Interaction of Tetrahydrocrysene Ketone with Estrogen Receptors α and β Indicates Conformational Differences in the Receptor Subtypes

Estrogen receptors (ER) α and β bind estradiol (E2) and other estrogenic ligands with different affinities. To measure the rate of E2 association with ERα and ERβ, we employed tetrahydrocrysene ketone (THCK), a fluorescent ligand that is an agonist with ERα and an antagonist with ERβ. We report that THCK binds E2-liganded and unliganded ERα and ERβ, indicating a THCK binding site(s) other than the E2 binding pocket. THCK fluorescence was greater for ligand-occupied ERβ than ERα, suggesting differences in the microenvironment of the THCK binding site(s). THCK fluorescence was also significantly greater for E2-, 4-hydroxytamoxifen-, and tamoxifen aziridine-liganded versus unliganded ER, allowing calculations of E2 association rate constants (ka) of 7.60 ± 0.75 and 5.12 ± 0.30 × 105 M−1 s−1 for E2-ERα and E2-ERβ, respectively. THCK did not affect ERα binding to estrogen response element (ERE) DNA, but decreased ERβ-ERE binding. We conclude that THCK binding site(s) on ERα versus ERβ are different and important for ER function.