Mutations targeted to a predicted helix in the extreme carboxyl-terminal region of the human estrogen receptor-alpha alter its response to estradiol and 4-hydroxytamoxifen.
The human estrogen receptor-alpha, a member of the nuclear receptor superfamily, is a ligand-regulated transcriptional modulator. Because comparatively little is known about the extreme carboxyl-terminal region of the estrogen receptor (F domain), we used secondary structure prediction to design mutations that delete the F domain (S554stop), disrupt a possible turn (G556L/G557L), and alter a predicted helix (S559A/E562A, Q565P), and we evaluated the effects of these mutations on hormone binding and transcription activation in response to estradiol and the mixed agonist/antagonist 4-hydroxytamoxifen. Mutations that deleted the F domain (S554stop) or targeted the predicted helix (S559A/E562A, Q565P) greatly reduced or eliminated the agonist activity of 4-hydroxytamoxifen. Deleting the F domain increased the affinity of the receptor for estradiol and decreased the antagonist activity of 4-hydroxytamoxifen. The Q565P mutant exhibited a non-cooperative hormone-binding mechanism, as well as an impaired response to estradiol and increased antagonist activity of 4-hydroxytamoxifen. Our results show that mutations in the F domain alter not only the response to estradiol, the affinity for hormone, and the interaction between receptor subunits but can uncouple the agonist and antagonist activities of 4-hydroxytamoxifen. These results suggest that the F domain modulates the activity of the estrogen receptor-alpha by multiple mechanisms.
Schwartz
Janice A
JA
Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA.
Zhong
Li
L
Deighton-Collins
Sarah
S
Zhao
Changqing
C
Skafar
Debra F
DF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004958:Estradiol / Q000494:pharmacology*; D047628:Estrogen Receptor alpha; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011960:Receptors, Estrogen / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology*
D017931:DNA Primers; D047628:Estrogen Receptor alpha; D011960:Receptors, Estrogen; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol