Journal: The Journal of Biological Chemistry

Article Title: A GluN2B disease-associated variant promotes the degradation of NMDA receptors via autophagy

doi: 10.1016/j.jbc.2026.111147

Figure Lengend Snippet: Molecular characterization of the disease-associated variant GluN2B_R519Q . A , structure of the rat tetrameric GluN1_GluN2B NMDA receptor in complex with glutamate, with the GluN1 subunit in gray , and the GluN2B subunit in blue (PDB: 9ARI ) . The amino-terminal domain (ATD), ligand-binding domain (LBD), and transmembrane domain (TMD) are shown in ribbon format with the R519 residue selected for study in yellow spheres. B , structural model displaying residues involved in the glutamate binding site of the WT GluN2B subunit are represented as sticks, with the glutamate ligand in pink ( upper ). In silico mutagenesis of the R519 residue to a glutamine eliminates the electrostatic interaction with glutamate ( lower ). C , effects of R519Q on total GluN2B protein expression levels 48 h post transient transfection of HEK293T with GluN1 and GluN2B constructs at a 1:1 ratio to express WT or GluN2B_R519Q variant NMDARs. β-actin serves as the soluble total protein loading control (n = 7). D , surface biotinylation assay to monitor the influence of the R519Q DAV on the surface expression of NMDARs 48 h post transient transfection. Na + /K + ATPase served as a membrane protein loading control (n = 8). E , HEK293T cells stably expressing WT or R519Q GluN2B NMDARs were subjected to cycloheximide (100 μg/ml) for the indicated times to determine the stability and rate of degradation. β-actin serves as the soluble total protein loading control (n = 5). Statistical significance was determined by a two-way repeated measures analysis of variance (ANOVA) followed by a post hoc Tukey test for comparison in multiple groups. F , immunofluorescence images of GluN2B ( green ) and calnexin ( red ), an ER marker to assess ER accumulation (scale bar = 10 μm). Pearson’s coefficients are reported (n > 30 cells) and statistical significance was determined using a Mann-Whitney test. All data are normalized to the appropriate loading control, and data are presented as mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test between two groups for ( C ) and ( D ). Significance level defined as ∗ p < 0.05, ∗∗∗∗ p < 0.0001.

Article Snippet: The pre-cleared lysates were incubated with 2 μg of mouse anti-GluN2B antibody (NeuroMab #75–097) for 1 h at 4 °C, and then 50 μl of Protein A/G-plus agarose beads were added and incubated overnight at 4 °C.

Techniques: Variant Assay, Ligand Binding Assay, Residue, Binding Assay, In Silico, Mutagenesis, Expressing, Transfection, Construct, Control, Surface Biotinylation Assay, Membrane, Stable Transfection, Comparison, Immunofluorescence, Marker, MANN-WHITNEY, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: A GluN2B disease-associated variant promotes the degradation of NMDA receptors via autophagy

doi: 10.1016/j.jbc.2026.111147

Figure Lengend Snippet: The GluN2B subunit undergoes degradation via autophagy . A , inhibition of the proteasome with MG132 (10 μM) and the lysosome with Bafilomycin-A1 (1 μM) for 6 h effect on the GluN2B subunit in HEK293T cells stably expressing recombinant WT or R519Q NMDARs. β-actin serves as the soluble total protein loading control (n = 5). B , effects of R519Q on total GluN2B protein expression levels 48 h post transient transfection with GluN1 and GluN2B constructs at a 1:1 ratio in ATG7 KO HEK293T to express WT or GluN2B_R519Q variant NMDARs. β-actin served as the soluble total protein loading control. (n = 6). C , surface biotinylation assay to monitor the influence of the R519Q DAV on the surface expression of NMDARs expressed in ATG7 KO HEK293T 48 h post transient transfection. Na + /K + ATPase served as a membrane protein loading control (n = 6). D , schematic of macroautophagy pathway, including induction, nucleation and formation of the isolation membrane, elongation, autophagosome formation, and fusion with the lysosome forming the autophagolysosome. Rapamycin is shown to inhibit mTOR, which results in autophagy activation, while 3-MA and Baf-A1 inhibit autophagy at different stages as shown. E , HEK293T cells stably expressing R519Q NMDARs were treated with autophagy activators (SMER28 10 μM, Rapamycin 100 nM) and inhibitors of autophagy (3-MA 50 mM and Baf-A1 20 nM) for 24 h. Changes to total GluN2B expression were monitored via immunoblot and p62 was used as a marker for autophagic flux. β-actin serves as the soluble total protein loading control (n = 3). F , siRNA knockdown of ATG7 effect on R519Q variant GluN2B expression and immunoblot was performed 48 h after knockdown. β-actin served as the soluble total protein loading control (n = 3). Non-targeting (NT) siRNA was used as a control for each condition, and two independent siRNA constructs were used to knockdown gene expression. G , siRNA knockdown of LC3b effects on R519Q variant GluN2B expression. Immunoblot performed 48 h after knockdown. β-actin served as the soluble total protein loading control (n = 3). Non-targeting siRNA was used as a control for each condition, and two independent siRNA constructs were used to knockdown gene expression. LC3-Pool contains two unique siRNAs for each of the LC3a, LC3b, and LC3c isoforms. All data are normalized to the appropriate loading control, and data are presented as mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test between two groups or an analysis of variance (ANOVA) followed by a post hoc Dunnett’s test for comparison in multiple groups. Significance level defined as ns, not significant, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The pre-cleared lysates were incubated with 2 μg of mouse anti-GluN2B antibody (NeuroMab #75–097) for 1 h at 4 °C, and then 50 μl of Protein A/G-plus agarose beads were added and incubated overnight at 4 °C.

