N-glycosylation of the AMPA-type glutamate receptor regulates cell surface expression and tetramer formation affecting channel function
N-glycosylation of the AMPA-type glutamate receptor regulates cell surface expression and tetramer formation affecting channel function
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DOI：
10.1111/jnc.14565
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发表时间：
2018-12-01
影响因子：
4.7
通讯作者：
Takamiya, Kogo
中科院分区：
文献类型：
--
作者：
Kandel, Munal Babu;Yamamoto, Saki;Takamiya, Kogo
AMPA型谷氨酸受体（AMPA-R）在主要兴奋性突触传递和许多神经元功能中起主要作用，包括作为学习和记忆基础的突触可塑性。N-糖基化是膜蛋白的主要翻译后修饰之一，但其在神经元中的具体作用仍不清楚。AMPA-R亚基在内质网和高尔基体系统的腔中生物合成期间在其胞外结构域处被N-糖基化。GluA 1是AMPA-R亚基的一个成员，其胞外区存在6个N-糖基化位点。我们观察到，在HEK 293 T细胞中，在N63/N363处缺乏N-聚糖的GluA 1突变体中，细胞内运输和细胞表面表达被强烈抑制。使用Blue Native-PAGE的多聚体分析显示糖基化突变体（N63 S和N363 S）中受损的四聚体形成，表明错误转运由受损的四聚体形成引起。N63 S和N363 S突变体主要通过溶酶体途径降解。Flag-taged N363 S GluA 1，而不是N63 S GluA 1，在原代皮层神经元培养制备GluA 1基因敲除小鼠的观察定位在细胞表面。在HEK 293 T细胞中，GluA 2的共表达部分挽救了四聚体的形成和N363 S GluA 1的细胞表面表达，但不挽救N63 S GluA 1的细胞表面表达。电生理学分析也证明了N363 S GluA 1与GluA 2的功能异聚体。这些数据表明，位点特异性的N-聚糖GluA 1亚基调节四聚体的形成，细胞内运输，和细胞表面的AMPA-R的表达。
The AMPA-type glutamate receptor (AMPA-R) plays a primary role in principal excitatory synaptic transmission and many neuronal functions including synaptic plasticity that underlie learning and memory. N-glycosylation is one of the major post-translational modifications of membrane proteins, but its specific roles in neurons remain largely unknown. AMPA-R subunits are N-glycosylated at their extracellular domains during their biosynthesis in the lumen of the endoplasmic reticulum and Golgi system. Six N-glycosylation sites are presumed to exist in the extracellular domain of GluA1, which is a member of the AMPA-R subunits. We observed that the intracellular trafficking and cell surface expression were strongly suppressed in the GluA1 mutants lacking N-glycans at N63/N363 in HEK293T cells. Multimer analysis using Blue Native-PAGE displayed the impaired tetramer formation in the glycosylation mutants (N63S and N363S), indicating that the mis-transport was caused by impaired tetramer formation. N63S and N363S mutants were primarily degraded via the lysosomal pathway. Flag-tagged N363S GluA1, but not N63S GluA1, expressed in primary cortical neuron cultures prepared from GluA1 knockout mice was observed to localize at the cell surface. Co-expression of GluA2 partially rescued tetramer formation and the cell surface expression of N363S GluA1 but not N63S GluA1, in HEK293T cells. Electrophysiological analysis also demonstrated functional heteromers of N363S GluA1 with GluA2. These data suggest that site-specific N-glycans on GluA1 subunit regulates tetramer formation, intracellular trafficking, and cell surface expression of AMPA-R.