Isoform-specific regulation of the inositol 1,4,5-trisphosphate receptor by O-linked glycosylation

Abstract

The inositol 1,4,5-trisphosphate receptor (InsP(3)R), an intracellular calcium channel, has three isoforms with >65% sequence homology, yet the isoforms differ in their function and regulation by post-translational modifications. We showed previously that InsP(3)R-1 is functionally modified by O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) (Rengifo, J., Gibson, C. J., Winkler, E., Collin, T., and Ehrlich, B. E. (2007) J. Neurosci. 27, 13813-13821). We now report the effect of O-GlcNAcylation on InsP(3)R-2 and InsP(3)R-3. Analysis of AR4-2J cells, a rat pancreatoma cell line expressing predominantly InsP(3)R-2, showed no detectable O-GlcNAcylation of InsP(3)R-2 and no significant functional changes despite the presence of the enzymes for addition (O-β-N-acetylglucosaminyltransferase) and removal (O-β-N-acetylglucosaminidase) of the monosaccharide. In contrast, InsP(3)R-3 in Mz-ChA-1 cells, a human cholangiocarcinoma cell line expressing predominantly InsP(3)R-3, was functionally modified by O-GlcNAcylation. Interestingly, the functional impact of O-GlcNAcylation on the InsP(3)R-3 channel was opposite the effect measured with InsP(3)R-1. Addition of O-GlcNAc by O-β-N-acetylglucosaminyltransferase increased InsP(3)R-3 single channel open probability. Incubation of Mz-ChA-1 cells in hyperglycemic medium caused an increase in the InsP(3)-dependent calcium release from the endoplasmic reticulum. The dynamic and inducible nature of O-GlcNAcylation and the InsP(3)R isoform specificity suggest that this form of modification of InsP(3)R and subsequent changes in intracellular calcium transients are important in physiological and pathophysiological processes.

FIGURE 1.

InsP₃R-3 is modified by O-GlcNAcylation. A and B, Western blot analysis of InsP₃R-3 immunoprecipitated (IP) from Mz-ChA-1 cell lysates using anti-InsP₃R-3 antibody or anti-O-GlcNAc antibody RL2, respectively. Cells were grown for 72 h in normal medium (5.6 mm glucose) or in medium supplemented with 8 mm GlcNAc as indicated. Membranes were probed with anti-InsP₃R-3 or anti-O-GlcNAc antibody, respectively. The arrows indicate the InsP₃R-3 band. Quantitation of bands was done as described under “Experimental Procedures.” O-GlcNAcylation after treatment with 8 mm GlcNAc was twice the amount in immunoprecipitates from untreated cell lysates (A). O-GlcNAcylation after treatment with 8 mm GlcNAc was enhanced by 1.6-fold in comparison with untreated InsP₃R-3 (B). IB, immunoblot.

FIGURE 2.

O-GlcNAcylation of InsP₃R-3 in Mz-ChA-1 cells occurs in vivo. A, Mz-ChA-1 cells were grown in medium supplemented with either 20 mm mannitol (Ma; control; n = 4) or 8 mm glucosamine (GA; n = 3) for 72 h and lysed, and 30 μg of protein was analyzed by Western blotting. B, to examine O-GlcNAcylation under physiological conditions, Mz-ChA-1 cells were grown for 72 h in medium supplemented with 20 mm mannitol or the physiological sugar glucose at a concentration of 20 mm (total concentration of 25.6 mm), and the whole cell lysate was analyzed by Western blotting (n = 4). Membranes were probed with anti-InsP₃R-3 or anti-O-GlcNAc antibody, respectively. Results are shown as averaged densitometry values normalized to control values (20 mm mannitol) per blot; graphs are means ± S.E. IP, immunoprecipitation; IB, immunoblot.

FIGURE 3.

