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J. Biol. Chem., Vol. 266, Issue 17, 10880-10887, Jun, 1991
JC Reese and BS Katzenellenbogen
We have carried out experiments to determine the role of the cysteines in
the hormone-binding domain (HBD) of the human estrogen receptor (ER) in
receptor function. In each mutant receptor, 1 of the 4 cysteines in the HBD
(cysteines 381, 417, 447, and 530) was changed by in vitro mutagenesis of
the ER cDNA (containing Gly400) from cysteine to alanine; Cys530 was also
mutated to a serine. The mutant and wild-type receptor cDNAs were expressed
in Chinese hamster ovary cells using an expression vector containing the
Rous sarcoma virus promoter. The mutant and wild-type receptors were
assayed for hormone binding and for their ability to activate
estrogen-responsive reporter plasmids. All ER mutants bound estradiol (E2)
with affinity similar to wild-type ER, displaying a Kd between 0.3 and 0.8
nM (wild-type ER Kd = 0.45 +/- 0.10 nM). All were capable of covalent
labeling by the affinity ligands ketononestrol aziridine, an estrogen
agonist, and tamoxifen aziridine, an antagonist. Since in previous work we
identified Cys530 as the site of covalent attachment of these ligands
(Harlow, K.W., Smith, D.N., Katzenellenbogen, J.A., Greene, G.L., and
Katzenellenbogen, B.S. (1989) J. Biol. Chem. 264, 17476-17485) it appears
that an alternate residue(s) can be labeled in the absence of a cysteine at
position 530; studies with methyl methanethiosulfonate, a cysteine-specific
reagent, suggest that this residue is probably another cysteine in the HBD.
The C381A, C417A, C530A and C530S ERs showed E2-stimulated transcriptional
activation profiles similar to wild-type ER whereas the dose response for
E2 for the C447A mutant was shifted to the right, requiring 50 x higher E2
concentrations to achieve half-maximal response. Tamoxifen aziridine
inhibited E2-stimulated transcription, and ketononestrol aziridine
stimulated transcription by wild-type, C530A, and C530S ER, but the
effectiveness of these covalently attaching ligands was altered in the
C530A and C530S mutants. Thus, these two mutant receptors are altered in
their transactivation response to agonist and antagonist affinity labeling
ligands but are unaltered in their response to reversibly binding estrogens
and antiestrogens. In addition, we show that a mutant ER (C447A) can have
an affinity for E2 similar to that of wild-type ER but differs in its
ability to activate transcription in response to E2, indicating a
decoupling of the hormone binding and transcriptional activation functions
in this receptor.
Mutagenesis of cysteines in the hormone binding domain of the human estrogen receptor. Alterations in binding and transcriptional activation by covalently and reversibly attaching ligands
Department of Physiology and Biophysics, University of Illinois, Urbana 61801.
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