Mutation of Leu-536 in human estrogen receptor-alpha alters the coupling between ligand binding, transcription activation, and receptor conformation.
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.
Zhao
Changqing
C
Department of Physiology, Wayne State University School of Medicine, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201, USA.
Koide
Akiko
A
Abrams
Judith
J
Deighton-Collins
Sarah
S
Martinez
Angela
A
Schwartz
Janice A
JA
Koide
Shohei
S
Skafar
Debra F
DF
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism; D047628:Estrogen Receptor alpha; D000077267:Fulvestrant; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D020849:Raloxifene Hydrochloride / Q000378:metabolism; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
D047628:Estrogen Receptor alpha; D008024:Ligands; D011960:Receptors, Estrogen; D018808:Transcription Factor AP-1; D013629:Tamoxifen; C016601:afimoxifene; D000077267:Fulvestrant; D020849:Raloxifene Hydrochloride; D004958:Estradiol; D004247:DNA; D007930:Leucine