Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore.
Neurochem Int. 2010 Mar;56(4):569-76. doi: 10.1016/j.neuint.2009.12.020. Epub 2010 Jan 12.
Rat pheochromocytoma (PC12) cells have been shown to lack functional NMDA receptors; yet, these cells express NR1 subunits of the NMDA receptor. The reason for the lack of functional receptors has been attributed to the absence of significant levels of NR2 subunits to co-assemble with NR1. It is known that PC12 expresses very low levels of NR2C, with complete absence of other types of NR2 subunits. The purpose of the present study is to describe the molecular mechanism of trafficking and degradation of unassembled NR1 subunits in PC12 cells. The localization of NR1 subunits in PC12 cells were evaluated by immunofluorescence and co-immunoprecipitation, which showed that NR1 was present in the endoplasmic reticulum and cis-middle compartments of the Golgi apparatus. Upon treatment with a proteasome inhibitor, MG132, the ubiquitinylated species of NR1 subunit were detected, suggesting that NR1 is being targeted for endoplasmic reticulum-associated proteasomal degradation. Our previous studies suggest that NR1 subunits from the Golgi do not proceed to trans-Golgi, hence they will require re-routing to the endoplasmic reticulum for degradation. Further investigations on the factors involved in the trafficking of NR1 from Golgi to endoplasmic reticulum were performed using co-immunoprecipitation and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. These revealed the co-association of NR1 with non-muscle myosin heavy chain II isoforms A and B. We also demonstrate the functional significance of this interaction through the use of a myosin inhibitor, blebbistatin, to disrupt brefeldin A-induced Golgi-to-endoplasmic reticulum trafficking of NR1. In conclusion, our results suggest that non-muscle myosin II is involved in the retrograde trafficking of NR1 subunits from the cis/middle-Golgi to the endoplasmic reticulum for proteasomal degradation in PC12.
大鼠嗜铬细胞瘤（PC12）细胞已被证明缺乏功能性 NMDA 受体；然而，这些细胞表达 NMDA 受体的 NR1 亚基。缺乏功能性受体的原因归因于缺乏大量的 NR2 亚基与 NR1 共同组装。已知 PC12 表达非常低水平的 NR2C，完全缺乏其他类型的 NR2 亚基。本研究的目的是描述 PC12 细胞中未组装的 NR1 亚基的运输和降解的分子机制。通过免疫荧光和共免疫沉淀评估 NR1 亚基在 PC12 细胞中的定位，结果表明 NR1 存在于内质网和高尔基体顺面中间隔室。用蛋白酶体抑制剂 MG132 处理后，检测到 NR1 亚基的泛素化形式，表明 NR1 被靶向内质网相关蛋白酶体降解。我们之前的研究表明，来自高尔基体的 NR1 亚基不会进入反式高尔基体，因此它们将需要重新定向到内质网进行降解。通过共免疫沉淀和基质辅助激光解吸/电离飞行时间质谱进一步研究 NR1 从高尔基体到内质网的运输所涉及的因素。这些揭示了 NR1 与非肌肉肌球蛋白重链 II 同工型 A 和 B 的共关联。我们还通过使用肌球蛋白抑制剂 blebbistatin 破坏 Brefeldin A 诱导的 NR1 从高尔基体到内质网的运输，证明了这种相互作用的功能意义。总之，我们的结果表明，非肌肉肌球蛋白 II 参与了 NR1 亚基从顺面/中间高尔基体到内质网的逆行运输，以便在 PC12 中进行蛋白酶体降解。
