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The Journal of Neuroscience, August 18, 2004, 24(33):7292-7304; doi:10.1523/JNEUROSCI.1261-04.2004

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Development/Plasticity/Repair
NMDA Receptor GluR/NR2 Subunits Are Essential for Postsynaptic Localization and Protein Stability of GluR1/NR1 Subunit

Manabu Abe,^1,5 * Masahiro Fukaya,^2 * Takeshi Yagi,³ Masayoshi Mishina,^4,5 Masahiko Watanabe,² and Kenji Sakimura^1,5

¹Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan, ²Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan, ³KOKORO Biology Group and Core Research for Evolutional Science and Technology, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan, ⁴Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan, and ⁵Solution Oriented Research for Science and Technology, Japan Science and Technology, Tokyo 113-0033, Japan

In NMDA receptors, GluR/NR2 subunits strictly require the GluR1/NR1 subunit to exit from endoplasmic reticulum (ER) to the cell surface in vitro and to the postsynapse in vivo, whereas C terminus-dependent self-surface delivery has been demonstrated for the GluR1 subunit in vitro. To test whether this leads to C terminus-dependent self-postsynaptic expression in neurons in vivo, we investigated the GluR1 subunit in cerebellar granule cells lacking two major GluR subunits, GluR1/NR2A and GluR3/NR2C. In the mutant cerebellum, synaptic labeling for the GluR1 subunit containing the C2 (GluR1-C2) or C2' (GluR1-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 GluR1-C2 and GluR1-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 GluR1-C2 and 17.0 ± 4.6% for GluR1-C2'. The GluR1 subunit carrying the C1 cassette (GluR1-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 GluR subunits in the mutant cerebellum, the severe reductions thus represent that the GluR1 subunit, by itself, is virtually unable to accumulate at postsynaptic sites, regardless of C-terminal forms. By protein turnover analysis, the degradation of the GluR1 subunit was accelerated in the mutant cerebellum, being particularly rapid for that carrying the C2 cassette. Therefore, accompanying expression of GluR subunits is essential for postsynaptic localization and protein stability of the GluR1 subunit.

Key words: NMDA receptor; mossy fiber-granule cell synapse; immunohistochemistry; immunoblot; protein turnover; cerebellum; knock-out mouse

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Received Oct 3, 2003; revised June 15, 2004; accepted June 18, 2004.

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