NR2C is phosphorylated on S1096 by PKB and phosphorylation is regulated by IGF-I and NMDA receptor activity. A, HEK-293 cells were co-transfected with NR2C WT or NR2C S1096A and vector, a constitutively active form of PKB (caPKB), or a dominant-negative form of PKB (dnPKB), and immunoblots of cell lysate were probed with either the NR2C antibody or NR2C S1096 phosphorylation-state specific antibody. B, HEK-293 cells expressing NR2C were treated for 10 min +/− wortmannin, and the cells were stimulated for 10 min +/− IGF-I in the continued presence or absence of wortmannin. The immunoblots of cell lysate were probed with either the NR2C antibody or NR2C S1096 phosphorylation-state specific antibody. A-B, The data were quantified by measuring phosphorylated NR2C/NR2C band intensity ratios using ImageJ software and normalizing to either NR2C with caPKB (A) or NR2C with IGF-I (B). Data represent means +/− SEM (n=3). C, cerebellum and hippocampus from a P12 mouse were solubilized, and NR2C was immunoprecipitated from lysates, resolved by SDS-PAGE and immunoblotted with antibodies as indicated. Recognition of phosphorylated NR2C was prevented following λ phosphatase treatment of immunoblots. D-F, Cultured cerebellar granule cells were treated +/− IGF-I (50 ng/ml), NMDA (50 μM) or APV (50 μM) for various times as indicated. NR2C was immunoprecipitated from lysates, resolved by SDS-PAGE and immunoblotted with either the NR2C antibody or the NR2C S1096 phosphorylation-state specific antibody. The data were quantified by measuring phosphorylated NR2C/input NR2C band intensity ratios using ImageJ software and normalizing to untreated NR2C WT control cultures. Data represent means +/− SEM (n=3) (*, P < 0.05).

Disruption of 14-3-3ε binding to NR2C reduces NR2C surface expression. Cerebelluar granule cells were co-transfected with NR2C containing an extracellular GFP protein tag (GFP-NR2C) or GFP-NR2C S1096A and Myc-14-3-3ε WT or Myc-14-3-3ε dn. Two days after transfection, cells were incubated with anti-GFP antibody for 30 min at room temperature. Cells were fixed and incubated with Alexa 647-conjugated (blue) anti-rabbit secondary antibody to visualize the surface receptors. Cells were then washed, permeabilized, and labeled with anti-Myc monoclonal antibody and Alexa 568-conjugated (red) anti-mouse secondary antibody to visualize the 14-3-3ε transfected cells. The Alexa 647 fluorescence is shown in green for visual effects. The right panels show higher magnification images of boxed regions. Scale bar = 10 μm. The data were quantified by measuring ratios of surface NR2C/total NR2C using Metamorph software. Data represent means +/− SEM (n=3) (*, P < 0.05).

NR2C specifically interacts with 14-3-3ε, and binding is regulated by S1096. A, Schematic diagram of NR2C including an alignment of NR2A (amino acids 1281-1300), NR2B (amino acids 1293-1312) and NR2C (amino acids 1086-1105). NR2C S1096 is indicated with an arrowhead, and NR2A S1291 and NR2B S1303 are indicated with asterisks. The shaded area indicates a.a. 1076 to a.a. 1250 of NR2C, which was used as bait in the yeast two-hybrid screen. B, Yeast were co-transformed with LexA-NR2C WT, LexA-NR2C S1096A or LexA-NR2C S1144A and either Gal4 vector or Gal4-14-3-3ε and growth was evaluated on appropriate yeast selection medium. Results shown are 10-fold serial dilutions of yeast cells. C, HEK-293 cells were transiently transfected with FLAG-NR2C or FLAG-NR2C S1096A and Myc-14-3-3ε with or without NR1-1a. Receptors were immunoprecipitated from cell lysates with anti-FLAG antibodies or IgG antibodies as a negative control. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-c-Myc antibodies. Input = 2% of total lysate. D, adult rat cerebella were solubilized, and 14-3-3ε was immunoprecipitated from lysates with anti-14-3-3 antibodies or IgG antibodies as a control. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-NR2C antibodies. Input = 5% of total lysate.

PKB phosphorylation of NR2C on S1096 increases NMDA receptor surface expression. A, HEK-293 cells were co-transfected with NR1-1a-IRES-DsRed, FLAG-NR2C-IRES-EGFP and vector, caPKB or dnPKB. Cells were collected and analyzed by FACS analysis (see Experimental Procedures). Bar plot showing the normalized ratio of NR2C, EGFP and DsRed expressed cells versus EGFP and DsRed expressed cells. Data represent means +/− SEM (n=3) (*, P < 0.05). B, HEK-293 cells expressing NR1-1a and NR2C WT or NR2C S1096A were incubated +/− IGF-I for 20 min before biotinylating surface proteins. Total lysates were compared to surface pools of proteins by immunoblotting with the NR2C antibody. The data were quantified by measuring surface NR2C/input NR2C band intensity ratios using ImageJ software and normalizing to untreated NR2C WT control cultures. Data represent means +/− SEM (n=3) (*, P < 0.05). C and D, Cultured cerebellar granule cells were treated +/− IGF-I (50 ng/ml) (C) or NMDA (50 μM) (D) before biotinylating surface proteins. Total lysates (lanes 1 and 3) were compared with surface pools of proteins (lanes 2 and 4) by immunoblotting with the NR2C antibody. Data was quantified using ImageJ software. Data represent means +/− SEM (n=3) (*, P < 0.05).

NR2C surface expression is neuroprotective. A, Cerebellar granule cells (DIV4) were co-transfected with DsRed and vector, GFP-NR2A, GFP-NR2B or GFP-NR2C. On DIV 6, time-lapse imaging was performed during exposure to NMDA (200 μM). DsRed was used to visualize the changes in cell morphology of transfected neurons. NMDA treatment resulted in cell body swelling and dendritic varicosities of neurons expressing DsRed alone, NR2A or NR2B. Scale bar = 20 μm. Data shown are representative of four independent experiments. B, Cerebellar granule cells (DIV4) were co-transfected with DsRed and GFP-NR2C WT or GFP-NR2C S1096A. Cells were fixed on DIV 8. The images shown are representative of healthy and sick neurons. The data were quantified by measuring percentage of NR2C-expressing neurons with dendritic fragmentation or varicosities. Data represent means +/− SEM (n=3) (*, P < 0.05).