內吞作用(endocytosis)在調控許多細胞表面接受器的下游訊號中扮演一個很重要的角色，例如凝血蛋白酶(thrombin)之接受器中的蛋白酶激活接受器一 (protease-activated receptor 1)。蛋白酶激活接受器一的內吞作用是經由凝血蛋白酶所誘導的一種不可逆的活化過程，被凝血蛋白酶活化的蛋白酶激活接受器一藉由此過程將凝血蛋白酶的訊號傳送到細胞內部。蛋白酶激活接受器一被活化後會經由克林斯林所組成的覆被孔(clathrin-coated pits)很快的進到細胞內並被運送到溶酶體(lysosome)中降解以終止訊號。中國倉鼠卵巢K1細胞(Chinese hamster ovary-K1 cells)是一種富含窖蛋白一(caveolin-1)的細胞，其蛋白酶激活接受器一的內吞作用之路徑目前仍然不清楚。本篇論文針對中國倉鼠卵巢K1細胞中蛋白酶激活接受器一的內吞作用之路徑與蛋白酶激活接受器一之激動劑所誘導之細胞外信號調節蛋白激酶(extracellular signal-regulated kinase)的活化加以探討。我發現當細胞過度表達顯性負動蛋白一突變K44A(dominant-negative dynamin-1 K44A)或是顯性負克林斯林軸突變(dominant-negative clathrin hub mutant)會抑制蛋白酶激活接受器一的內吞作用。此外在中國倉鼠卵巢K1細胞中，表達蛋白酶激活接受器一相較於不表現蛋白酶激活接受器一的細胞其激動劑所誘導細胞外信號調節蛋白激酶之活化是顯著的。因此，中國倉鼠卵巢K1細胞中蛋白酶激活接受器一的內吞作用需要動蛋白一(dynamin-1)以及克林斯林(clathrin)，而且其細胞中蛋白酶激活接受器一會刺激細胞外信號調節蛋白激酶的活化。

Endocytosis plays an important role in down-regulation of several cell-surface receptors such as protease-activated receptor 1 (PAR1), a thrombin receptor. The endocytosis of PAR1 is triggered by irreversible activation induced by thrombin, and then the activated PAR1 transduces thrombin signals into cells. Activated PAR1 is rapidly internalized via clathrin-coated pits and sorted to lysosomes for degradation to terminate thrombin signals. As for Chinese hamster ovary-K1 (CHO-K1) cells, a kind of caveolin-1-enriched cells, the endocytic pathway of PAR1 is still unknown. In this study, the endocytic pathway of PAR1 and its agonist-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation in CHO-K1 cells were investigated. The endocytosis of PAR1 was found to be blocked in cells overexpressing dominant-negative dynamin-1 K44A or dominant-negative clathrin hub mutant. In addition, the levels of agonist-induced transient ERK1/2 activation were significantly increased in CHO-K1 cells stably expressing FLAG-tagged PAR1 compared to those in CHO-K1 cells not expressing FLAG-tagged PAR1. These results indicated those the endocytic pathway of PAR1 is clathrin-dependent and dynamin-dependent in CHO-K1 cells and that activation of PAR1 can stimulate ERK1/2 activation in CHO-K1 cells.

Content
Abstract vi
Abstract (Chinese) vii
Acknowledgement viii
Content ix
List of Figures xi
Abbreviations xii
Introduction 1
The object of this work 5
Materials and methods 6
Materials 6
Cell culture and cell lines 7
DNA and transfection 7
Immunofluorescence microsopy 9
Internalization assay 10
Western blotting 11
Statistical analysis 12
Results 14
Dynamin-mediated endocytosis of PAR1 in CHO-K1 cells 14
Clathrin-mediated endocytosis of PAR1 in CHO-K1 cells 17
PAR1-mediated ERK activation in CHO-K1 cells 18
Discussion 20
References 24
Figures 28

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