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

J Biol Chem. 2003 Jul 18;278(29):27278-86. doi: 10.1074/jbc.M303840200. Epub 2003 May 6.

Abstract

The estrogen receptor (ER), of which there are two forms, ERalpha and ERbeta, 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 ERalpha 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 ERalpha, as well as in the ability of antagonists to block the activity of ERalpha. We found that certain mutations of Leu-536 increased the ligand-independent activity of ERalpha although greatly reducing or eliminating the agonist activity of 17beta-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 ERalpha 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 ERalpha and increased the reactivity of the raloxifene- or ICI 182,780-bound ERalpha, with probes that recognize the 4OHT-bound ERalpha 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 ERalpha.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Base Sequence
  • Binding Sites / genetics
  • DNA / genetics
  • Estradiol / analogs & derivatives*
  • Estradiol / metabolism
  • Estrogen Receptor alpha
  • Fulvestrant
  • HeLa Cells
  • Humans
  • In Vitro Techniques
  • Leucine / chemistry
  • Ligands
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Promoter Regions, Genetic
  • Protein Conformation
  • Raloxifene Hydrochloride / metabolism
  • Receptors, Estrogen / chemistry*
  • Receptors, Estrogen / genetics
  • Receptors, Estrogen / metabolism*
  • Tamoxifen / analogs & derivatives*
  • Tamoxifen / metabolism
  • Transcription Factor AP-1 / metabolism
  • Transcriptional Activation
  • Two-Hybrid System Techniques

Substances

  • Estrogen Receptor alpha
  • Ligands
  • Receptors, Estrogen
  • Transcription Factor AP-1
  • Tamoxifen
  • afimoxifene
  • Fulvestrant
  • Raloxifene Hydrochloride
  • Estradiol
  • DNA
  • Leucine