(A, B) Glutamate-induced change of NR2A (A) and NR2B (B) palmitoylation. Cultured cortical neurons were treated with 10 μM glutamate for 1 hr at 37°C. A representative example of an IP with each anti-NR2 antibody and [³H]palmitate incorporation (top) or IP and WB with each anti-NR2 antibody (middle) were shown. Quantified data of ratio of [³H]palmitate-labeled NR2 to total NR2 protein amount (bottom, left, n = 4, respectively, Student t-test, *p < 0.05, **p < 0.01 compared with control) and immunoprecipitated (IP) NR2 protein amount (bottom, right, n = 4, respectively). (C, D) Effects of treatment with tetrodotoxin (TTX) and bicuculline on NR2A (C) and NR2B (D) palmitoylation. Cultured cortical neurons were treated with 1 μM TTX or 20 μM bicuculline for 30 min at 37°C. A representative example of an in vitro acyl-biotinyl exchanged (ABE) sample, followed by purification with streptavidin-conjugated beads and WB with each anti-NR2 antibody is shown (top). Total protein expression (cell lysate) of each protein was confirmed by WB (middle). Quantified data of ratio of palmitoylated NR2 to total NR2 protein amount (bottom, left, n = 4, respectively, Student t-test, **p < 0.01, ***p < 0.001 compared with control) and total NR2 protein amount (bottom, right, n = 4, respectively).

(A) Palmitoylation of NR2A C-terminus in NR1/NR2A and NR1/NR2A 3CS by cotransfected palmitoyl acyl transferase GODZ or its inactive mutant GODZ(DHHS). (B) Palmitoylation of NR2A in NR1/NR2A and NR1/NR2A 4CS by cotransfected GODZ or GODZ(DHHS). (C) Palmitoylation of NR2A in NR1/NR2A and NR1/NR2A 7CS by cotransfected GODZ or GODZ(DHHS). (D) Palmitoylation of NR2B in NR1/NR2B, NR1/NR2B 3CS, NR1/NR2B 5CS and NR1/NR2B 8CS by cotransfected GODZ or GODZ(DHHS). Transfected HEK 293T cells were labeled with [³H]-palmitate and were then immunoprecipitated with anti-NR2A or anti-NR2B antibodies. The incorporated [³H]-palmitate signals were shown (upper top). Immunoprecipitation (IP, upper bottom) and total protein expression (cell lysate) of each protein (lower panels) were confirmed by western blotting (WB).

(A) Phosphorylation at Tyr1472 of GFP-NR2B WT and GFP-NR2B 3CS transfected cultured cortical neurons. (B) Representative patterns of surface expression of GFP-NR2B WT and GFP-NR2B 3CS mutant in transfected cultured cortical neurons. Total GFP-NR2B distribution (top) and surface expression of GFP-NR2B (bottom) were shown. (C) Ratio of surface to total expression of GFP-NR2B at 11 DIV (WT and 3CS, n = 16 neurons respectively). Student t-test. **p < 0.01 compared with WT. (D) Ratio of surface to total expression of GFP-NR2B at 22 DIV (WT and 3CS, n = 16 neurons respectively). Student t-test. **p < 0.01 compared with WT. (E) Regulation of NR2B constitutive internalization by its palmitoylation on Cys cluster I. The effect of Src family PTK-specific inhibitor PP2 (20 μM) or the inactive structural analog PP3 (20 μM) on internalization of GFP-NR2B WT (WT) and GFP-NR2B 3CS (3CS) in transfected cultured cortical neurons at 11 DIV (n = 12 neurons respectively). Cultured cortical neurons were treated with PP2 or PP3 for 30 min and the ratio of fluorescence intensities of internalized anti-GFP antibodies to surface GFP expression of GFP-NR2B were examined. ***p < 0.001. (F, G) Effects of treatment with Src family PTK specific inhibitor PP2 (20 μM) and its inactive analog PP3 (20 μM) for 30 min on steady state surface expression of GFP-NR2B WT and GFP-NR2B 3CS in transfected cultured cortical neurons were examined. (F) Ratio of surface to total GFP expression of GFP-NR2B at 11 DIV (four bars (WT+PP3, WT+PP2, 3CS+PP3, 3CS+PP2), n = 12 neurons respectively, F = 9.837, p < 0.0001, ANOVA). *p < 0.05 compared with PP3 treatment. (G) Ratio of surface to total GFP expression of GFP-NR2B at 22 DIV (four bars (WT+PP3, WT+PP2, 3CS+PP3, 3CS+PP2), n = 12 neurons respectively, F = 27.90, p < 0.0001, ANOVA). ***p < 0.001 compared with PP3 treatment. Error bars indicate sem.

