Molecular and Cellular Endocrinology I I5 (1995) 65-72 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ t!!i acular and lulmr Endwmksy Binding properties of coumestrol to expressed human estrogen receptor Suzanne Scarlata*a, Richard Miksicekb zyxwvutsrqponmlkjihgfedcbaZYXWVUT “Department of Physiology and Biophysics State University of New York at Stony Brook, Stony Brook NY 11794-8661, USA bDepartment of Pharmacology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA Received I June 1995; accepted 28 August 1995 - zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Abstract We have studied the binding of coumestrol, an inherently fluorescent analog of 17/I-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/?-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 C53OW 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 C53OW mutant. Keywords: Coumestrol; Estrogen receptor; Fluorescence; Ligand binding; Estrogen response element 1. Introduction The presence of estrogen receptor (ER) in breast carcinoma cells is an important predictor of patient response to hormonal therapy. Currently, ER is deter- mined by measuring the binding of isotopically labeled estrogen or by the use of ER-specific antibodies in immunohistochemical assays. However, it is difficult to assess the heterogeneity of receptor expression in tumor biopsy samples. One promising method for obtaining improved information about the amount and distribu- tion of ER present in individual cells is by using fluorescence microscopy of the receptor with inherently fluorescent analogs of 17/?-estradiol bound to the re- ceptor, or by using fluorescent antibodies to the recep- tor (Pertschuk et al., 1981, 1994; Ozzello et al., 1991; Miksicek, 1993). Only a limited number of fluorescent ligands have been described that interact with ER (Lee et al., 1977; Roa et al., 1980; Lee 1980; Fevig et al. 1987; Katzenellenbogen et al., 1986; Anstead and Katz, Abbreviations: hER, human estrogen receptor; ERE estrogen re- sponse element; dFl. normalized change in coumestrol fluorescence intensity of hER-transfected cells relative to mock-transfected cells: as defined in Eq. I. * Corresponding author, Phone: 516 444 3071; Fax: 516 444 3432. 1988; Bindal and Katzenellenbogen, 1986) and many of these display a significant reduction in affinity for the receptor compared with 17/3-estradiol or permeability problems that prevent their use in intact cells. Many years ago the fluorescence properties of a naturally occurring plant estrogen called coumestrol were ex- plored (Lee et al., 1977). These investigators found that association of coumestrol with ER in calf uterine ex- tracts produced observable changes in the fluorescence properties of this probe that could be readily reversed by the addition of 17/?-estradiol. Furthermore, they estimated that coumestrol binds to this receptor with an affinity only five- to ten-fold less than that of 17/?-estra- diol itself. Coumestrol has the potential disadvantage that its excitation wavelength (340 nm) is close to that of other cellular components such as nicotinamide adenine dinucleotides. However, its high quantum yield and strong affinity for ER coupled with improvements in optical components and computerized image en- hancement methods provides a strong impetus for a better understanding of the interaction of coumestrol with ER and the effects that association with the recep- tor has on the fluorescence properties of this probe. In this study we have used fluorescence spectroscopy to directly measure the binding of coumestrol to recom- 0303-7207/95/$09.50 0 1995 - Elsevier Science Ireland Ltd. Ail rights reserved SSDlO303.7207(95)0367 I -S