BINDING-PROPERTIES OF COUMESTROL TO EXPRESSED HUMAN ESTROGEN-RECEPTOR

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