Molecular Endocrinology, Vol 9, 457-466, Copyright © 1995 by Endocrine Society

ARTICLES

Estrogen receptor mutants which do not bind 17 beta-estradiol dimerize and bind to the estrogen response element in vivo

Y Zhuang, BS Katzenellenbogen and DJ Shapiro
Department of Biochemistry, University of Illinois, Urbana 61801, USA.

To investigate the stage in estrogen receptor (ER) action at which hormone functions, we prepared human ER mutants unable to bind 17 beta- estradiol. In transfected Chinese Hamster Ovary (CHO) cells, two of the ER mutants exhibited less than 5% of the ability to activate transcription shown by wild type ER. Immunoprecipitation followed by Western blotting with monoclonal antibodies was used to examine the ability of the ER mutants to form heterodimers with a truncated form of wild type ER. The non-hormone-binding mutants formed heterodimers with the truncated ER as efficiently as wild type ER. We used a promoter interference assay to measure the interaction of the ER with the estrogen response element (ERE) in vivo. Expression plasmids encoding the ER mutants and wild type ER were transfected into CHO cells across a range of concentrations, resulting in both high and low levels of promoter interference. The ER mutants and wild type ER elicited similar levels of promoter interference, indicating that although they were unable to bind ligand, the ER mutants bound to the ERE in vivo as effectively as wild type ER. Additional evidence that the non-hormone- binding ER mutants are not in a functionally inactive complex comes from their ability to suppress the activity of wild type ER, when they were coexpressed in the same cells. These data support a model for ER action in which the unliganded ER is free to dimerize and bind to the ERE. In this model, the primary role of 17 beta-estradiol in ER action is to induce a conformational change which activates the ligand- dependent transactivation domain.

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