Use of suppressor mutants to probe the function of estrogen receptor-p160 coactivator interactions

Estrogen-dependent recruitment of coactivators by estrogen receptor alpha ( ER alpha) represents a crucial step in the transcriptional activation of ta rget genes. However, studies of the function of individual coactivators has been hindered by the presence of endogenous coactivators, many of which ar e potentially recruited in the presence of agonist via a common mechanism. To circumvent this problem, we have generated second-site suppressor mutati ons in the nuclear receptor interaction domain of p160 coactivators which r escue their binding to a transcriptionally defective ER alpha that is refra ctory to wild-type coactivators. Analysis of these altered-specificity rece ptor-coactivator combinations, in the absence of interference from endogeno us coregulators, indicated that estrogen-dependent transcription from repor ter genes is critically dependent on direct recruitment of a p160 coactivat or in mammalian cells and that the three p160 family members serve function ally redundant roles. Furthermore, our results suggest that such a change-o f-specificity mutation may act as a transposable protein-protein interactio n module which provides a novel tool with which to dissect the functional r oles of other nuclear receptor coregulators at the cellular level.