First, we assayed chemicals to determine their ability to modulate direct interaction between the hER[Alpha] and the steroid receptor coactivator-1 (SRC-1) and in a competition binding assay to displace 17[Beta]-estradiol ([E.sub.2]).
This assay is based on the direct ligand-dependent interaction between the ER and the transcriptional steroid receptor coactivator-1 (SRC-1) after activation of the receptor, which results from a ligand-induced conformational change.
In the CARLA test, none of the xenobiotics studied was able to induce interaction between ER and SRC-1; this is similar to results with the antiestrogens ICI 182,780 and hydroxytamoxifen.
The pGEX GST-hER and pSG5 SRC-1 plasmids (34) were a gift from M.
Beads were incubated with the test compounds ([E.sub.2], DES, xenobiotic) and radiolabeled SRC-1 (produced in vitro using a coupled transcription/translation rabbit reticulocyte lysate system (TNT; Promega, Madison, WI).
This assay is based on the ligand-induced binding of SRC-1 to nuclear hormone receptors (34).
SRC-1 was expressed and labeled with [[sup.35]S]-methionine, and the LBD of ER was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST).
This property was used in the CARLA to identify potential ER ligands with a specific interaction between the coactivator SRC-1 and the fusion protein GST-ER[Alpha] LBD.
Our results demonstrate that, in contrast to [E.sub.2], none of the environmental pollutants tested was able to induce an interaction between SRC-1 and the ER[Alpha] LBD.