Evidence Denies the Presence of O-GlcNAcylation on Mouse Glucocorticoid Receptor and Its Potential Involvement in Receptor Transcriptional Activity

Abstract

O-linked N-acetylglucosamine (GlcNAc) glycosylation (O-GlcNAcylation) is commonly found on many cytoplasmic and nuclear proteins. It can play a role in protein trafficking, signal transduction, and in some nuclear proteins it is involved in the control of gene expression. The steroid receptor family consists of proteins that have similar domain architecture including individual DNA and hormone-binding domains that have closely related three-dimensional structures. The discovery of O-linked GlcNAc on both androgen and estrogen receptors and the realization that the GlcNAc plays a role in the transcriptional activity of these receptors raise the possibility that this glycosylation is a common mechanism involved in transcriptional modulation in all members of the steroid receptor family. To test this hypothesis, we affinity purified the mouse glucocorticoid receptor from cell lines engineered to overexpress the receptor and used GlcNAc-specific lectin chromatography, lectin-blotting analysis, and galactosylation assay to assess the presence of GlcNAc modification. All three techniques were found to be highly sensitive when used with proteins known to harbor GlcNAc yet they failed to show the presence of GlcNAcylation on the mouse GR. We also determined the effect of mutation at seven major potential glycosylation sites of the receptor on its transcriptional activity. We conclude that either the mouse GR is not modified by GlcNAc or that the amount of the modification is so low that it cannot be detected. Therefore, the O-GlcNAcylation appears not to be a common mechanism used to modify the activity of all steroid receptors.