DIFFERENT RESIDUES OF THE HUMAN ESTROGEN-RECEPTOR ARE INVOLVED IN THERECOGNITION OF STRUCTURALLY DIVERSE ESTROGENS AND ANTIESTROGENS

We have previously examined, by alanine scanning mutagenesis, amino ac ids 515-535 of the estrogen receptor (ER) ligand binding domain to det ermine which of these residues are important in estradiol binding, Mut ation at four sites that potentially lie along one face of an alpha-he lix, Gly(521), His(524), Leu(525), and Met(528), all significantly imp aired estradiol binding by the ER (Ekena, K., Weis, K. E., Katzenellen bogen, J. A., and Katzenellenbogen, B. S. (1996) J. Biol. Chem. 271, 2 0053-20059), In this report, we compare the pattern of residues that a re important in the recognition of several structurally diverse estrog en agonists and antagonists (the synthetic nonsteroidal agonist hexest rol, an agonist derived from the mold metabolite zearalenone, P1496, a nd the partial agonist antagonist trans-hydroxytamoxifen) with those t hat are predicted to contact estradiol in the receptor-ligand complex, Although there are some similarities in the pattern of residue recogn ition among all four ligands, each ligand showed distinct differences as well, Interestingly, alanine substitution at only one residue, the leucine at position 525, was found to inhibit binding of all the ligan ds tested, Another residue, His(524), was found to be important in the recognition of three different agonists but not trans-hydroxytamoxife n (the only ligand lacking a second hydroxyl group), The recognition o f estradiol and another agonist, P1496, was impaired by the G521A muta tion, whereas ligand-induced activity by the two compounds that lack B - and C-rings, hexestrol and trans-hydroxytamoxifen, was unaffected, O ur findings demonstrate that these ligands fit into the ER ligand bind ing pocket differently and that each contacts a distinct set of amino acids, The smaller ligands (estradiol and hexestrol) have a narrower f ootprint of interacting residues than the larger ligands (P1496 and tr ans hydroxytamoxifen). This pattern of interaction is most consistent with the amino acids within this region being in contact with the port ion of these ligands that corresponds to the D-ring end of estradiol, The interplay between the shape of an ER ligand and the residues that support its binding to ER may potentially underlie the selective actio ns of different ER ligands in various cell and promoter contexts.