In vivo composition of NMDA receptor signaling complexes differs between membrane subdomains and is modulated by PSD-95 and PSD-93

Abstract

Lipid rafts are dynamic membrane microdomains enriched in cholesterol and sphingolipids involved in the compartmentalization of signaling pathways, trafficking and sorting of proteins. At synapses, the glutamatergic NMDA receptor and its cytoplasmic scaffold protein PSD-95 move between postsynaptic density (PSD) and rafts following learning or ischemia. However it is not known whether the signaling complexes formed by these proteins are different in rafts nor the molecular mechanisms that govern their localization. To examine these issues in vivo we used mice carrying genetically encoded tags for purification of protein complexes and specific mutations in NMDA receptors, PSD-95 and other postsynaptic scaffold proteins. Isolation of PSD-95 complexes from mice carrying tandem affinity purification tags showed differential composition in lipid rafts, postsynaptic density and detergent-soluble fractions. Raft PSD-95 complexes showed less CaMKIIalpha and SynGAP and enrichment in Src and Arc/Arg3.1 compared with PSD complexes. Mice carrying knock-outs of PSD-95 or PSD-93 show a key role for PSD-95 in localizing NR2A-containing NMDA receptor complexes to rafts. Deletion of the NR2A C terminus or the C-terminal valine residue of NR2B, which prevents all PDZ interactions, reduced the NR1 association with rafts. Interestingly, the deletion of the NR2B valine residue increased the total amount of lipid rafts. These data show critical roles for scaffold proteins and their interactions with NMDA receptor subunits in organizing the differential expression in rafts and postsynaptic densities of synaptic signaling complexes.

Figure 1.

Composition of the PSD-95-NMDAR complex in lipid rafts and PSD fractions. A, Mouse cortices were extracted in 1% Triton X-100 and separated on a density gradient. Five fractions (from top to bottom of the gradient) were obtained from each gradient and equal volumes of these fractions, were immunoblotted for the indicated proteins. Fractions were also assayed for the sphingolipid GM1 (dot-blot assay using cholera toxin) and cholesterol (colorimetric assay). B, Lipid rafts and PSD-enriched fractions were isolated from wild-type (WT) and PSD-95^TAP mice (TAP). PSD-95 complexes were captured from these fractions with anti-Flag antibody and subsequently released by TEV protease cleavage. The eluted complexes were analyzed by Western blot for the indicated proteins. The densitometry readings of the blots were normalized against the optical density of PSD-95, except the phosphorylation of PY1252 in NR2B (PY-NR2B) which was normalized by the reading of NR2B. Graphs show the normalized results (mean ± SEM) expressed as the ratio lipid rafts/PSD for at least 4 independent experiments. C, Detergent-soluble extract (Sol) and lipid rafts (Rafts) and PSD fractions (PSD) were immunoblotted against the indicated proteins. Protein amounts loaded: Raft, 3.2 μg; PSD, 3.2 μg; Sol, 192 μg. D, TAP PSD-95 complexes were isolated from lipid rafts (Rafts), PSD fractions (PSD) and detergent-soluble extract (Sol) from wild-type (WT) and PSD-95^TAP (TAP) mice with anti-Flag antibody and subsequently released by TEV protease cleavage. The eluted complexes were immunoblotted against indicated proteins. **p < 0.01, *p < 0.05.

Figure 2.

Analysis of PSD-95^−/− knock-out mice. Cortices of wild-type and PSD-95^−/− litter-matched mice were dissected and the lipid rafts were isolated by density gradient centrifugation. A, Equal amounts of proteins from Lipid raft (fraction 2) were analyzed by Western blot for indicated proteins; equal volumes of the fractions were analyzed for GM1 amount (dot blot) (left panel) and cholesterol (spectrophotometer determination). B, Equal amounts of proteins from the PSDs (pellets) were analyzed by Western blot for the indicated proteins. C, Total proteins in crude homogenate were analyzed by Western blot. D, Concentrations of the indicated proteins in the detergent-soluble extract were analyzed by Western blot. Graphics show densitometry analysis of blots (mean ± SEM) expressed as the ratio of immunoreactivity (PSD-95^−/− mice over litter-matched wild-type) for five independent experiments, except (D) detergent-soluble extract (4 litter matched pairs of animals), **p < 0.01, *p < 0.05.

Figure 3.

A–D, Analysis of PSD-93^−/− knock-out mice. Cortices of wild-type (WT) and PSD-95^−/− litter-matched mice were dissected and the lipid rafts isolated by density gradient. Equal amounts of proteins of lipid raft (A) and PSD (B) were analyzed by Western blot for indicated proteins. C, Total proteins in crude homogenate were analyzed by Western blot. D, Total proteins in the detergent-soluble extract were analyzed by Western blot. Graphics show densitometry analysis of blots (mean ± SEM) expressed as the ratio of immunoreactivity (PSD-93^−/− mice over litter-matched wild-types) for five independent experiments, except (D) detergent-soluble extract (4 litter matched pairs of animals), **p < 0.01, *p < 0.05. (KO) PSD-95^−/− knock-out mice; (WT) wild-type mice.

Figure 4.

Analysis of mice carrying a C-terminal truncation of NR2A subunits (NR2A^ΔC/ΔC). Cortices of wild-type and NR2A^ΔC/ΔC litter-matched mice were dissected and lipid rafts were isolated by density gradient. A, Equal amounts of proteins from lipid raft fractions were analyzed by Western blot for indicated proteins and equal volumes were analyzed for GM1 (dot blot) and cholesterol concentration (spectrophotometric determination). B, Equal amounts of protein from the PSD fraction were analyzed by Western blot for the indicated proteins. C, Total proteins in crude homogenate were analyzed by Western blot. D, Total proteins in the detergent-soluble extract were analyzed by Western blot. Graphics show densitometry analysis of blots (mean ± SEM) expressed as the ratio of immunoreactivity (NR2A^ΔC/ΔC mice over wild types) for six independent pairs of litter-matched mice, except (D) detergent-soluble extract (4 litter matched pairs of animals), **p < 0.01, *p < 0.05.

Figure 5.

Analysis of carrying a punctual deletion of the Valine 1482 of NR2B subunits (NR2B ^ΔV/ΔV) Cortices of wild-type and NR2B ^ΔV/ΔV litter-matched mice were dissected and lipid rafts were isolated by density gradient. A, Equal volumes of soluble and insoluble Triton X-100 extract (resuspended in lyses buffer), and lipid rafts and PSD fractions were analyzed for GM1 (dot blot), cholesterol (colorimetric assay), total protein (Bradford), or Flotillin-1 (Western blot). B, C, Equal amounts of proteins from lipid rafts and PSD fractions were analyzed by Western blot for the indicated proteins. D, Expression of proteins in crude homogenized tissue was analyzed by Western blot. E, Concentration of the indicated proteins in the detergent-soluble extracted was analyzed by Western blot. Graphics show spectrophotometric reading and densitometry analysis of blots (mean ± SEM) expressed as the ratio of immunoreactivity (KO mice over wild types) for six independent experiments, except (D) detergent-soluble extract (4 litter matched pairs of animals), **p < 0.01, *p < 0.05.