中文摘要
凝血蛋白酶激活接受器1（protease-activated receptor 1）是ㄧ個G蛋白連結接受器 (GPCR)，會被凝血蛋白酶(thrombin) 切除接受器N端的胺基酸而活化。ㄧ旦凝血蛋白酶激活接受器1被激活後，就會藉著G蛋白將訊息傳遞至細胞內。因為凝血蛋白酶激活接受器1活化的過程是不可逆的，所以必須透過迅速的與G蛋白分離、被吞噬進入細胞、及送至溶小體分解等方式結束訊號。最近發現凝血蛋白酶激活接受器1透過Src的活化會引起細胞增生，過度表現的凝血蛋白酶激活接受器1也會引起細胞癌化並造成細胞移轉。在乳癌細胞中發現過度表現的凝血蛋白酶激活接受器1會導致細胞移轉，可能是因為其被吞噬作用緩慢並且無法被溶小體分解，所以其傳遞下游訊號通常都無法被結束。由此可知，凝血蛋白酶激活接受器1在細胞中的路徑、活化、及訊號的結束，都是對此接受器造成細胞增生與移轉是ㄧ個重要的機制。
細胞膜上的細微坑洞(caveolae) 為細胞膜上一個特別的區塊屬於脂筏(lipid rafts) 的ㄧ種，不同於脂筏的是細胞膜上的細微坑洞含有一個和膽固醇結合的窖蛋白1(caveolin-1)。ㄧ些G蛋白連結接受器會經由細胞膜上的細微坑洞被吞噬及調控其訊號。除此之外，細胞膜上的細微坑洞也和細胞遷移有關。至於細胞膜上的細微坑洞是不是和凝血蛋白酶激活接受器1的被吞噬、訊號的傳遞及其造成的細胞移動有關，這些部份都尚未被研究。所以在本論文中，細胞膜上的細微坑洞是否參與凝血蛋白酶激活接受器1的分佈及訊號調控先被研究。由不連續性蔗糖密度離心分析顯示，部分的凝血蛋白酶激活接受器1位於細胞膜上的細微坑洞。接下來將膽固醇抽出破壞細微坑洞來研究是否細胞膜上的細微坑洞參與凝血蛋白酶激活接受器1吞噬作用。結果顯示凝血蛋白酶激活接受器1的吞噬作用並不經由細微坑洞。那麼細微坑洞是否參與凝血蛋白酶激活接受器1訊號傳遞。我發現凝血蛋白酶激活接受器1會促使窖蛋白1的酪氨酸14號被磷酸化。這個磷酸化過程是透過一個Gi 連結的Src 酵素及p38 肌細胞絲裂原活化蛋白激酶(p38 mitogen-activated protein kinase)活化而形成的。這個磷酸化的窖蛋白1可以透過和一個抑制Src活性的蛋白質Csk結合，進而抑制了凝血蛋白酶激活接受器1刺激的Src活性。所以磷酸化窖蛋白是凝血蛋白酶激活接受器1用來調控Src活性的一個分子。最後在乳癌細胞中研究是否細微坑洞參與凝血蛋白酶激活接受器1所導致的細胞遷移。破壞了細微坑洞就破壞了乳癌細胞本身及凝血蛋白酶激活接受器1所導致的細胞遷移。凝血蛋白酶激活接受器1下游分子，Gi蛋白、ERK1/2 及 PI3K是否位也於細微坑洞及參與細微坑洞所參與的凝血蛋白酶激活接受器1所導致的細胞移動也被調查。結果顯示Gi蛋白在凝血蛋白酶激活接受器1活化前或活化後均存在細微坑洞。當凝血蛋白酶激活接受器1被活化後ㄧ部份胞外訊號調節活化酶(ERK1/2)會重新分怖於細微坑洞。而PI3K不論在凝血蛋白酶激活接受器1未活化前或活化後不存在細微坑洞。抑制了Gi蛋白及胞外訊號調節活化酶(ERK1/2)的活性，而非PI3K的活性，可以抑制了凝血蛋白酶激活接受器1所導致的細胞遷移。這些結果顯示位於細微坑洞中的訊號分子Gi蛋白及ERK1/2參與凝血蛋白酶激活接受器1所導致的細胞遷移。由上述的結果中可以知，細胞膜上的細微坑洞參與凝血蛋白酶激活接受器1的訊號的傳遞及其造成的細胞遷移。這些研究結果可對凝血蛋白酶激活接受器1所造成的細胞癌化有更深一層的了解。

Abstract
Protease-activated receptor 1 (PAR1), a G protein-coupled receptor, is activated by thrombin cleavage at the amino-terminal domain of the receptor. Upon activation, PAR1 couples to several G proteins to transduce signals from cell membrane to cells. Since the activation is irreversible, PAR1 has to be rapidly terminated its signaling by uncoupling from G proteins, internalized to endosome and degraded in lysosomes. PAR1-induced cell proliferation has been reported to be mediated by Src kinase activation. In addition, overexpression of PAR1 can induce cell transformation and cell invasion in several cells. In breast carcinoma cells, PAR1 is overexpressed and fails to be degraded, which contributes prolonged signaling and cell invasion. Thus, the regulation of PAR1 trafficking, activation, and downregulation has critical roles in PAR1-induced cell proliferation and cell invasion.
