Evaluation of Conformational Changes in hER-HBD by Pharmacological Dissection of Hormone Dissociation Rates in a Homogeneous Hormone-Binding Assay
Hormone dissociation rates can be used as a surrogate parameter to monitor changes in conformational states, as the hormone binding affinity is directly related to the dissociation rate and is dependent on receptor conformation. High-resolution studies of dissociation rates are tedious because the bound radioligand has to be separated from unbound. In contrast, homogeneous radioligand binding assays are rapid and have high temporal resolution.The homogeneous assay format allows rapid mixing, followed by continuous measurement of bound radioactivity. We describe a homogeneous hormone-binding assay for human estrogen-receptor hormone-binding domain (hER-HBD) produced in a yeast expression system. The binding assay was used for quantitation of dissociation rates for [3H]-estradiol (E2) by addition of different estrogens and anti-estrogens. Our results revealed that different compounds caused significantly different monophasic dissociation rates (expressed as min) for [3H]-E2: DES (0.046 min−1) > >TAM (0.040) >E3 (0.032) ≈ E2 (0.029). However, a closer examination of the concentration dependence showed that at higher concentrations certain nonsteroidal compounds induced biexponential dissociation curves. We propose that, depending on the concentration and identity of the chaser used, different conformational states of the hER-HBD are induced. These different states have different affinities for the hormone.
KeywordsEstrogen Estradiol Diethylstilbestrol Estriol
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