Characterization of rodent constitutively active estrogen receptor α variants and their constitutive transactivation mechanisms

Estrogen receptor α (ERα) mRNAs exhibit remarkable heterogeneity owing to complicated alternative splicing. Some encode C-terminally-truncated ERα proteins, which display ligand-independent transactivation or dominant-negative activity. We previously characterized C-terminally-truncated ERα mRNA variants with cryptic sequences in humans and mice, and demonstrated that helices in the ligand-binding domains (LBDs) of ERα variants contribute to ligand-independent transcriptional activity. However, existence of non-conventional coding exons and generation of constitutively active ERα variants have remained to be examined in rats. To comparatively analyze modular organization and splicing profiles of the ERα genes, the range of C-terminally-truncated ERα variants was explored in rats and mice using rapid amplification of cDNA ends and RT-PCR cloning. Furthermore, their functions were determined in transiently transfected cells using expression constructs and luciferase reporter assays. Multiple cryptic exons and C-terminally-truncated ERα variant mRNAs were identified in rats and mice. Naturally occurring and artificially truncated variants/constructs lacking helix 5 potentially exhibited gain-of-function in transfected cells. Although cryptic exons and splicing profiles were poorly conserved among humans, mice, and rats, constitutively active variants were generated from the ERα genes. Moreover, the primary mechanism of ligand-independent activation by C-terminally-truncated ERα variants is C-terminus to helix 5 deletion in the LBD. This comparative study documented the complexity of ERα genes, mRNAs, and proteins, and further determined the underlying structural basis of ligand-independent activation by C-terminally-truncated ERα variants.