Binding properties of coumestrol to expressed human estrogen receptor

We have studied the binding of coumestrol, an inherently fluorescent analog of 17 beta-estradiol, to human estrogen receptor (hER) in extracts of transfected cultured cells. The binding of coumestrol to the hER as well as its dissociation from the receptor can be directly determined by the change in fluorescence intensity of the probe. In agreement with previous studies using calf uterine extracts, we find that coumestrol binds to the receptor with a ten-fold lower affinity than 17 beta-estradiol. However, the rate of dissociation appears to be close to that of the native ligand. Coumestrol can accept energy from Trp residues in the excited state and, using a C530W hER mutant, we have confirmed that residue 530 is close to the ligand-binding pocket. We also find that the presence of saturating amounts of the specific DNA binding sites (ERE) did not significantly alter the binding affinity of coumestrol to either wild type hER or the C530W mutant.