Functional characteristics of a novel murine estrogen receptor-beta isoform, estrogen receptor-beta 2

We have isolated a highly expressed splice variant mRNA of murine estrogen receptor-P (ERP), mER beta2, containing an in-frame 54 nucleotide insertion between exons 5 and 6 of wild-type mER beta1. The predicted ER beta2 prote in contains 18 amino acids inserted in the ligand binding domain of mER bet a1. Recombinant protein generated by in vitro transcription/translation sho wed that mER beta2 had markedly reduced ligand binding (K-D = 17.7 +/- 4.7 nM, mean +/- S.E.M., n= 3) compared with mER beta1-bound H-3-estradiol (K-D = 0.56 +/- 0.19nM, mean +/-s.E.M, n=3). Both receptors bound similarly to palindromic estrogen responsive elements (EREs) in vitro and in vivo, and s imilarly bent DNA. Transcriptional activity was assessed using transient tr ansfection analysis into a homologous murine cell line, NIH 3T3 cells. mER beta1 transactivated ERE-tk-CAT reporter genes similarly to mER alpha, wher eas mER beta2 had little activity except at high ligand concentrations. How ever, under conditions in which mER beta2 is unlikely to be ligand saturate d, co-transfected mER beta2 inhibited activity of mER alpha and possibly mE R beta1 on ERE-tk- CAT genes. Using a 'novel raloxifene responsive' gene re porter system (TGF-beta3-CAT), we found the ability of estradiol and LY1170 18 to activate both mER alpha and mER beta1 on this promoter was identical, and mER beta2 activity in the presence of either estradiol or LY117018 was only slightly less than that observed with either mER beta1 or mERa. Both mER beta1 and mER beta2 when liganded with LY117018 inhibited transcription at a classical ERE-regulated promoter under these transfection conditions, which was in marked contrast to their stimulatory effect at the transformi ng growth factor-beta3 promoter. These data suggest that responsiveness of gene expression to a relatively highly expressed variant murine ERP isoform , mER beta2, is both ligand and promoter specific. Determination of the rel ative level of expression of mER beta1 mRNA and mER beta2 mRNA in mouse tis sues indicated predominance of mER beta2 mRNA in some but not all tissues. These data suggest that the mER beta2 may have some tissue-specific and pro moter-specific modulatory effects.