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

Abstract

Estrogen receptor alpha (ERalpha) is a ligand-inducible transcription factor that mediates the biological effects of estrogens and antiestrogens. Many point mutations in the human ERalpha 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 ERalpha 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 ERalpha expression plasmids were constructed by site-directed mutagenesis. With gel-shift assays using in vitro translated ERalpha proteins, binding to the consensus estrogen response element (ERE) was observed for the mutated ERalpha proteins except ERalpha (G160C) and ERalpha (411fsX7), the binding of which was comparable with that of the wild type. Western blot analyses showed that ERalpha (G160C) could not be efficiently translated with the in vitro transcription/translation system and that ERalpha (411fsX7) produced a truncated protein. To investigate the transactivation potency, wild-type or mutated ERalpha expression plasmids were co-transfected with pGL3-3EREc38 reporter plasmid into human breast adenocarcinoma MDA-MB-435 cells. The concentration-response curves (10pM-100nM E2) of the mutant ERalpha proteins except ERalpha (E353V) and ERalpha (411fsX7) were similar to that of wild-type ERalpha. However, at a low level of E2 (100pM), the mutants ERalpha (N69K), ERalpha (L296P), ERalpha (S309F), and ERalpha (M396V) showed a significant decrease of transactivation compared with that of the wild-type ERalpha. The mutants ERalpha (E353V) and ERalpha (411fsX7) did not show responsiveness to E2 and antiestrogens, 4-hydroxytamoxifen (4OHT) and ICI 182,780. The mutant ERalpha (S309F) showed decreased responsiveness for the antiestrogenicity of 4OHT. In conclusion, we found that some of the naturally occurring human ERalpha mutants with amino acid changes may have an altered responsiveness to estrogen and antiestrogens.

Comparison of chemical binding to recombinant fathead minnow and human estrogen receptors alpha in whole cell and cell-free binding assays

“…It has been established that a single key amino acid substitution in the ER ligand binding domain can alter receptor binding capacity [3,4]. Notably, at least one key amino acid in the hER, which interacts hydrophobically with E2 (L349), is different in many fish receptors, including fathead minnow and rainbow trout [23].…”

“…It has been established that a single key amino acid substitution in the ER ligand binding domain can alter receptor binding capacity [3,4]. Notably, at least one key Chemical binding to fish and human estrogen receptors Environ.…”

“…All chemical stock solutions were made up in 100% ethanol except cadmium chloride, which was made up in distilled, deionized water. Radiolabeled [2,4,6,7,16,…”

“…It has been shown that single amino acid substitutions in the human estrogen receptor (hER) can have dramatic effects on receptor function [3,4]. Some piscine ligand‐binding domains have only 60% homology to hER or other nonpiscine vertebrate receptors [5,6].…”

Cellular recruitment and cytokine generation in a rat model of allergic lung inflammation are differentially modulated by progesterone and estradiol

“…Control groups consisted of Ovx-allergic rats injected with the corresponding volumes of the hormone vehicles (corn oil for 17␤-estradiol or distilled water for progesterone). One group of otherwise intact allergic rats was treated, 24 h before the antigen challenge, with a single injection of ICI 182,780 (500 g ip), an estradiol receptor antagonist (20,29) devoid of agonist effects (18).…”