Tamoxifen and Raloxifene Differ in Their Functional Interactions with Aspartate 351 of Estrogen Receptor α

Abstract

The bulky side chains of antiestrogens hinder folding of the ligand binding domain (LBD) of estrogen receptors (ERs) into a transcriptionally active conformation. The presence of a tertiary amine in the side chain of raloxifene, which interacts with a negatively charged residue in helix H3 of the ER LBD [Asp351 in human (h)ER␣], is important for antiestrogenicity in animal and cellular models. To better understand the molecular basis of the differential activity of tamoxifen and raloxifene, we have examined the influence of tertiary amine substituents and of mutations at position 351 in hER␣ on the activity profiles of tamoxifen derivatives. Results obtained in several cellular model systems suggest that the degree of antagonist activity of tamoxifen derivatives does not strictly correlate with the basicity of the side chain but depends on an optimal spatial relationship between the tertiary amine of these antiestrogens and the negative charge at position 351. Although altering the position of the negative charge at residue 351 (mutation D351E) had little effect on transcriptional activity in the presence of tamoxifen, it drastically increased the partial agonist activity of a tamoxifen derivative with improved antagonist activity as well as that of raloxifene. Our results suggest that contrary to raloxifene, tamoxifen and most of its derivatives do not interact with Asp351 in an optimal manner, although this can be improved by modifying tertiary amine substituents.

Transactivation Function-2 of Estrogen Receptor α Contains Transactivation Function-1-regulating Element

“…Previously, Mak et al (17) reported that the I362D mutation disrupted the recruitment of p160/SRC1 to the LBD; however, replacement to alanine (I362A) did not affect p160/SRC1 recruitment and transcription activity. There have been reports that altering the surface charge of H3 causes the antagonist reversal (22)(23)(24). The mutation of aspartic acid 351 on human ER␣ corresponding to mouse Asp-355 to tyrosine (hER␣-D351Y) enhances agonist activity of 4OHT and alters raloxifene from an antagonist to a partial agonist (23).…”

“…Replacement of Asp-351 to alanine (hER␣-D351A) attenuated ligand (E2 and raloxifene)-mediated transactivation. In contrast, replacement to glutamic acid (hER␣-D351E) attenuated the E2-mediated transactivation but did not affect raloxifene-mediated transcription (24). Importantly, the residues of mouse I362 corresponding to human Ile-358 and human Asp-351 are localized on the same surface of H3 forming a highly conserved region between mouse and human ER␣.…”

Antiestrogens: structure-activity relationships and use in breast cancer treatment

“…Mutation D351G abrogated induction of expression of the estrogen target gene TGFA by tamoxifen in transfected MDA-MB-231 cells (MacGregor Schafer et al . 2000), and mutation D351A abolished partial activity of ERα on a reporter gene in the presence of tamoxifen in HepG2 cells (Dayan et al . 2006), consistent with a role of Asp351 in mediating the partial agonist activity of SERMs in the absence of optimal interaction with their side chain tertiary amines.…”

The 2.0 Å crystal structure of the ERα ligand‐binding domain complexed with lasofoxifene

“…In support of this mechanism, modifications to the pendant side chain of tamoxifen that alter the nature of the azacycle, while preserving the tertiary amine functionality, show dramatic differences in the level of transcriptional activation in cell-based assays (Dayan et al 2006). Abolition of the negative charge at Asp 351, by mutation of this residue to Ala or Val, greatly reduced the propensity for transcriptional activation in tamoxifen derivatives, regardless of the chemical structure of the terminal azacycle (Dayan et al 2006). Similarly, Webb et al (2003) have reported differential effects of tamoxifen and raloxifene on recruitment of corepressor proteins.…”

“…Such a repositioning of AF-2 has been demonstrated to be critical for corepressor recruitment in the case of PPARa bound to a potent antagonist (Xu et al 2002), and Nettles and Greene (2005) have suggested a similar mechanism at work in ERa. In support of this mechanism, modifications to the pendant side chain of tamoxifen that alter the nature of the azacycle, while preserving the tertiary amine functionality, show dramatic differences in the level of transcriptional activation in cell-based assays (Dayan et al 2006). Abolition of the negative charge at Asp 351, by mutation of this residue to Ala or Val, greatly reduced the propensity for transcriptional activation in tamoxifen derivatives, regardless of the chemical structure of the terminal azacycle (Dayan et al 2006).…”