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

Estrogen binds to estrogen receptors in cells, thereby activating the receptors and eliciting biological effects. One of the best characterized effects of estrogen receptor-alpha (ERalpha) is transcriptional activation that regulates selected target genes in the nucleus. Work from several laboratories has documented a Ca2+-dependent interaction between ERalpha and calmodulin. In addition, we previously showed that antagonism of calmodulin function in cells prevented estradiol from inducing ERalpha transcriptional activity, suggesting that association of ERalpha with calmodulin participates in ERalpha function. In this study we adopted a multifaceted approach to directly address this hypothesis. The calmodulin binding domain on ERalpha was identified and several mutant ERalpha constructs unable to bind calmodulin were generated. Elimination of calmodulin binding prevented estradiol from stimulating ERalpha 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 ERalpha transcriptional activity. These data indicate that binding to calmodulin is required for normal transcriptional function of ERalpha.