Interaction of tetrahydrocrysene ketone with estrogen receptors alpha and beta indicates conformational differences in the receptor subtypes

Estrogen receptors (ER) alpha and beta bind estradiol (E-2) and other estro genic ligands with different affinities. To measure the rate of E-2 associa tion with ER alpha and ER beta, we employed tetrahydrocrysene ketone (THCK) , a fluorescent ligand that is an agonist with ER alpha and an antagonist w ith ER beta, We report that THCK binds E-2-liganded and unliganded ER alpha and ER beta, indicating a THCK binding site(s) other than the E-2 binding pocket. THCK fluorescence was greater for ligand-occupied ER beta than ER a lpha, suggesting differences in the microenvironment of the THCK binding si te(s), THCK fluorescence was also significantly greater for E-2-, 4-hydroxy tamoxifen-, and tamoxifen aziridine-liganded versus unliganded ER, allowing calculations of E-2 association rate constants (k(a)) of 7.60 +/- 0.75 and 5.12 +/- 0.30 x 10(5) M-1 s(-1) for E-2-ER alpha and E-2-ER beta, respecti vely, THCK did not affect ER alpha binding to estrogen response element (ER E) DNA, but decreased ER beta-ERE binding. We conclude that THCK binding si te(s) on ER alpha versus ER beta are different and important for ER functio n. (C) 2000 Academic Press.