Techniques: Inhibition, Stable Transfection, Expressing, Recombinant, Control, Transfection, Construct, Variant Assay, Surface Biotinylation Assay, Membrane, Isolation, Activation Assay, Western Blot, Marker, Knockdown, Gene Expression, Two Tailed Test, Comparison

Journal: The Journal of Biological Chemistry

Article Title: A GluN2B disease-associated variant promotes the degradation of NMDA receptors via autophagy

doi: 10.1016/j.jbc.2026.111147

Figure Lengend Snippet: The ER-phagy receptor CCPG1 interacts with the R519Q GluN2B variant and promotes its degradation through ER-to-lysosome-associated degradation . A , essential membrane-bound ER-phagy receptors. CCPG1, SEC62, and FAM134b are generally located in the ER sheets, while RTN3L, TEX264, and ATL3 are in the ER tubules. All ER-phagy receptors contain at least one LIR, represented as red circles, but induce degradation of distinct ER subdomains and client proteins. B , co-immunoprecipitation of HEK293T cells exogenously expressing R519Q GluN2B variants was done to measure interaction with ER-phagy receptors present in ER sheets CCPG1, FAM134b, and SEC62. Cells were treated with Baf-A1 (20 nM for 24 h), 24 h post transient transfection to enrich autophagy proteins for interaction. Quantification of the ratio of the protein of interest to GluN2B post co-IP is shown in the right panel (n = 3). C , siRNA knockdown of CCPG1 effects on GluN2B expression in HEK293T cells stably expressing R519Q NMDARs. Immunoblots performed 48 h after knockdown. Non-targeting siRNA was used as a control for each condition, and four independent siRNA constructs were used to knockdown gene expression. β-actin served as the soluble total protein loading control (n = 3). D , Dose–response overexpression of CCPG1 cDNA in HEK293T cells stably expressing R519Q GluN2B variants NMDARs. Cells were harvested 24 h after transient transfection. β-actin served as the soluble total protein loading control (n = 3). All data are normalized to the appropriate loading control, and data are presented as mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test between two groups or an analysis of variance (ANOVA) followed by a post hoc Dunnett’s test for comparison in multiple groups. Significance level defined as ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The pre-cleared lysates were incubated with 2 μg of mouse anti-GluN2B antibody (NeuroMab #75–097) for 1 h at 4 °C, and then 50 μl of Protein A/G-plus agarose beads were added and incubated overnight at 4 °C.

Techniques: Variant Assay, Membrane, Immunoprecipitation, Expressing, Transfection, Co-Immunoprecipitation Assay, Knockdown, Stable Transfection, Western Blot, Control, Construct, Gene Expression, Over Expression, Two Tailed Test, Comparison

Journal: The Journal of Biological Chemistry

Article Title: A GluN2B disease-associated variant promotes the degradation of NMDA receptors via autophagy

doi: 10.1016/j.jbc.2026.111147

Figure Lengend Snippet: The GluN2B LC3-interacting region (LIR) motif is highly conserved . A , canonical LC3-interacting motif consensus sequence pointing to the LIR motif identified in the GluN2B subunit and subsequent alanine substitution. Aromatic residue is shown in red, hydrophobic residue in blue. B , sequence alignment of Homo sapiens GluN2A and GluN2B CTD amino acid sequence in the region of interest. The LIR motif is in red in the GluN2B subunit. The serine residue that is phosphorylated by CAMKII is shown in green for each subunit. Asterisks mark conserved residues, colons mark conserved residues with similar properties, and a period denotes residues that are semi-conservative. C , amino acid sequence alignment of mammalian species demonstrating species conservation of the GluN2B CTD region of interest surrounding the LIR motif. D , effects of LIR motif disruption on WT, WT_F1307A and R519Q, R519Q_F1307A total GluN2B protein expression levels 48 h post transient transfection of HEK293T cells transfected with a 1:1 ratio of GluN1 and GluN2B constructs to express WT or GluN2B_R519Q variant NMDARs. β-actin served as the soluble total protein loading control (n = 4). E , surface biotinylation assay to monitor the influence of the LIR domain disruption on the WT and R519Q DAV surface expression of NMDARs 48 h post transient transfection Na + /K + ATPase served as a membrane protein loading control (n = 3). F , effects of LIR motif disruption on total expression of GluN2B subunits expressed in ATG7 KO cells. 48 h post transient transfection of GluN1 and GluN2B constructs with a 1:1 ratio of GluN1 and GluN2B constructs to express WT, WT_F1307A, R519Q, R519Q_F1307A variant NMDARs. β-actin served as the soluble total protein loading control (n = 4). G , surface biotinylation assay to monitor the influence of the LIR motif disruption on WT and the R519Q variant GluN2B subunits on the surface expression of NMDARs in ATG7 KO cells 48 h post transient transfection. Na + /K + ATPase served as a membrane protein loading control (n = 4). All data are normalized to the appropriate loading control, and data are presented as mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test between two groups or an analysis of variance (ANOVA) followed by a post hoc Tukey test for comparison in multiple groups. Significance level defined as ns, not significant, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The pre-cleared lysates were incubated with 2 μg of mouse anti-GluN2B antibody (NeuroMab #75–097) for 1 h at 4 °C, and then 50 μl of Protein A/G-plus agarose beads were added and incubated overnight at 4 °C.

