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.
Department of Physiology, Wayne State University School of Medicine, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201, USA.
Changqing
C
Zhao
Akiko
A
Koide
Judith
J
Abrams
Sarah
S
Deighton-Collins
Angela
A
Martinez
Janice A
JA
Schwartz
Shohei
S
Koide
Debra F
DF
Skafar
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