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研究生:李珮宜
研究生(外文):Pei-Yi Lee
論文名稱:泛素黏合酶c-Cbl參與蛋白酶激活接受器一所引起酪氨酸蛋白激酶Src的降解
論文名稱(外文):c-Cbl-mediated protease-activated receptor 1-induced degradation of Src
指導教授:傅化文傅化文引用關係
指導教授(外文):Hua-Wen Fu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子與細胞生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:34
中文關鍵詞:蛋白酶激活接受器一酪氨酸蛋白激酶泛素黏合酶
外文關鍵詞:PAR1Srcc-Cbl
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蛋白酶激活接受器一 (protease-activated receptor 1, PAR1) 是一個被凝血蛋白酶 (thrombin) 所激活的G蛋白連結接受器(G protein-coupled receptor)。酪氨酸蛋白激酶Src 除了會被連接G蛋白的蛋白酶激活接受器一活化外,還會藉由��-制動素 (��-arrestin) 結合到蛋白酶激活接受器一進而傳遞此接受器的下游訊號。接著酪氨酸蛋白激酶Src會和活化的蛋白酶激活接受器一結合並一起到溶酶體 (lysosome) 降解。然而,蛋白酶激活接受器一所引起的酪氨酸蛋白激酶Src及此接受器被降解的機制仍然不清楚。有研究文獻指出,泛素黏合酶c-Cbl (ubiquitin E3 ligase c-Cbl) 會參與活化的酪氨酸蛋白激酶Src降解反應。為了探討在蛋白酶激活接受器一活化後,泛素黏合酶c-Cbl是否會參與酪氨酸蛋白激酶Src及此接受器的降解反應,我將有穩定表現FLAG標幟之蛋白酶激活接受器一的中國倉鼠卵巢細胞 (CHO-K1) 短暫表現優勢陰性型突變 (dominant negative) 的泛素黏合酶c-Cbl,並且測試此突變的泛素黏合酶c-Cbl對於蛋白酶激活接受器一所引起酪氨酸蛋白激酶Src降解反應的影響。我發現溶酶體抑制劑—氯化喹啉 (chloroquine) 會抑制蛋白酶激活接受器一活化所引起的酪氨酸蛋白激酶Src及此接受器的降解。又此缺少環指狀功能區域 (RING-finger domain) 之優勢陰性突變的泛素黏合酶c-Cbl (�嵇F c-Cbl) 會完全抑制蛋白酶激活接受器一引起的酪氨酸蛋白激酶Src降解,且只會部分抑制蛋白酶激活接受器一的降解。然而,此缺少環指狀功能區域之優勢陰性突變的泛素黏合酶c-Cbl卻完全不影響蛋白酶激活接受器一的胞飲作用。綜合上述結果可知,在蛋白酶激活接受器一被活化後,泛素黏合酶c-Cbl參與酪氨酸蛋白激酶Src及蛋白酶激活接受器一的降解。
Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor for thrombin. In addition to active Src by G protein, PAR1 recruits Src by ��-arrestin to transduce signals. Src is then sorted to lysosomes with the activated PAR1 for degradation. However, the mechanism by which PAR1-induced degradation of Src and the receptor itself is still unclear. It has been reported that degradation of active Src is mediated by c-Cbl, an ubiquitin E3 ligase. To determine whether c-Cbl mediates the degradation of Src and PAR1 after receptor activation, CHO-K1 cells stably expressing FLAG-tagged PAR1 were transiently transfected with a dominant negative c-Cbl mutant to examine its effect on the degradation of Src and PAR1. I found that stimulation of PAR1 induced degradation of Src. The degradation of Src was blocked by chloroquine, a lysosomal inhibitor. The dominant negative c-Cbl mutant lacking the RING-finger domain (�嵇F c-Cbl) inhibited PAR1-induced degradation of Src and partially inhibited the degradation of PAR1. However, �嵇F c-Cbl did not affect the endocytosis of PAR1. Taken together, these results indicated that c-Cbl mediated lysosomal degradation of Src and PAR1 after the receptor activation.
Abstract ii
Abstract (Chinese) iii
Acknowledgment iv
Content v
List of Figures vii
Abbreviations viii
Introduction 1
Materials and Methods 8
Results 14
Discussion 25
References 30
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