Techniques: Sequencing, Residue, Disruption, Expressing, Transfection, Construct, Variant Assay, Control, Surface Biotinylation Assay, Membrane, Two Tailed Test, Comparison

Journal: The Journal of Biological Chemistry

Article Title: A GluN2B disease-associated variant promotes the degradation of NMDA receptors via autophagy

doi: 10.1016/j.jbc.2026.111147

Figure Lengend Snippet: Effects of LIR motif disruption on GluN2B autophagic degradation . A , inhibition of the proteasome with MG132 (10 μM) and the lysosome with Baf-A1 (1 μM) for 6 h effect on the WT ( left ) and WT_F1307A ( right ) GluN2B subunit in HEK293T cells 48 h after transient transfection. β-actin served as the soluble total protein loading control (n = 5). B , inhibition of the proteasome with MG132 (10 μM) and the lysosome with Baf-A1 (1 μM) for 6 h effect on the R519Q ( left ) and the R519Q_F1307A ( right ) GluN2B subunit in HEK293T cells 48 h after transient transfection. β-actin served as the soluble total protein loading control (n = 5). C , ratio of the normalized accumulation of R519Q/WT GluN2B ( left ) and the ratio of the normalized accumulation of WT_F1307A/WT GluN2B ( right ) upon treatment with Baf-A1 and MG132. D , ratio of the normalized accumulation of R519Q_F1307A/WT_F1307A GluN2B ( left ) and the ratio of the normalized accumulation of R519Q_F1307A/R519Q GluN2B ( right ) upon treatment with Baf-A1 and MG132. E , co-immunoprecipitation of HEK293T cells exogenously expressing WT, R519Q, or R519Q_F1307A NMDARs with GluN2B variants was done to measure interaction with key autophagy proteins. Cells were treated with Baf-A1 (20 nM for 24 h), 24 h post transient transfection to enrich autophagy proteins for interaction. Quantification of the ratio of the protein of interest to GluN2B post co-IP is shown in the right panel (n = 3). All data are normalized to the appropriate loading control, and data are presented as mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test between two groups or an analysis of variance (ANOVA) followed by a post hoc Tukey test for comparison in multiple groups. Significance level defined as ns, not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The pre-cleared lysates were incubated with 2 μg of mouse anti-GluN2B antibody (NeuroMab #75–097) for 1 h at 4 °C, and then 50 μl of Protein A/G-plus agarose beads were added and incubated overnight at 4 °C.

Techniques: Disruption, Inhibition, Transfection, Control, Immunoprecipitation, Expressing, Co-Immunoprecipitation Assay, Two Tailed Test, Comparison

Journal: The Journal of Biological Chemistry

Article Title: A GluN2B disease-associated variant promotes the degradation of NMDA receptors via autophagy

doi: 10.1016/j.jbc.2026.111147

Figure Lengend Snippet: Proposed mechanism of autophagic clearance of the GluN2B R519Q variant . The GluN2B R519Q variant fails quality control measures in the ER, resulting in its retention within the ER lumen. CCPG1, shown in red , present in the ER sheets, can recognize the misfolded and accumulating R519Q variant, blue , and targets it for autophagic degradation via recruitment of autophagy machinery via interactions with the LIR, shown as yellow circles , and FIR domains, shown in neon green , of CCPG1. It is also possible that the LIR domain present on the cytoplasmic CTD of the GluN2B subunit is able to interact with autophagy machinery directly.

Article Snippet: The pre-cleared lysates were incubated with 2 μg of mouse anti-GluN2B antibody (NeuroMab #75–097) for 1 h at 4 °C, and then 50 μl of Protein A/G-plus agarose beads were added and incubated overnight at 4 °C.

Techniques: Variant Assay, Control