Endocrine Abstracts

Oestrogen receptor (ER) activity has a wide spectrum of functions in the human body, including the development, growth and maintenance of the reproductive system. There are two ER subtypes, ERα and ERβ. The latter exists as multiple splice variants (ERβ1, ERβ2 and ERβ5) some of which lack a steroid ligand-binding domain. ERs act as steroid-ligand activated transcription factors. Epidermal growth factor (EGF) acting via the MAP kinase pathway has been shown to induce phosphorylation of serine residues within the N-terminal domain of ERα.

In the present study we have used fluorescence recovery after photobleaching (FRAP) and an ERE-luciferase reporter assay to investigate the impact of steroid ligands and EGF on the sub-nuclear mobility and transcriptional activity of ERs. ERα, ERβ1 (full length, wild type) and ERβ5 (C-terminal splice variant) proteins tagged with yellow fluorescent protein (YFP) were cloned into adenoviral vectors and used to infect MDA or Ishikawa cells.

Expression of all YFP-ERs was restricted to the nuclear compartment: FRAP analysis revealed that all receptors were highly mobile in the absence of ligand. Addition of 10⁻⁷ M oestradiol resulted in a re-distribution of ERα from a diffuse to a punctate pattern and a marked reduction in intra-nuclear mobility of both ERα and ERβ1. Addition of E2 had no impact on the mobility of ERβ5. Results obtained with an ERE-reporter assay mirrored these findings with increased expression following addition of E2 to cells containing ERα or ERβ1 but no induction in the presence of ERβ5. Addition of EGF to Ishikawa cells containing YFP-ERα or -ERβ also had an impact on sub-nuclear mobility; ERβ5 is currently being investigated. In summary, FRAP provides a rapid, visual, analysis of the behaviour of ERs in live cells in the presence of different stimuli including steroid ligand(s) or growth factor(s).