Ligand Requirements for Estrogen Receptor Function and the Actions of Antiestrogens

Abstract

We have undertaken studies to examine the ligand requirements for the DNA binding and functional activity of human estrogen receptor (ER) within intact cells. By employing an assay using competitive binding of ER with basal transcription factors on a constitutive promoter (CMV-(HRE)n-CAT, containing hormone response element(s) between the TATA-box and the start site of transcription of the Cytomegalovirus promoter upstream of the chloramphenicol acetyltransferase gene) we were able to examine the DNA binding ability of the human ER in whole cells. We used this promoter interference assay to examine the DNA binding of ER in cell lines containing high and low levels of endogenous ER, as well as in Chinese Hamster Ovary (CHO) cells expressing wild-type and mutant ERs from cotransfected expression vectors. We have found that the ER is capable of binding to reporter templates within intact cells in the absence of ligand; however, ligand enhances this interaction. We provide some evidence that this DNA binding may result in ligand-independent transactivation of transgenes by the receptor, and that DNA binding alone is not sufficient for full receptor activity. Likewise, receptors occupied with estradiol or two classes of antiestrogens were also capable of DNA binding. Receptors occupied with the pure antiestrogen ICI 164,384 bound to DNA in whole cells and were in sufficient quantity to occupy receptor binding sites on reporter templates, indicating that neither a loss of DNA binding of ICI-receptor complexes or reduction of ER protein in target cells could fully explain the antagonistic nature of this compound. Through the analysis of a temperature-sensitive ER mutant (C447A), we provide evidence that the ligand-independent DNA binding observed for the ER is the basis of functional differences observed between the ER and other members of the steroid receptor family.