Enrichment of N-methyl-D-aspartate NR1 splice variants and synaptic proteins in rat postsynaptic densities.
Previous studies have suggested that the localization of the NMDA receptor NR1 subunit may be determined by the splice variant form of NR1 present. Functional studies have also supported selective targeting of NR2A and NR2B to synaptic and extrasynaptic populations, respectively. We set out to determine whether rat cortical and cerebellar NR1 splice variants and NR2 subunits are differentially localized to the postsynaptic density. Using western blot techniques, we measured the percentage of NR1 containing each cassette and the enrichment of the different cassettes and other proteins in the preparations. The results indicate that: (1) no single cassette of NR1 is differentially enriched in the postsynaptic densities and (2) the NR2A and NR2B subunits are similarly enriched at the synapse. The enrichment profiles of postsynaptic density-associated proteins demonstrated similar enrichment levels for postsynaptic density (PSD)-95, the NMDA receptor subunits, chapsyn-110, and the CaMKII alpha subunit. However, synaptophysin, SAP-102, and the GABA(A) receptor beta subunit exhibited lower enrichment levels compared to PSD-95. Additionally, cerebellar but not cortical PSDs exhibited significantly lower enrichment of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluR1. Thus, although postsynaptic densities are highly enriched in synaptic proteins, there appears to be no selective incorporation of specific NR1 splice variants or NR2 subunits into this structure.
Interdisciplinary Program in Neuroscience, Georgetown University Medical Center, Washington DC 20007, USA. alhallar@georgetown.edu
R A
RA
Al-Hallaq
R P
RP
Yasuda
B B
BB
Wolfe
D017398:Alternative Splicing; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002531:Cerebellum / Q000737:chemistry / Q000378:metabolism; D002540:Cerebral Cortex / Q000737:chemistry / Q000378:metabolism; D000076122:Disks Large Homolog 4 Protein; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000378:metabolism*; D009928:Organ Specificity; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D013569:Synapses / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C501451:Mpp2 protein, rat; C409466:NR1 NMDA receptor; C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; D009419:Nerve Tissue Proteins; D016194:Receptors, N-Methyl-D-Aspartate; C033652:postsynaptic density proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases