Binding of estrogen receptor with estrogen conjugated to bovine serum albumin (BSA)

BACKGROUND: The classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA (E(2)-BSA) has been widely used as an agent to study these novel membrane-associated ER events. However, a recent report suggests that E(2)-BSA does not compete for E(2 )binding to purified ER in vitro. To resolve this apparent discrepancy, we performed competition studies examining the binding of E(2 )and E(2)-BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E(2)-BSA preparations with unconjugated E(2 )(usually between 3–5%), the latter was carefully removed by ultrafiltration. RESULTS: As previously reported, a 10-to 1000-fold molar excess of E(2)-BSA was unable to compete with (3)H-E(2 )binding to ER when added simultaneously. However, when ER was pre-incubated with the same concentrations of E(2)-BSA, the binding of (3)H-E(2 )was significantly reduced. E(2)-BSA binding to a putative membrane-associated ER was directly visualized using fluorescein labeled E(2)-BSA (E(2)-BSA-FITC). Staining was restricted to the cell membrane when E(2)-BSA-FITC was incubated with stable transfectants of the murine ERα within ER-negative HeLa cells and with MC7 cells that endogenously produce ERα. This staining appeared highly specific since it was competed by pre-incubation with E(2 )in a dose dependent manner and with the competitor ICI-182,780. CONCLUSIONS: These results demonstrate that E(2)-BSA does bind to purified ER in vitro and to ER in intact cells. It seems likely that the size and structure of E(2)-BSA requires more energy for it to bind to the ER and consequently binds more slowly than E(2). More importantly, these findings demonstrate that in intact cells that express ER, E(2)-BSA binding is localized to the cell membrane, strongly suggesting a membrane bound form of the ER.