Abstract
Estrogen receptors (ER) are ligand-inducible transcription factors regulated by Ser(Thr)-O-phosphorylation. Many transcription factors and eukaryotic RNA polymerase II itself are also dynamically modified by Ser(Thr)-O-linked N-acetylglucosamine moieties (O-GlcNAc). Here we report that subpopulations of murine, bovine, and human estrogen receptors are modified by O-GlcNAc. O-GlcNAc moieties were detected on insect cell-expressed, mouse ER (mER) by probing with bovine milk galactosyltransferase, followed by structural analysis. Wheat germ agglutinin-Sepharose affinity chromatography also readily detected terminal GlcNAc residues on subpopulations of ER purified from calf uterus, from human breast cancer cells (MCF-7), or from mER produced by in vitro translation. These data suggest that greater than 10% of these populations of estrogen receptors bear O-GlcNAc. Site mapping of insect cell expressed mER localized one major site of O-GlcNAc addition to Thr-575, within a PEST region of the carboxyl-terminal F domain. Based upon their relative resistance to both hexosaminidase and to in vitro galactosylation, O-GlcNAc moieties appear to be largely buried on native mER. This dynamic saccharide modification, like phosphorylation, may play a role in modulating the dimerization, stability, or transactivation functions of estrogen receptors.
Highlights
O-GlcNAc moieties were detected on insect cell-expressed, mouse estrogen receptor (ER) by probing with bovine milk galactosyltransferase, followed by structural analysis
Western blot analysis demonstrates that Mr ϭ 66,000 and a proteolytic fragment Mr ϭ 50,000 were recognized by the monoclonal antibody H222 (Fig. 2B), generated against the human ER
About 10% [3H]tamoxifen-labeled hER binds WGA-Sepharose and is eluted with GlcNAc. These data indicate that a significant subpopulation of both calf ER and hER are modified by O-GlcNAc. These studies demonstrate that a significant subpopulation (10% or more) of estrogen receptors from mouse, bovine, or human sources are modified by Ser(Thr)-O-GlcNAc, a highly dynamic form of intracellular glycosylation that is often reciprocal with Ser(Thr)-O-phosphorylation
Summary
We report that subpopulations of murine, bovine, and human estrogen receptors are modified by O-GlcNAc. O-GlcNAc moieties were detected on insect cell-expressed, mouse ER (mER) by probing with bovine milk galactosyltransferase, followed by structural analysis. Wheat germ agglutinin-Sepharose affinity chromatography readily detected terminal GlcNAc residues on subpopulations of ER purified from calf uterus, from human breast cancer cells (MCF-7), or from mER produced by in vitro translation. We show that the estrogen receptors from murine, bovine, and human sources are all modified by OGlcNAc and that a major O-GlcNAc site (Thr-575) occurs within a PEST sequence of the carboxyl-terminal F domain.
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