NMDA Receptor GluRϵ/NR2 Subunits Are Essential for Postsynaptic Localization and Protein Stability of GluRζ1/NR1 Subunit

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Abstract
In NMDA receptors, GluRϵ/NR2 subunits strictly require the GluRζ1/NR1 subunit to exit from endoplasmic reticulum (ER) to the cell surfacein vitroand to the postsynapsein vivo, whereas C terminus-dependent self-surface delivery has been demonstrated for the GluRζ1 subunitin vitro. To test whether this leads to C terminus-dependent self-postsynaptic expression in neuronsin vivo, we investigated the GluRζ1 subunit in cerebellar granule cells lacking two major GluRϵ subunits, GluRϵ1/NR2A and GluRϵ3/NR2C. In the mutant cerebellum, synaptic labeling for the GluRζ1 subunit containing the C2 (GluRζ1-C2) or C2′ (GluRζ1-C2′) cassette was reduced at mossy fiber-granule cell synapses to the extrasynaptic level. The loss was not accompanied by decreased transcription and translation levels, increased extrasynaptic labeling, or ER accumulation. Quantitative immunoblot revealed substantial reductions in the mutant cerebellum of GluRζ1-C2 and GluRζ1-C2′. The most severe deficit was observed in the postsynaptic density (PSD) fraction: mutant levels relative to the wild-type level were 12.3 ± 3.3% for GluRζ1-C2 and 17.0 ± 4.6% for GluRζ1-C2′. The GluRζ1 subunit carrying the C1 cassette (GluRζ1-C1) was, although low in cerebellar content, also reduced to 12.7 ± 3.5% in the mutant PSD fraction. Considering a trace amount of other GluRϵ subunits in the mutant cerebellum, the severe reductions thus represent that the GluRζ1 subunit, by itself, is virtually unable to accumulate at postsynaptic sites, regardless of C-terminal forms. By protein turnover analysis, the degradation of the GluRζ1 subunit was accelerated in the mutant cerebellum, being particularly rapid for that carrying the C2 cassette. Therefore, accompanying expression of GluRϵ subunits is essential for postsynaptic localization and protein stability of the GluRζ1 subunit.