, thereby activating the receptors and eliciting biological effects. One of the best characterized effects of estrogen receptor-α (ERα) is transcriptional activation that regulates selected target genes in the nucleus. Work from several laboratories has documented a Ca2+-dependent interaction between ERα and calmodulin. In addition, we previously showed that antagonism of calmodulin function in cells prevented estradiol from inducing ERα transcriptional activity, suggesting that association of ERα with calmodulin participates in ERα function. In this study we adopted a multifaceted approach to directly address this hypothesis. The calmodulin binding domain on ERα was identified and several mutant ERα constructs unable to bind calmodulin were generated. Elimination of calmodulin binding prevented estradiol from stimulating ERα transcriptional activation. Essentially identical results were obtained when intracellular Ca2+ was chelated with the cell-permeable chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM). Moreover, CaM1234, a calmodulin mutant that is unable to bind Ca2+, functioned as a dominant negative construct. Transfection of cells with CaM1234 reduced estradiol-stimulated ERα transcriptional activity. These data indicate that binding to calmodulin is required for normal transcriptional function of ERα.