Abstract
A new level of complexity has recently been added to estrogen signaling with the identification of a second estrogen receptor, ERβ. By screening a rat prostate cDNA library, we detected ERβ as well as a novel isoform that we termed ERβ2. ERβ2 contains an in-frame inserted exon of 54 nucleotides that results in the predicted insertion of 18 amino acids within the ERβ hormone-binding domain. We also have evidence for the expression of both ERβ1 and ERβ2 in human cell lines. Competition ligand binding analysis of bacterially expressed fusion proteins revealed an 8-fold lower affinity of ERβ2 for 17β-estradiol (E2)[ dissociation constant (Kd ∼ 8 nm)] as compared with ERβ1 (Kd ∼ 1 nm). In vitro transcribed and translated ERβ1 and ERβ2 bind specifically to a consensus estrogen responsive element in a gel mobility shift assay. Furthermore, we show heterodimerization of ERβ1 and ERβ2 with each other as well as with ERα. In affinity interaction assays for proteins that associate specifically with the hormone-binding domain of these receptors, we demonstrate that the steroid receptor coactivator SRC-1 interacts in an estrogen-dependent manner with ERα and ERβ1, but not with ERβ2. In cotransfection experiments with expression plasmids for ERα, ERβ1, and ERβ2 and an estrogen-responsive element-containing luciferase reporter, the dose response of ERβ1 to E2 was similar to that of ERα although the maximal stimulation was approximately 50%. In contrast, ERβ2 required 100- to 1000-fold greater E2 concentrations for maximal activation. Thus, ERβ2 adds yet another facet to the possible cellular responses to estrogen.
In VitroBinding of the Purified Hormone-Binding Subunit of the Estrogen Receptor to Oligonucleotides Containing Natural or Modified Sequences of an Estrogen-Responsive Element
