Association of SR to brain membranes inhibits D-serine synthesis. (A) Western blot analysis showing the presence of SR in rat brain homogenate (H) and synaptosomal plasma membrane (SM). SR is not released from SM by washing once (1T) or twice (2T) with buffer containing 0.5% Triton X-100 (Upper). Middle and lower panels show the postsynaptic density protein 95 (PSD-95) and synaptophysin (Syn) levels. (B) SR is not removed from the membrane by high-salt washing. Fraction 1T was incubated in buffer containing 10 mM Tris-HCl (pH 7.4) and increasing concentrations of NaCl. Membrane-bound SR was recovered by centrifugation and analyzed by Western blot. (C) Membrane-bound SR is mostly inactive. Cytosolic (SR cyt) and membrane-bound (SR memb) SR were separated by centrifugation of cerebral cortex of adult rats (Left), primary neuronal cultures (Middle), and SH-SY5Y neuroblastoma cells overexpressing HA-SR (Right). The values of D-serine synthesis were normalized to the amounts of SR as determined by densitometric analysis of the Western blot with SR antibody. The results in A and B are representative of 3–5 experiments with different preparations. The values in C represent the average ± SEM of 5–6 experiments done in triplicates. ***Different from control at P < 0.001.

NMDAR activation elicits SR translocation to the membrane in neurons. (A) Addition of 100 μM NMDA to primary cortical neurons promotes a time-dependent increase in the levels of membrane-bound SR and a simultaneous decrease in the levels of the cytosolic SR. (B) The increase in the membrane-bound SR upon NMDA activation is blocked by 40 μM MK-801 (Upper) added 20 min before and during the NMDAR stimulation. (C) The increase in the membrane-bound SR upon NMDA activation is blocked by preincubation with 1 μM Ro-25–6981, but not by 0.2 μM PPPA. (D) Levels of membrane-bound SR after a 30-min stimulation with NMDA (100 μM) glutamate (40 and 500 μM), KCl (20 mM), or forskolin (Forsk) (50 μM). (E) Addition of 40 μM MK-801 prevents the increase in membrane-bound SR promoted by 500 μM glutamate. (F) NO scavenging by 20 μM oxyhemoglobin or NO release by 200 μM GSNO do not affect membrane-bound SR levels in the absence or presence of NMDA. (G) Omission of external Ca²⁺ during NMDAR stimulation inhibits NMDA effect on membrane-bound SR. (H) Densitometric analysis of membrane-bound SR in neurons after a 30-min incubation with 100 μM NMDA, 100 μM NMDA plus 40 μM MK-801, 500 μM glutamate, 500 μM glutamate plus 40 μM MK-801, 40 μM glutamate, 100 μM kainate, 100 μM t-ACPD, 20 mM KCl, 50 μM forskolin, 1 μM A23187, or 100 μM NMDA in the absence of external Ca²⁺. The results represent the average ± SEM of 4–22 experiments done in duplicates with different primary cultures. (I) Western blot showing lack of reversibility of the NMDAR-elicited SR translocation. After a 30-min stimulation with 100 μM NMDA, the neurons were washed twice and subsequently incubated for up to 2 h in fresh medium containing 100 μM APV to block NMDARs. (J) NMDAR stimulation increases membrane-bound SR levels in neuronal processes. Primary neuronal cultures were incubated with or without 100 μM NMDA for 30 min and subsequently treated for 1 min on ice with HBSS buffer containing 0.5% Triton X-100 before fixation, which removes most cytosolic SR. After fixation with 4% paraformaldehyde, SR localization was revealed by immunocytochemistry. The pictures are representative of 30 fields of different cultures containing approximately the same number of neuronal cell bodies. (K) NMDAR stimulation promotes a prolonged inhibition of D-serine synthesis in primary neurons. D-serine synthesis was assayed for 2 h following NMDA removal. The results represent the average ± SEM of 5 experiments with different culture preparations done in sextuplicate. Loading controls in (A–F) and (G) were monitored with anti-TfR (membrane fraction) or anti-actin (cytosolic fraction). *Different from control at P < 0.05; **Different from control at P < 0.01; ***Different from control at P < 0.001.

