Human estrogen receptor mutants with altered estrogen and antiestrogen ligand discrimination

A structural characteristic of many antiestrogens (AEs) is a bulky side chain with basic or polar functional groups that may interact with charged and polar amino acids near the hormone-binding site in the estrogen receptor. Recently, we have identified Cys⁵³⁰ as the residue of the human estrogen receptor that is the site of covalent labeling by aziridine analogs of estrogens (Es) and AEs (Harlow, K. W., Smith, D. N., Katzenellenbogen, J. A., Greene, G. L., and Katzenellenbogen, B. S. (1989) J. Biol. Chem. 264, 17476-17485). Since the aziridine function is on the bulky side chain of these ligands, Cys⁵³⁰ must be at or near the site of those side chain interactions. To probe these interactions, we have, by site-directed mutagenesis, made mutant human estrogen receptors in which charged and polar amino acids near Cys⁵³⁰ are changed (Glu⁵²³ to Gln, Lys⁵²⁹ and Lys⁵³¹ to Gln, Asn⁵³² to Asp, and Asp⁵³⁸ to Asn) so as to alter charge with minimal steric alteration. These receptors were expressed in mammalian (Chinese hamster ovary or COS-1) cells and assayed for their binding affinity for Es and AEs, their interaction with estrogen-responsive element DNA, and their ability to activate or suppress transcription of estrogen-responsive reporter genes. Two of the estrogen receptor mutants, KKN-QQD (mutation of Lys⁵²⁹, Lys⁵³¹, and Asn⁵³² to Gln⁵²⁹, Gln⁵³¹, and Asp⁵³², respectively) and KK-QQ (mutation of Lys⁵²⁹ and Lys⁵³¹ to Gln⁵²⁹ and Gln⁵³¹, respectively), in which the local charge is changed from +2 to -1 or 0, respectively, display an affinity for estradiol (E₂) 5-10 times lower than that of the wild-type receptor, which is attributable to an enhanced rate of E₂ dissociation. Although these mutant receptors have reduced affinity for a variety of Es, they retain unaltered affinity for AE. The profiles of transcriptional activation of reporter genes by various concentrations of E₂ show that these two mutants (KKN-QQD and KK-QQ) require 40- and 15-fold higher E₂ concentrations, respectively, to achieve half-maximal activity. In contrast, mutants E523Q and D538N, with changes at amino acids further from Cys⁵³⁰, were unaltered in their hormone binding and transactivation activity by E or AE. Interestingly, the AEs 4-hydroxytamoxifen, LY 117018, U 23469M, and ICI 164384 were 15-30-fold more effective in inhibiting E₂-stimulated transcription by mutants KKN-QQD and KK-QQ compared to the wildtype receptor. These mutants bind to estrogen-responsive element DNA as well as does the wild-type estrogen receptor, as demonstrated by gel retardation assays; but the mobilities of mutant receptor•E₂•DNA and mutant receptor•AE•DNA complexes are different from that of the wild type receptor. These results show that changes of specific amino acids in the hormone-binding domain near Cys⁵³⁰ can result in changes in binding affinity of the receptor for one category of ligands (Es) but not for another (AEs), resulting in a greatly reduced potency of Es in transactivation and a greatly enhanced sensitivity to AEs in suppression of E-stimulated transcription. The findings suggest that these amino acids near Cys⁵³⁰ are involved in receptor discrimination between Es and AEs.