DETERMINANTS OF LIGAND SPECIFICITY OF ESTROGEN RECEPTOR-ALPHA - ESTROGEN VERSUS ANDROGEN DISCRIMINATION

We have been interested in understanding how the estrogen receptor (ER ) binds estrogens and discriminates between different classes of stero ids with closely related structures, Using insights from our prior stu dies on ER and from sequence comparisons of steroid receptors, we iden tified three residues in the hormone-binding domain of the human ER, L eu(345), Thr(347), and Glu(353), that we considered were likely to be involved in steroid A-ring recognition and therefore estrogen versus a ndrogen discrimination. We then tested the effect on ER activity of mu tating these ER residues to the corresponding androgen receptor residu es, Specifically, we examined the ability of the mutant receptors to b ind and be activated by 17 beta-estradiol and three different androgen s, No change in receptor activity was observed with the T347N mutation , while the L345S mutation greatly reduced ER activity in response to all ligands. Interestingly, the E353Q substitution behaved as expected , causing a 9-fold reduction in the transactivation potency of estradi ol and a concomitant 10-140-fold increase in the transactivation poten cy of different androgens, These reciprocal changes in the transcripti onal effectiveness of estrogens and androgens correlated with a decrea sed affinity of the E353Q ER for estradiol binding and an increased af finity for androgen binding, Therefore, amino acid Glu(353) appears to be playing a significant role in binding the A-ring phenolic group of estradiol and in receptor discrimination between estrogens and the mo st closely structurally related steroids, androgens. Based on this dat a and our earlier observations, we propose a model for the orientation of ligand within the binding pocket of ER in which the A-ring 3-pheno l of estradiol is hydrogen bonded to Glu(353) in helix-3 and the 17 be ta-hydroxyl of estradiol is hydrogen bonded to His(524) in helix-11. O ur findings with estrogen and androgen suggest that this orientation o f the steroid in the ligand-binding pocket, with the steroid A-ring in contact with helix-3 and the D-ring in contact with helix-11 residues , is likely to be general for all the steroid hormone receptors.