Estrogen Receptors Beta4 and Beta5 Are Full Length Functionally Distinct ERβ Isoforms: Cloning from Human Ovary and Functional Characterization

Abstract

We describe here the cloning and functional characterization of two unique ER isoforms, ERbeta4 and ERbeta5. The full length ERbeta4 and ERbeta5 were identified by asymmetric PCR using human ovary cDNA, cloning, and sequence analyses. Both receptors share identical sequences with ERbeta1 from exon 1 to exon 7. In the place of exon 8, ERbeta4 has unique sequences arising from a region downstream of the ERbeta gene and upstream of the SYNE2 gene. ERbeta5 has sequences arising from retention of the 5' end of the intron between exon 7 and 8. Both receptors bind promoter sequences on DNA but do not bind estrogen. They translocate to the nucleus and exhibit three to four times higher estrogen-independent transcriptional activity than ERbeta1. When co-transfected with ERalpha, they predominantly form heterodimers and negatively regulate its transcriptional activity. Estrogen-independent transcriptional activity of ERbeta5, but not ERbeta4, was inhibited by ERalpha, demonstrating for the first time that ERalpha regulates ERbeta. Tissue-specific expression of ERbeta4 and ERbeta5, together with their ligand-independent transcriptional properties and ERalpha modulating activities, could have a number of implications in seemingly unlinked biological processes regulated by estrogen.

The importance of ERβ signalling in the ovary

“…While this isoform has not been detected in humans, shortened transcripts and alternatively spliced forms of ERb have been reported in normal ovary and ovarian tumours (Maruyama et al 1998, Moore et al 1998, Ogawa et al 1998. These forms designated ERb1, ERb2 (also known as ERbcx), ERb3, ERb4, and ERb5 (Ogawa et al 1998, Inoue et al 2000, Poola et al 2005) each produce a full-length transcript despite truncations at the 3 0 -end (Fig. 3B).…”

“…Multiple isoforms of the ERb subtype have now been described (Chu & Fuller 1997, Petersen et al 1998, Matthews & Gustafsson 2003, Poola et al 2005, Zhao et al 2005, although it is not clear whether these forms are all biologically active. Chu & Fuller (1997) reported the existence of a 54 nucleotide insert (Fig.…”

“…3B). Initially, it was thought that ERb4 and ERb5 existed only as truncated transcripts but this has proven not to be the case (Poola et al 2002(Poola et al , 2005.…”

Nuclear and Cytoplasmic Expression of ERβ1, ERβ2, and ERβ5 Identifies Distinct Prognostic Outcome for Breast Cancer Patients

“…ERh exists as five full-length versions, ERh1 to ERh5 (8,9), each formed by alternative splicing of the last coding exon. ERh1 is the only fully functional isoform (10).…”

“…ERh2 is identical to ERhcx (11). ERh3 seems to be testis specific (8) and ERh4 is not found in breast tissue (9). Although ERh2 and ERh5 cannot bind ligand, transient transfection studies suggest that they may antagonize ERa function through heterodimerization (11 -13).…”