THE KEY TO THE ANTIESTROGENIC MECHANISM OF RALOXIFENE IS AMINO-ACID 351 (ASPARTATE) IN THE ESTROGEN-RECEPTOR

The crystallization of the ligand-binding domain (LBD) of the estrogen receptor (ER) with 17 beta-estradiol and raloxifene [A. M. Brzozowski et al,, Nature (Lond.), 389: 753-758, 1997] now provides a molecular basis for the biological activity of complexes as either agonists or a ntagonists. It is well established that the critical structural featur e of antiestrogens is a correctly positioned alkylaminoethoxy side cha in. The X-ray crystallography clearly shows that the alkylaminoethoxy side chain of raloxifene causes a specific and inappropriate molecular perturbation of the LBD and that the nitrogen in the side chain must hydrogen bond with aspartate 351 in the LBD of ER. We previously ident ified and characterized a naturally occurring mutation in the ER from a tamoxifen-stimulated transplantable human breast tumor line. The mut ation is at AA351 of LBD, where the aspartate is changed to tyrosine ( Asp351Tyr). In this report, we compared and contrasted the pharmacolog y of raloxifene to block or induce E-2-stimulated increase in TGF-alph a mRNA in stable transfectants of ER-negative human breast cancer cell s with the cDNAs from wild-type, mutant-amino acid (AA) 400 ER and mut ant-AA 351 ER. Our results show that the mutation at AA 351 that repla ces aspartate by tyrosine specifically changes the pharmacology of ral oxifene from an antiestrogen to an estrogen. By contrast, a mutation a t A4 400 does not, and the antiestrogenic properties of raloxifene are retained. These data and the fact that the nitrogen in the side chain must specifically interact with aspartate 351 makes this the key to t he antiestrogenic activity of raloxifene.