MECHANISTIC ASPECTS OF ESTROGEN-RECEPTOR ACTIVATION PROBED WITH CONSTITUTIVELY ACTIVE ESTROGEN-RECEPTORS - CORRELATIONS WITH DNA AND COREGULATOR INTERACTIONS AND RECEPTOR CONFORMATIONAL-CHANGES

The estrogen receptor (ER) belongs to a large family of nuclear recept ors, many of whose members function as ligand-dependent transcriptiona l activators. The mechanism by which the receptor is converted from an inactive into an activated state is not yet completely understood. To investigate the kind of changes in receptor conformation and interact ions that are involved in this activation, we have used the wild type ER and a set of constitutively active ER point mutants that show from 20% to nearly 100% activity in the absence of estrogen. These mutants are of particular interest as they could mimic, in the absence of liga nd, the activated state of the wild type receptor. We have analyzed se veral transcriptional steps that could be involved in the activation: the ability of these receptors 1) to interact with several coactivator s (steroid receptor coactivator-1, SRC-1; transcription intermediary f actor-1, TIF-1; and estrogen receptor-associated protein 140, FRAP 140 ) and with members of the preinitiation complex [TATA box-binding prot ein (TBP), transcription factor IIB (TFIIB)]; 2) to exhibit conformati onal changes revealed by proteolytic digest patterns similar to those observed for the wild type hormone-occupied ER; and 3) to bend estroge n response element-containing DNA, which is thought to be one of the i mportant phenomena triggering transcriptional activation. Our results demonstrate that the interaction of these mutant receptors with coacti vators is likely to be one of the features of the activated step, as t he mutant receptors interacted with some coactivators in a ligand-inde pendent manner in proportion to their extent of constitutive activity. However, the different degrees of ligand-independent interaction of t he mutant ERs with the three coactivators suggest that SRC-1, TIF-1, a nd FRAP 140 may play different Poles in receptor activity. Limited pro teolytic digest experiments reveal that the activated state of the rec eptor corresponds to a particular conformation of the receptor, which is fully observed with the mutant ER showing the highest activity in t he absence of estrogen. Finally, it appears that in inactive or active states, the receptor exhibits distinctly different DNA-bending abilit ies. Addition of estradiol is able to modify the bending ability of on ly the wild type receptor, whereas estradiol has no influence on the c onstitutive receptors, which exhibited the same bending ability as tha t observed for the ligand-occupied wild type receptor. These data docu ment that the ER undergoes major changes in its conformation and also in its functional properties when it is turned from an inactive into a n active state and that mutational changes in the ER protein that resu lt in constitutive, hormone-independent activation mimic many of the c hanges in ER properties that are normally under hormone regulation.