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 am ino 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 a ssays, 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 l7bet a-estradiol dose-response curves for transactivation. In studies carri ed 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 transactiv ation. A 2-3-fold increase in transactivation by XER/2AH was also obse rved using synthetic promoters in which the two EREs exhibit synergist ic interactions with the NF1, AP1, or vitellogenin activator upstream activator sequences. Using a promoter interference assay to investigat e intracellular interactions between the estrogen receptor and the ERE , we showed that binding of wild-type XER to the ERE was strongly estr ogen-dependent. In the presence of 17beta-estradiol, XER/2AH and wild- type XER exhibited similar promoter interference curves. In the absenc e of 17beta-estradiol, the expression plasmid encoding the XER/2AH mut ant achieved levels of promoter interference with 0.25-0.5 mug of tran sfected DNA that were similar to those observed with 5-10 mug of the e xpression plasmid encoding wild-type XER. The ability of the XER/2AH m utant to activate transcription in the absence of estrogen therefore i s likely to be related to the approximately 20-fold increase in its ap parent ability to bind to the ERE. Since XER/2AH was unable to activat e transcription from a glucocorticoid response element, enhanced bindi ng of XER/2AH to the ERE did not result from a general increase in bin ding to DNA. The XER/2AH mutant appears to be the first nuclear recept or mutant to retain hormone-dependent transactivation and to exhibit e nhanced hormone-independent binding to its hormone response element.