Decreased responsiveness of naturally occurring mutants of human estrogen receptor α to estrogens and antiestrogens

Estrogen receptor a (ER alpha) is a ligand-inducible transcription factor that mediates the biological effects of estrogens and antiestrogens. Many point mutations in the human ER alpha gene have been reported to be associated with breast cancer, endometrial cancer, and psychiatric diseases. However, functional analyses for most mutants with amino acid changes are still lacking. In the present study, to investigate the effects of point mutations on the function, gel-shift assays and luciferase assays were performed for eight kinds of mutated ER alpha proteins, including a single nucleotide change of C207G (N69K), G478T (G160C), T887C (L296P), A908G (K303R), C926T (S309F), A1058T (E353V), A1186G (M396V), and G1231deletion (411fsX7). The mutated ER alpha expression plasmids were constructed by site-directed mutagenesis. With gel-shift assays using in vitro translated ER alpha proteins, binding to the consensus estrogen response element (ERE) was observed for the mutated ER alpha proteins except ER alpha (G160C) and ERa (411 fsX7), the binding of which was comparable with that of the wild type. Western blot analyses showed that ER alpha (G160C) could not be efficiently translated with the in vitro transcription/translation system and that ER alpha (411 fsX7) produced a truncated protein. To investigate the transactivation potency, wild-type or mutated ER alpha expression plasmids were co-transfected with pGL3-3EREc38 reporter plasmid into human breast adenocarcinoma MDA-MB-435 cells. The concentration-response curves (10 pM-100 nM E2) of the mutant ER alpha proteins except ER alpha (E353V) and ER alpha (411fsX7) were similar to that of wild-type ER alpha. However, at a low level of E2 (100pM), the mutants ER alpha (N69K), ER alpha (L296P), ER alpha (S309F), and ER alpha (M396V) showed a significant decrease of transactivation compared with that of the wild-type ER alpha. The mutants ER alpha (E353V) and ER alpha (411fsX7) did not show responsiveness to E2 and antiestrogens, 4-hydroxytamoxifen (40HT) and ICI 182,780. The mutant ER alpha (S309F) showed decreased responsiveness for the antiestrogenicity of 40HT. In conclusion, we found that some of the naturally occurring human ERa mutants with amino acid changes may have an altered responsiveness to estrogen and antiestrogens. (c) 2006 Elsevier Ltd. All rights reserved.