(A) Surface expression of endogenous NR2A in GODZ-GFP transfected cultured cortical neurons. Typical expression patterns of surface NR2A and GODZ-GFP or GODZ(DHHS)-GFP were shown (left). Quantified data of surface expression of endogenous NR2A in transfected cultured cortical neurons at 21 DIV (right, n = 10 neurons respectively, Student t-test. ***p < 0.001 compared with control. (B) Ratio of surface to total GFP expression of GFP-NR2B at 11 DIV (WT and 5CS, n = 16 neurons respectively). (C) Ratio of surface to total GFP expression of GFP-NR2B at 22 DIV (WT and 5CS, n = 16 neurons respectively). Student t-test. *p < 0.05 compared with WT. (D) Effect of myc-GODZ or myc-GODZ(DHHS) inactive mutant on surface expression of GFP-NR2B in transfected cultured cortical neurons at 11 DIV. Ratio of surface to total GFP expression of GFP-NR2B at 11 DIV (WT and 5CS, n = 16 neurons respectively). Student t-test. *p < 0.05 compared with WT. Error bars indicate the sem.

(A) Palmitoylation of endogenous GluR2/3, NR2A and NR2B was examined in metabolically labeled cultured cortical neurons. Immunoprecipitation (IP) and detection of incorporated [³H]-palmitate into GluR2/3 and NR2A and NR2B (top). IP and expression of each protein was confirmed by western blotting (WB) (bottom). (B) Hydroxylamine treatment of palmitoylated NR2A and NR2B to confirm thioester linkage to intracellular cysteines. [³H]-palmitate content of Tris-HCl-treated (top) and hydroxylamine (NH₂OH)-treated (middle) IP samples are shown. IP of each NR2 protein from cell lysates of metabolically labeled cultured cortical neurons was confirmed by WB (bottom). (C) Palmitoylation of endogenous NR2A and NR2B was examined in cultured cortical neurons using a biotin switch assay for palmitoylation (Wan et al., 2007: see Experimental Procedures). Palmitoylated (Acyl-Biotinyl Exchanged) NR2 (top) and total protein expression (cell lysate) of NR2 protein (bottom) were confirmed by WB.

(A) C-terminal amino acid sequences of NMDA receptor subunits NR2A and NR2B, containing consensus cysteine clusters. Boxes indicate conserved cysteine residues (Cys cluster I and II). Transmembrane domain (TMD) 4 is underlined. Dotted box indicates AP-2 binding motif (YXXΦ). (B–E) Identification of NR2A and NR2B palmitoylation sites using a series of CS mutants. NR1/NR2A wild type and NR1/NR2A 7CS (B), or NR1/NR2A 3CS, NR1/NR2A C1214SC1217S, NR1/NR2A C1236SC1239S, NR1/NR2A 4CS (C), or NR1/NR2A C848S, NR1/NR2A C853S, NR1/NR2A C870S, NR1/NR2A C848SC853S, NR1/NR2A 3CS (D) and NR1/NR2B wild type and NR1/NR2B 3CS (E) were cotransfected into HEK 293T cells. Transfected cells were metabolically labeled with [³H]-palmitate and then solubilized and lysates were immunoprecipitated with anti-NR2A or anti-NR2B antibodies. The incorporated [³H]-palmitate signals were shown (upper top). Immunoprecipitation (IP, second panels) and total protein expression (cell lysate) of each protein (third and fourth panels) was confirmed by western blotting (WB).

