探討ANKS1B蛋白在N-甲基-D天門冬氨酸受體子單元蛋白細胞內分佈所扮演之角色

Abstract

Ankyrin repeat and sterile alpha motif domain containing 1B （ANKS1B）是一個會與amyloid precursor protein （APP）產生交互作用的蛋白，然而ANKS1B以及ANKS1B與APP之間交互作用的功能目前仍不清楚。研究指出ANKS1B會與N-甲基-D-天門冬氨酸受體 （NMDAR）中的NR2子單元蛋白組合在一起並促進NMDAR內質網到細胞表面的運輸。ANKS1B被指出會與膜相關鳥苷酸激酶（MAGUK）蛋白產生交互作用，加上MAGUK 蛋白被指出會和NR2 子單元蛋白產生交互作用並促進NMDAR的運輸，因此ANKS1B可能是藉由MAGUK 蛋白來與NMDAR結合。此外，APP也被指出會與NR1子單元蛋白產生交互作用並促進NMDAR從內質網到細胞表面的運輸。基於前人的研究，我們的假說是ANKS1B、APP以及NMDAR會在內質網結合形成一個複合體並一起運輸到細胞表面，而若破壞ANKS1B與APP之間的交互作用可能會破壞這個複合體的生成影響到NMDAR的運輸，進而改變NMDAR在細胞內的分佈。我們利用過度表現野生型（wild type）與突變體（mutant）ANKS1B的SH-SY5Y細胞作為研究材料。在此研究中，我們先建立了exon14-ANKS1B 這個ANKS1B突變體的質體。接著偵測到了穩定細胞株（stable cell）中所表現的野生型以及突變體ANKS1B。在確認了NMDAR 子單元蛋白的蛋白質表現量在各組穩定細胞株中沒有明顯差異後，我們利用非連續性蔗糖梯度離心（discontinuous sucrose gradient centrifugation）來分離細胞中不同的胞器並分析NR1、NR2A和NR2B蛋白在細胞中分布的情形。在各穩定細胞株中，NR1、NR2A、NR2B在高密度的部分有較高的表現量，根據各胞器的標誌（marker）來推斷，NMDAR子單元蛋白可能與細胞膜或初期內體（early endosome）有所重疊。結果顯示ANKS1B與APP之間的交互作用有可能會影響NR1和NR2A的細胞分佈情形，另外，表現ANKS1B時會影響NR2B的細胞分佈情形且此影響與ANKS1B和APP之間的交互作用可能有關。然而免疫螢光染色（immunofluorescence staining）的結果顯示所有的穩定細胞株中NR1, NR2A 和NR2B都與EEA1出現在相同區域而沒有與CD71共定位（colocalize）且在各細胞株中沒有明顯差別。總結來說，ANKS1B和ANKS1B與APP之間的交互作用可能會影響NMDAR子單元蛋白細胞分佈的情形，然而因免疫螢光染色的結果顯示NMDAR子單元蛋白與各胞器標誌共定位的現象沒有明顯的改變，因此仍需要後續的研究來了解ANKS1B以及ANKS1B與APP之間的交互作用對NMDAR子單元蛋白細胞分佈的詳細影響。

Ankyrin repeat and sterile alpha motif domain containing 1B (ANKS1B) is a protein shown to interact with amyloid precursor protein (APP). However, the function of ANKS1B and the ANKS1B-APP protein- protein interaction (PPI) are not fully understood. ANKS1B was shown to associate with N-methyl-D-aspartic acid receptor (NMDAR) NR2 subunits and facilitate the transport of NMDAR subunits from endoplasmic reticulum (ER) to the cell surface. The association between ANKS1B and NMDAR subunits may be facilitated by membrane-associated guanylate kinases (MAGUK) proteins since MAGUK proteins were shown to interact with NR2 subunits and facilitate the transport of NMDARs. Moreover, APP was demonstrated to interact with NR1 subunits of NMDAR at ER and enhanced cell surface transport of NMDAR. Based on results of previous researches, we hypothesized that ANKS1B, APP and NMDAR subunits may form a complex at ER and transport to cell surface together and disrupting ANKS1B-APP PPI may affect the formation of the complex influencing the transport of NMDAR and further change the cellular distribution of NMDAR subunits. Wild type ANKS1B and mutant ANKS1B expressing SH-SY5Y cell line were used in this research. In this research, exon14-ANKS1B, a mutant ANKS1B, plasmid was constructed. Next, wildtype and mutant ANKS1Bs were detected in SH-SY5Y stable cells. After confirming the consistent protein expression of NR subunits in ANKS1B expressing stable cell clones, cellular distribution was determined using sucrose gradient centrifugation cellular fractionation. NR subunits were distributed at higher density fractions which overlapped with the plasma membrane marker CD71 or early endosome marker EEA1 using organelle markers. Results showed that the interaction between ANKS1B and APP affected the distribution pattern of NR1 and NR2A. As for NR2B, the expression of ANKS1B influenced NR2B distribution pattern in which ANKS1B-APP PPI was indespencible. However, immunofluorescence staining showed that NR1, NR2A and NR2B subunits colocalized with EEA1 but not CD71, which showed no difference in all cell groups. In conclusion, ANKS1B and ANKS1B-APP PPI may play a role in NR subunit subunits. However, the exact change of NR subunits cellular distribution still need to be determined since no significant colocalization change was observed in immunofluorescence staining results.