Roles of palmitoylation and phosphorylation in SR binding to the membrane. (A) Palmitoylation inhibition blocks SR translocation in primary neuronal cultures. The neuronal cultures were preincubated for 2.5 h in NB medium containing 7 mg/ml fatty-acid-free albumin and in the presence or in the absence of 100 μM 2-Br-palmitate. Subsequently, 100 μM NMDA was added for 30 min and the levels of membrane-bound SR were monitored by Western blotting (Upper). Lower panel depicts β-tubulin levels in each sample. (B) Treatment with 100 μM 2Br-palmitate for 12 h decreases the amount of membrane-bound SR (Mem) but does not change the cytosolic levels (Cyt) of SR in transfected SH-SY5Y cells. Statistical analysis shows a 2-fold increase in cytosol/membrane SR ratio (Right; n = 6). Cytosolic fractions were analyzed using 5 μg protein, whereas membrane fractions contained 50 μg protein. (C) Phosphorylation of Thr-227 is exclusively found in the membrane-bound SR. Cytosolic and membrane-bound HA-SR from SH-SY5Y cells were purified, in-gel digested with trypsin, and the peptides analyzed by LC-MS (see SI Methods). The extracted ion chromatogram (m/z 737.1 ± 0.1) of the membrane-bound HA-SR shows elution of the monophosphorylated peptide 222–241 after 28.8 min (Left), whereas no corresponding peptide peak was detected in the cytosolic HA-SR (Right). (D) Mutation of Thr-227 to Ala (T227A) does not affect the acylation levels of HA SR by [³H]palmitate in transfected HEK 293 cells (Upper). Lower panel depicts the SDS-PAGE loading control. (E) T227A SR mutant exhibits lower binding to the membrane in transfected HEK293 cells. Statistical analysis shows a 4- to 5-fold increase in cytosol/membrane ratio when T227A is compared with wild-type SR (n = 6). (F) Levels of membrane-bound SR under basal (−NMDA) or stimulated (+NMDA) conditions in primary neuronal culture transduced with recombinant lentivirus containing either HA-SR WT (Left) or HA-SR T227A (Right). HA-SR was detected with anti-HA. Lower panel depicts TfR loading control in the membrane fraction. **Different from control at P < 0.01; ***Different from control at P < 0.001.

SR localization at dendritic processes. Localization of SR (A) and transferrin receptor (TfR) (B) in primary neurons. (C) Colocalization of SR and TfR in a dendritic process. Inset shows colocalization in the soma and process of a neuron. SR (D) and PSD-95 (E) do not significantly colocalize (F and Inset). (Scale bar, 2.5 μm [F Inset].) SR (G) and GM130 (H) expression exhibit limited overlap in the soma of 30% of the neurons, but not in dendrites (I). SR (J) and KDEL (K) exhibited limited colocalization with SR in the soma of only 10% of examined cells, but not in dendrites (L Inset). SR (M) and synaptophysin (Syn) (N) display no overlap (O). The pictures are representative of 4–12 different experiments. (Scale bar, 25 μm [A–O].)

Acylation of SR by oxyester formation. (A) SH-SY5Y cells overexpressing hemaglutinin-tagged SR (HA-SR) were labeled with 1 mCi/ml [³H]palmitic acid and HA-SR was immunoprecipitated with anti-HA affinity matrix. The immunoprecipitates were treated either with 1 M Tris-HCl (pH 7.4) or 1 M hydroxylamine (pH 7.4) for 2 h, and acylation was monitored by fluorography (Upper) of the SDS-PAGE. Pretreatment of the cell culture with 100 μM 2Br-palmitate abolishes HA-SR acylation, whereas hydroxylamine has no effect (fluorogram, Upper). Lower panel depicts immunoprecipitated HA-SR by analyzing 1/10th of the immunoprecipitate by Western blotting. (B) Acylation of HA-GAD65 in SH-SY5Y cells is sensitive to both hydroxylamine and 2Br-palmitate. Lower panel depicts immunoprecipitated HA-GAD65 by Western blot. (C) Acylation of SR is not affected by treatment with 200 mM DTT for 5 min at 100 °C. Lower panel depicts the loading control in the SDS-PAGE. (D) Acylation of HA-GAD65 is sensitive to DTT. Lower panel depicts the loading control in the SDS-PAGE. (E) HA-SR acylation is decreased by incubating the immunoprecipitate for 1 h with 50 mM NaOH in 90% ethanol. Lower panel depicts the SDS-PAGE, showing equal loading. (F) Comparison of SR acylation by [³H]palmitic acid and [³H]octanoic acid. The experiment was carried out as in (A) except that the cells were incubated with fatty acids using 4× less total radioactivity (250 μCi/ml), which gives a lower signal. Lower panel depicts the loading control in the SDS-PAGE.