Estrogen binds to estrogen receptors in cells, thereby activating the receptors and eliciting biological effects. One of the best characterized effects of estrogen receptor-␣ (ER␣) is transcriptional activation that regulates selected target genes in the nucleus. Work from several laboratories has documented a Ca 2؉ -dependent interaction between ER␣ and calmodulin. In addition, we previously showed that antagonism of calmodulin function in cells prevented estradiol from inducing ER␣

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... onal activity, suggesting that association of ER␣ with calmodulin participates in ER␣ function. In this study we adopted a multifaceted approach to directly address this hypothesis. The calmodulin binding domain on ER␣ was identified and several mutant ER␣ constructs unable to bind calmodulin were generated. Elimination of calmodulin binding prevented estradiol from stimulating ER␣ transcriptional activation. Essentially identical results were obtained when intracellular Ca 2؉ 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 Ca 2؉ , functioned as a dominant negative construct. Transfection of cells with CaM1234 reduced estradiolstimulated ER␣ transcriptional activity. These data indicate that binding to calmodulin is required for normal transcriptional function of ER␣.