(A) Fyn-mediated tyrosine phosphorylation of NR2A in NR1/NR2A and NR1/NR2A 3CS receptors. HEK 293T cells were cotransfected with NR2A or NR2A 3CS and NR1 in the presence or absence of Fyn. Transfected cells were solubilized and then immunoprecipitated with anti-NR2A antibodies, followed by probing with phosphotyrosine antibody (PY20) (upper top panel). Immunoprecipitation (IP, upper bottom) and total protein expression (cell lysate) of each protein (lower panels) were confirmed by western blotting (WB). (B) Fyn-mediated tyrosine phosphorylation of NR2B and NR2B 3CS. The phosphotyrosine signals (PY20) were shown (upper top). IP of NR2B (top), site-specific tyrosine phosphorylation (NR2BpY1472 and NR2BpY1252, middle) and total protein expression (cell lysate) of each protein (bottom) were confirmed by WB. (C) Effect of Fyn on NMDA receptor endocytosis in transfected HEK 293T cells. Representative patterns of total and constitutive internalized NR1 were shown (top). The ratio of fluorescence intensities of internalized anti-NR1 antibodies to total NR1 expression in transfected HEK 293T cells (bottom, four bars (NR1/NR2A, NR1/NR2A/Fyn, NR1/NR2A 3CS, and NR1/NR2A 3CS/Fyn), n = 22, n = 28, n = 29, n = 34 cells, respectively, F = 5.276, p < 0.002, ANOVA). ***p < 0.001 compared with combination without Fyn. Error bars indicate sem.

(A) Colocalization of NR2A with Golgi-apparatus marker GM-130 in HEK 293T cells cotransfected with NR1 and NR2A with or without myc-GODZ or myc-GODZ(DHHS) (upper panels) and with NR1 and NR2A 4CS with or without myc-GODZ or myc-GODZ(DHHS) (lower panels). (B) Colocalization of NR2A with GODZ in HEK 293T cells cotransfected with NR1 and NR2A with myc-GODZ or myc-GODZ(DHHS) (upper panels) and with NR1 and NR2A 4CS with myc-GODZ or myc-GODZ(DHHS) (lower panels). (C) Colocalization of NR2B with Golgi-apparatus marker GM-130 in HEK 293T cells cotransfected with NR1 and NR2B with or without myc-GODZ or myc-GODZ(DHHS) (upper panels) and with NR1 and NR2B 5CS with or without myc-GODZ or myc-GODZ(DHHS) (lower panels). (D) Colocalization of NR2B with GODZ in HEK 293T cells cotransfected with NR1 and NR2B with myc-GODZ or myc-GODZ(DHHS) (upper panels) and with NR1 and NR2B 5CS with myc-GODZ or myc-GODZ(DHHS) (lower panels).

(A) GODZ increases palmitoylation of NMDA receptors on the NR2A/2B subunit Cys cluster II region ((II) palm-NR2) in Golgi apparatus. Depalmitoylation of the NR2A/2B subunit Cys cluster II may regulate release of NMDA receptors from the Golgi for surface delivery as shown by black arrow. (B) Palmitoylation of the NMDA receptor on the NR2A/2B subunit Cys cluster I ((I) palm-NR2) ensures proper surface delivery and increases the stability of NMDA receptors on the cell surface via tyrosine phosphorylation of tyrosine based internalization motifs (pY). In contrast, depalmitoylated forms of surface NMDA receptors are less stably associated with the plasma membrane. Palmitoylation is reversible between these two forms as shown by white arrows. (C) Palmitoylation on Cys cluster I ((I) palm-NR2) allows rapid constitutive internalization from surface in developing neurons as shown by slashed arrow.