Addition and removal of O-GlcNAc has subtype-specific effects on channel open probability of the InsP₃R. A, the removal of O-GlcNAc decreases and addition increases the channel open probability of InsP₃R-3 inserted into planar lipid bilayers. All channel recordings were performed in the presence of 2 mm ATP and 8 μm ruthenium red at pCa 6.8. Four representative traces of recordings of one InsP₃R-3 from the same experiment are shown. First trace, in the absence of InsP₃, no channel activity was measured. Second trace, the addition of 5 μm InsP₃ evoked channel activity. Third trace, the addition of OGA to the cis-side of the channel and subsequent de-O-GlcNAcylation decreased channel open probabilities. Fourth trace, the addition of OGT to the cis-side of the channel, causing O-GlcNAcylation, reversed the effect of OGA and increased channel open probabilities. Channel openings are defined as downward deflections from the base line. B, the removal and addition of O-GlcNAc has reciprocal effects on the channel open probability of InsP₃R-3. Recordings were performed in the presence of 5 μm InsP₃, 2 mm ATP, and 8 μm ruthenium red at pCa 6.8. The open probability of untreated channels incorporated in lipid membranes was measured (mean ± S.E., n = 4). Treatment with OGA, causing removal of O-GlcNAc, decreased open probability (mean ± S.E., n = 4), whereas following OGT treatment, the addition of O-GlcNAc increased channel activity (mean, n = 2). C, InsP₃-dependent measurement of channel open probability. The channel open probabilities of untreated and OGA-treated (deglycosylated) InsP₃R-3 are shown for different InsP₃ concentrations. Data points were normalized to averaged values of untreated channels stimulated with 5 μm InsP₃. D, the channel open probabilities of OGA-treated (deglycosylated) and OGT-treated (O-GlcNAcylated) InsP₃R-1 compared with previously published data for untreated WT InsP₃R-1 (67) are shown for different InsP₃ concentrations. Data points were normalized to averaged values of channels stimulated with 2 μm InsP₃. Data were taken from at least three independent experiments for each [InsP₃].

FIGURE 4.

O-GlcNAcylation alters intracellular calcium transients due to extracellular stimulation. Shown are imaging data of Mz-ChA-1 cells grown in different sugar media and stimulated with Mg-ATP. A, representative single traces showing the time course for single cells stimulated with 1 μm Mg-ATP and 10 μm for the second stimulus. B, percentage of cells responding to stimulation with 0.5, 1.0, 2.0, and 5.0 μm Mg-ATP after incubation in different media as indicated. C, peak heights of cell calcium response to Mg-ATP. Cells were grown in different media as indicated. *, p < 0.05.

FIGURE 5.

InsP₃R-2 in AR4-2J cells is not O-GlcNAcylated. A, Western blot analysis of AR4-2J cell lysates incubated for 72 h in 20 mm mannitol or 20 mm glucose as indicated. Membranes were probed for O-GlcNAc and InsP₃R-2, respectively. The arrow indicates the height of the InsP₃R-2 band, which was not recognized by antibody RL2, showing that InsP₃R-2 in AR4-2J cells is not glycosylated. B, Western blot analysis of AR4-2J cell lysates incubated for 72 h in 20 mm mannitol (control (CTL)) or grown in normal medium and incubated with 10 mm streptozotocin (STZ) for 60 min. C, immunoprecipitation of InsP₃R-2 with anti-InsP₃R-2 antibody (lane 1), the control (CTL) with anti-HSP90 antibody (lane 2), and O-GlcNAcylated proteins with anti-O-GlcNAc antibody RL2 (lane 3) from AR4-2J lysates from cells incubated in 100 μm PUGNAc for 24 h ahead of lysis. Western blot analysis was performed, and membranes were probed with anti-InsP₃R-2 and anti-O-GlcNAc antibodies, respectively. The arrow indicates the expected height of the InsP₃R-2 band. D, AR4-2J cells were grown on coverslips and incubated in different sugar-containing media for 72 h as described. Cells were stimulated with 100 nm carbachol, and single calcium transients were measured. ns, not significant; IB, immunoblot.