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研究生:李沈洋
研究生(外文):Shen-Yang Lee
論文名稱:探討腺苷酸A2a亞型受體與TranslinX在功能上的交互作用
論文名稱(外文):Functional Characterization of the Interaction between A2a adenosine receptoer and Translin X.
指導教授:陳儀莊陳儀莊引用關係
指導教授(外文):Yijuang Chern
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:神經科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
中文關鍵詞:腺苷酸A2a亞型受體Translin X蛋白質運輸作用
外文關鍵詞:A2a adenosine receptorTranslin Xnucleocytoplasmic transport
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腺苷酸A2A亞型受體 (A2A adenosine receptor ; 簡稱A2A-R) 在腦中很多區域皆有分佈,並且其能調控神經細胞的生理功能。 實驗室過去同仁利用A2A-R 的C端胺基酸序列當作引誘餌,在yeast two hybrid的實驗系統中發現到一個會與A2A-R交互作用的新蛋白―Translin X (TraX)。 TraX最初是以會與Translin相結合蛋白的面貌被發現 被認為可能參與DNA的修復與重組機制。 為了了解A2A-R和TraX分別是利用哪一段區域進行交互作用,便設計四個TraX的截斷型態蛋白 (TraX81-290,TraX150-290,TraX1-120和TraX1-210) 突變株。 這四個TraX片段突變株利用生物體外蛋白質合成系統和pull down實驗分析方式,與A2A-R的C端胺基酸區域進行交互作用反應。 根據結果推論,TraX的第1~80胺基酸對於TraX是否能與A2A-R進行交互作用有著關鍵的決定性。 為了尋找A2A-R是以哪一段區域與TraX進行交互作用,利用四個A2A-R的C端片段蛋白 (A2A291-410,A2A310-410,A2A322-410和A2A371-410) 以GST-pull down的實驗方法解決這個問題。 實驗結果顯示,A2A-R第322~371胺基酸對於與TraX進行交互作用扮演著重要的角色。 使用細胞染色的方式,發現TraX不論是在未分化型態或是在NGF誘導成已分化型態的PC12細胞中,其表達的位置大部分都集中在細胞核內。 然而,若是將PC12細胞短時間 (15分鐘) 暴露於含有H2O2 (100 μM) 的環境下,便會造成TraX在細胞中表達的位置轉變成集中在細胞質。 由此推測,在PC12細胞內的TraX可能是會對於壓力產生反應的一種蛋白。 即使在未分化型態和用H2O2處理的PC12細胞,發現使用CGS21680去活化A2A-R後皆可以改變TraX在細胞中的位置。 推論TraX的功能有可能是A2A-R下游的一個新的訊號因子,而且參與在氧化性傷害時A2A-R保護作用的機制中。
A2A adenosine receptors (A2A-R) are located in many different areas of the brain and have been implicated in modulating neuronal functions. By using the C terminus domain of A2A-R as the bait in the yeast two-hybrid system, my colleagues previously identified a novel A2A-R-interacting protein, Translin X (TraX). TraX was first identified as an associating protein of translin, which has been implicated in DNA repair and recombination. To identify the interacting domains of these two molecules, I designed four truncation mutants of TraX (designated TraX81-290, TraX151-290, TraX1-120, TraX1-210) mutants. These TraX-truncated mutants were used in the in vitro pull down analysis to examine their interaction with the C terminus of A2A-R. My data suggest that amino acids 1~80 of TraX might be important for the interaction to A2A-R. To characterize the region of A2A-R for the binding of TraX, various A2A-R C-terminal fragments were used in the GST-pull down assays. Data analyses indicate that amino acid 322~371 of A2A-R might play a critical role in the binding of TraX. Moreover, immunocytochemical staining of TraX showed that TraX was enriched in the nucleus of both naïve and NGF-differentiated PC12 cells. In contrast, a brief exposure (15 min) of PC12 cells to H2O2 (100 μM) caused TraX to be enriched mostly in the cytoplasm of PC12 cells. TraX therefore might serve as a stress-response protein in PC12 cells. Most importantly, stimulation of A2A-R by CGS21680 altered the localization of TraX in both naïve and H2O2-treated PC12 cells. Taken together, TraX might function as a novel downstream effector of A2A-R and might contribute to the protective effect of A2A-R on oxidant injury
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