Functional analysis of a novel estrogen receptor-beta isoform

A new level of complexity has recently been added to estrogen signaling wit h the identification of a second estrogen receptor, ER beta. By screening a rat prostate cDNA library, we detected ER beta as well as a novel isoform that we termed ER beta 2. ER beta 2 contains an in-frame inserted exon of 5 4 nucleotides that results in the predicted insertion of 18 amino acids wit hin the ER beta hormone-binding domain. We also have evidence for the expre ssion of both ER beta 1 and ER beta 2 in human cell lines. Competition liga nd binding analysis of bacterially expressed fusion proteins revealed an 8- fold lower affinity of ER beta 2 for 17 beta-estradiol (E-2) [dissociation constant (K-d similar to 8 nM)] as compared with ER beta 1 (K-d similar to 1 nM). In vitro transcribed and translated ER beta 1 and ER beta 2 bind spe cifically to a consensus estrogen responsive element in a gel mobility shif t assay. Furthermore, we show heterodimerization of ER beta 1 and ER beta 2 with each other as well as with ER alpha. In affinity interaction assays f or 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 ER alpha and ER beta 1, but not with ER beta 2. In cotransfection experiments with expression plasmids for ER alpha, ER beta 1, and ER beta 2 and an estrogen-responsive element- containing luciferase reporter, the dose response of ER beta 1 to E-2 was s imilar to that of ER alpha although the maximal stimulation was approximate ly 50%. In contrast, ER beta 2 required 100- to 1000-fold greater E-2 conce ntrations for maximal activation. Thus, ER beta 2 adds yet another facet to the possible cellular responses to estrogen.