Identification of a Structural Determinant Necessary for the Localization and Function of Estrogen Receptor α at the Plasma Membrane

Abstract

Estrogen receptors (ER) have been localized to the cell plasma membrane (PM), where signal transduction mediates some estradiol (E2) actions. However, the precise structural features of ER that result in membrane localization have not been determined. We obtained a partial tryptic peptide/mass spectrometry analysis of membrane mouse ER␣ protein. Based on this, we substituted alanine for the determined serine at amino acid 522 within the E domain of wild-type (wt) ER␣. Upon transfection in CHO cells, the S522A mutant ER␣ resulted in a 62% decrease in membrane receptor number and reduced colocalization with caveolin 1 relative to those with expression of wt ER␣. E2 was significantly less effective in stimulating multiple rapid signals from the membranes of CHO cells expressing ER␣ S522A than from those of CHO cells expressing wt ER␣. In contrast, nuclear receptor expression and transcriptional function were very similar. The S522A mutant was also 60% less effective than wt ER␣ in binding caveolin 1, which facilitates ER transport to the PM. All functions of ER␣ mutants with other S-to-A substitutions were comparable to those of wt ER, and deletion of the A/B or C domain had little consequence for membrane localization or function. Transfection of ER␣ S522A into breast cancer cells that express native ER downregulated E2 binding at the membrane, signaling to ERK, and G 1 /S cell cycle events and progression. However, there was no effect on the E2 transactivation of an ERE-luciferase reporter. In summary, serine 522 is necessary for the efficient translocation and function of ER␣ at the PM. The S522A mutant also serves as a dominant-negative construct, identifying important functions of E2 that originate from activating PM ER.

The localization and non‐genomic function of the membrane‐associated estrogen receptor in oligodendrocytes

“…Using antibodies directed against different epitopes of the nuclear ER, many laboratories have demonstrated that the ER can locate in the region of the cell membrane (Razandi et al, 2003;Simoncini et al, 2003;Song et al, 2002). However, the structure of mERs and whether they span the plasma membrane or contain an extracellular ligand binding region is controversial.…”

“…Using antibodies directed against different epitopes of nuclear ER, many laboratories have demonstrated that some forms of ER can locate in the region of the cell membrane (Razandi et al, 2003;Simoncini et al, 2003;Song et al, 2004). Toran-Allerand et al (2002) described a novel mER, designated ER-X, which is enriched in the brain, uterus, and lung of the postnatal rodent and prefers 17a-estradiol to 17b-estradiol as the ligand.…”

Caveolin proteins and estrogen signaling in the brain

“…Mutation of mouse serine 522 to an alanine reduces ERα binding to CAV1 by ~60% in CHO cells and reduces membrane-localization of ERα by a similar percentage [72]. The S522A mutation also acts as a dominant-negative in relation to membrane estrogen receptor signaling [73].…”

Crosstalk between steroid receptors and the c-Src-receptor tyrosine kinase pathways: implications for cell proliferation

“…ERa forms that have been engineered to remain in the cytoplasm and/or to be transcriptionally inactive can still stimulate intracellular signaling pathways such as MAPK and stimulate DNA synthesis in ER-negative fibroblast cells (Castoria et al, 1999). Mutations that diminish the cytoplasmic/membrane localization severely impair these responses in other ER-negative model systems (Razandi et al, 2003a). Others have reported that transcriptionally inactive and cytoplasmic forms of ERs and the androgen receptor are fully functional in activating ERK signaling through a Shc/c-Src-mediated pathway and inhibiting apoptosis in bone cells (Kousteni et al, 2001).…”