An estrogen receptor mutant exhibiting hormone-independent transactivation and enhanced affinity for the estrogen response element.

To study transactivation by the Xenopus laevis estrogen receptor (XER), we inserted one or two copies of a synthetic amphipathic helix at amino acid 276 of the XER. The XER mutants containing one or two copies of the amphipathic helix (XER/1AH and XER/2AH, respectively) and wild-type XER were expressed at similar levels. In transient transfection assays, XER/1AH exhibited only a modest, promoter-specific increase in transactivation. Constitutive (estrogen-independent) transcription of a synthetic promoter containing two estrogen response elements (EREs) was approximately 10-fold higher for the XER/2AH mutant than for wild-type XER. The XER/2AH mutant and wild-type XER exhibited similar 17 beta-estradiol dose-response curves for transactivation. In studies carried out over a broad range of DNA concentrations using the simple 2ERE-TATA promoter or a complex vitellogenin-derived promoter, the XER/2AH mutant exhibited an estrogen-dependent 2-3-fold increase in transactivation. A 2-3-fold increase in transactivation by XER/2AH was also observed using synthetic promoters in which the two EREs exhibit synergistic interactions with the NF1, AP1, or vitellogenin activator upstream activator sequences. Using a promoter interference assay to investigate intracellular interactions between the estrogen receptor and the ERE, we showed that binding of wild-type XER to the ERE was strongly estrogen-dependent. In the presence of 17 beta-estradiol, XER/2AH and wild-type XER exhibited similar promoter interference curves. In the absence of 17 beta-estradiol, the expression plasmid encoding the XER/2AH mutant achieved levels of promoter interference with 0.25-0.5 microgram of transfected DNA that were similar to those observed with 5-10 micrograms of the expression plasmid encoding wild-type XER. The ability of the XER/2AH mutant to activate transcription in the absence of estrogen therefore is likely to be related to the approximately 20-fold increase in its apparent ability to bind to the ERE. Since XER/2AH was unable to activate transcription from a glucocorticoid response element, enhanced binding of XER/2AH to the ERE did not result from a general increase in binding to DNA. The XER/2AH mutant appears to be the first nuclear receptor mutant to retain hormone-dependent transactivation and to exhibit enhanced hormone-independent binding to its hormone response element.