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Functional Analysis of a Novel Estrogen Receptor- b Isoform

A new level of complexity has recently been added to estrogen signaling with the identification of a second estrogen receptor, ER b . By screening a rat prostate cDNA library, we detected ER b as well as a novel isoform that we termed ER b 2. ER b 2 contains an in-frame inserted exon of 54 nucleotides that results in the predicted insertion of 18 amino acids within the ER b hormone-binding domain. We also have evidence for the expression of both ER b 1 and ER b 2 in human cell lines. Competition ligand binding analysis of bacterially expressed fusion proteins revealed an 8-fold lower affinity of ER b 2 for 17 b -estradiol (E 2 ) [dissociation constant (K d ; 8 n M )] as compared with ER b 1 (K d ; 1 n M ). In vitro transcribed and translated ER b 1 and ER b 2 bind specifically to a consensus estrogen responsive element in a gel mobility shift assay. Furthermore, we show heterodimerization of ER b 1 and ER b 2 with each other as well as with ER a . 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-de-pendent manner with ER a and ER b 1, but not with ER b 2. In cotransfection experiments with expression plasmids for ER a , ER b 1, and ER b 2 and an estrogen-responsive element-containing luciferase reporter, the dose response of ER b 1 to E 2 was similar to that of ER a although the maximal stimulation was approximately 50%. In contrast, ER b 2 required 100- to 1000-fold greater E 2 concentrations for maximal activation. Thus, ER b 2 adds yet another facet to the possible cellular responses to estrogen. (Molecular Endocrinology