Regulation Of Inositol 1,4,5-Trisphosphate Receptor Isoforms By O-Linked Glycosylation

Abstract

The inositiol 1,4,5 trisphospate receptor (InsP3R), an intracellular calcium channel, is a family of three isoforms. All three isoforms display a significant level of sequence identity yet they differ in expression level, localization and many functional aspects. We previously showed that InsP3R type 1 is modified by O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylated). Through this dynamic and inducible modification a single monosaccharide is covalently attached to serine and threonine residues of the protein backbone, providing protein regulation similar to O-phosphorylation. We also reported that increased O-GlcNAcylation of the InsP3R type 1 reduced the percent of cells that responded to addition of extracellular agonists and those that did respond had a decreased InsP3 dependent calcium release from the endoplasmic reticulum (ER). We now report that the InsP3R type 3 is also O-GlcNAcylated. Interestingly, the functional impact of O-GlcNAcylation on InsP3R type 3 channel is opposite to the effect measured with the InsP3R type 1. Human cholangiocytoma cells (MzChA-1) contain >90% InsP3R type 3. When these cells were incubated in hyperglycemic media there was an increase in the percent cells responding to InsP3 generating stimuli and there was an increase in the InsP3 dependent calcium release from the ER. A difference in functional response between InsP3R isoforms was reported previously for phosphorylation by cyclic AMP dependent protein kinase (PKA). In contrast, the InsP3R type 2 showed no detectable O-GlcNAc glycosylation and no significant functional changes even though the enzymes necessary for both the addition and removal of the monosccharide are present in all cell types tested. The dynamic and inducible nature of O-GlcNAcylation and the isoform specificity suggests that this form of modification of the InsP3R and subsequent changes in intracellular calcium transients will be important in physiological and pathophysiological processes.