Abe Manabu, Fukaya Masahiro, Yagi Takeshi, Mishina Masayoshi, Watanabe Masahiko, Sakimura Kenji
Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan.
J Neurosci. 2004 Aug 18;24(33):7292-304. doi: 10.1523/JNEUROSCI.1261-04.2004.
In NMDA receptors, GluRepsilon/NR2 subunits strictly require the GluRzeta1/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 GluRzeta1 subunit in vitro. To test whether this leads to C terminus-dependent self-postsynaptic expression in neurons in vivo, we investigated the GluRzeta1 subunit in cerebellar granule cells lacking two major GluRepsilon subunits, GluRepsilon1/NR2A and GluRepsilon3/NR2C. In the mutant cerebellum, synaptic labeling for the GluRzeta1 subunit containing the C2 (GluRzeta1-C2) or C2' (GluRzeta1-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 GluRzeta1-C2 and GluRzeta1-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 GluRzeta1-C2 and 17.0 +/- 4.6% for GluRzeta1-C2'. The GluRzeta1 subunit carrying the C1 cassette (GluRzeta1-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 GluRepsilon subunits in the mutant cerebellum, the severe reductions thus represent that the GluRzeta1 subunit, by itself, is virtually unable to accumulate at postsynaptic sites, regardless of C-terminal forms. By protein turnover analysis, the degradation of the GluRzeta1 subunit was accelerated in the mutant cerebellum, being particularly rapid for that carrying the C2 cassette. Therefore, accompanying expression of GluRepsilon subunits is essential for postsynaptic localization and protein stability of the GluRzeta1 subunit.
在N-甲基-D-天冬氨酸（NMDA）受体中，谷氨酸受体亚基ε（GluRepsilon）/NR2亚基在体外从内质网（ER）转运至细胞表面以及在体内转运至突触后位点时，严格需要谷氨酸受体ζ1（GluRzeta1）/NR1亚基，而体外实验已证明GluRzeta1亚基存在C末端依赖性的自身表面转运。为了检测这是否会导致体内神经元中C末端依赖性的自身突触后表达，我们研究了缺乏两种主要的GluRepsilon亚基，即GluRepsilon1/NR2A和GluRepsilon3/NR2C的小脑颗粒细胞中的GluRzeta1亚基。在突变型小脑中，含有C2（GluRzeta1-C2）或C2'（GluRzeta1-C2'）盒的GluRzeta1亚基在苔藓纤维-颗粒细胞突触处的突触标记减少至突触外水平。这种减少并非伴随着转录和翻译水平的降低、突触外标记的增加或内质网积累。定量免疫印迹显示突变型小脑中GluRzeta1-C2和GluRzeta1-C2'大幅减少。在突触后致密物（PSD）组分中观察到最严重的缺陷：相对于野生型水平，GluRzeta1-C2的突变型水平为12.3±3.3%，GluRzeta1-C2'为17.0±4.6%。携带C1盒的GluRzeta1亚基（GluRzeta1-C1），尽管在小脑中含量较低，但在突变型PSD组分中也降至12.7±3.5%。考虑到突变型小脑中存在微量的其他GluRepsilon亚基，如此严重的减少表明，无论C末端形式如何，GluRzeta1亚基自身实际上无法在突触后位点积累。通过蛋白质周转分析，突变型小脑中GluRzeta1亚基的降解加速，携带C2盒的GluRzeta1亚基降解尤其迅速。因此，GluRepsilon亚基的伴随表达对于GluRzeta1亚基的突触后定位和蛋白质稳定性至关重要。
