Mutation of leu-536 in human estrogen receptor-α alters the coupling between ligand binding, transcription activation, and receptor conformation

Abstract

The estrogen receptor (ER), of which there are two forms, ERα and ERβ, is a ligand-modulated transcription factor important in both normal biology and as a target for agents to prevent and treat breast cancer. Crystallographic studies of the ERα ligand-binding domain suggest that Leu-536 may be involved in hydrophobic interactions at the start of a helix, "helix 12," that is crucial in the agonist-stimulated activity of ERα, as well as in the ability of antagonists to block the activity of ERα. We found that certain mutations of Leu-536 increased the ligand-independent activity of ERα although greatly reducing or eliminating the agonist activity of 17β-estradiol (E2) and 4-hydroxytamoxifen (4OHT), on an estrogen response element-driven and an AP-1-driven reporter. The mutations impaired the interaction of the ER ligand-binding domain with the SRC1 receptor-interacting domain in a mammalian two-hybrid system. When tested in the yeast two-hybrid system, mutation of Leu-536 increased the basal reactivity of ERα to probes that recognize the agonist-bound conformation but did not significantly alter its reactivity to these probes in the presence of E2. Most interestingly, mutation of Leu-536 reduced the interaction of the 4OHT-bound ERα and increased the reactivity of the raloxifene- or ICI 182,780-bound ERα, with probes that recognize the 4OHT-bound ERα conformation in a yeast two-hybrid system. These results show that Leu-536 is critical in coupling the binding of ligand to the modulation of the conformation and activity of ERα.