In vivo composition of NMDA receptor signaling complexes differs between membrane subdomains and is modulated by PSD-95 and PSD-93.
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.
Delint-Ramirez
Ilse
I
Genes to Cognition Programme, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, United Kingdom.
Fernández
Esperanza
E
Bayés
Alex
A
Kicsi
Emese
E
Komiyama
Noboru H
NH
Grant
Seth G N
SG
D000818:Animals; D002772:Cholera Toxin; D000076122:Disks Large Homolog 4 Protein; D016537:Gangliosidosis, GM1 / Q000378:metabolism; D051528:Guanylate Kinases; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000172:deficiency / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation / Q000235:genetics; D054731:PDZ Domains / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014633:Valine / Q000235:genetics
D000076122:Disks Large Homolog 4 Protein; C501447:Dlg4 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D002772:Cholera Toxin; C501445:Dlg2 protein, mouse; D051528:Guanylate Kinases; D014633:Valine