A TRANSCRIPTIONALLY ACTIVE ESTROGEN-RECEPTOR MUTANT IS A NOVEL TYPE OF DOMINANT-NEGATIVE INHIBITOR OF ESTROGEN ACTION

We have characterized a human estrogen receptor (ER) mutant, V364E, wh ich has a single amino acid substitution in its hormone-binding domain . This ER mutant is fully active or even superactive at saturating lev els of estradiol (10(-8) M E(2)) yet has the capacity to act as a stro ng dominant negative inhibitor of the wild type ER. In transient trans fection assays using ER-negative Chinese hamster ovary (CHO) cells and two different estrogen response element (ERE)-containing promoter rep orter genes, V364E treated with 10(-8) M E(2) exhibited similar to 250 % and 100% of the activity of the wild type ER with these two promoter contexts, respectively. Despite the high activity of V364E when prese nt alone in cells, coexpression of both V364E and wild type ER causes a significant decrease in overall ER-mediated transcriptional activity . On the TATA promoter, where V364E was more inhibitory, estrogen-stim ulated activity was reduced by approximately 50% at a 1:1 ratio of mut ant to wild type ER expression vector, and at a 10:1 ratio, 75% of ER activity was inhibited. V364E was expressed at lower levels than wild type ER and has a similar to 40-fold lower affinity for E, compared wi th wild type ER. In promoter interference assays, V364E exhibited a st rict dependence upon E, for binding to an ERE. Surprisingly, even when V364E was unable to bind to ERE DNA (i.e. either at low E, concentrat ion or by mutation of its DNA-binding domain), this mutant retained fu ll dominant negative activity. This highly active ER mutant is, thus, able to repress ER-mediated transcription when the mutant and wild typ e ER are present together in cells, even without DNA binding. Since co mpetition for ERE binding and the formation of inactive heterodimers c annot fully account for the dominant negative activity of V364E, it is probable that altered interactions with proteins important in DR-medi ated transcription play a key role in the repression of transcription by V364E. The properties and probable mechanism of action of V364E dis tinguish it from other previously described dominant negative inhibito rs, in which competition for cis-acting DNA elements by transcriptiona lly inactive receptors played a large role in the resultant dominant n egative phenotype.