Caveolae, specialized plasmalemmal microdomains, belong to one subset of lipid rafts but can be distinguished by the presence of the cholesterol binding protein caveolin-1. Several G protein-coupled receptors are internalized via caveolae and transduce their signals in caveolae. In addition, caveolae play a role in cell migration. However, the involvement of caveolae in PAR1 trafficking, PAR1-mediated signaling and PAR1-induced cell migration remained unclear. In this study, the involvement of caveolae in both the localization and internalization of PAR1 were first examined. Analysis of the cells by discontinuous-sucrose gradient centrifugation shows that PAR1 was partially localized in the caveolin-1-enriched fraction. However, cholesterol extraction by methyl-□-cyclodextrin to disrupt caveolae could not inhibit PAR1 internalization, indicating that caveolae are not involved in PAR1 internalization. Since some receptors can induce caveolin-1 phosphorylation on tyrosine 14, the involvement of caveolae in PAR1-induced phosphorylation of caveolin-1 was then examined. It was found that caveolin-1 was phosphorylated at tyrosine 14 by a Gi-linked Src kinase and p38 mitogen-activated protein kinase pathways. Phosphocaveolin-1 but not caveolin-1 with mutation at tyrosine 14 could bind to c-terminal Src kinase (Csk). The recruitment of Csk by phosphocaveolin-1 further resulted in a rapid decrease in Src kinase activity, indicating that phosphocaveolin-1 represents a novel effector of PAR1 to downregulate Src kinase activity. Moreover, the involvement of caveolae in PAR1-induced migration has been investigated by wound healing assay in MDA-MB-231 cells. Cholesterol extraction by methyl-□-cyclodextrin to disrupt caveolae inhibited PAR1-induced cell migration and cholesterol repletion restored PAR1-induced cell migration, indicating that caveolae are involved in PAR1-induced cell migration. The localization of Gi proteins, ERK1/2 and PI3K, downstream signaling molecules of PAR1, in caveolae and their involvement in PAR1-induced cell migration were also examined by discontinuous-sucrose gradient centrifugation and wound healing assay. A small portion of Gi proteins were localized in caveolin-1-enriched fraction before and after PAR1 activation. Of interest, activation of PAR1 resulted in the redistribution of a portion of phosphorylated ERK1/2 to caveolae. However, PI3K did not appear at caveolin-1-enriched fraction before and after PAR1 activation. Inactivation of signaling molecules localized in caveolae, Gi proteins or ERK1/2, but not PI3K inhibited PAR1-induced cellular migration. PAR1-induced ERK1/2 activation was also partially inhibited by disruption of caveolae, indicating PAR1-induced ERK1/2 activation was partially mediated by caveolae. These results suggest that caveolae and PAR1’s downstream signaling molecules, Gi and ERK1/2, localized in caveolae are involved in PAR1-induced cell migration.
Taken together, PAR1-induced Src activity is regulated by phosphocaveolin-1, implicating that caveolae can regulate PAR1-induced cell proliferation. In addition, caveolae and signaling molecules downstream of PAR1 concentrated in caveolae are involved in PAR1-induced cell migration, indicating that caveolae regulate PAR1-induced cell migration. These results show that caveolae are involved in regulation of PAR1-mediated signaling and PAR1-induced cell migration. The findings also provide new insight of how caveolae play a role in PAR1-induced tumor formation.

Table of Contents
Abstract v
中文摘要 viii
致謝 ix
Abbreviation x
Table of Contents xii
Chapter 1 1
Introduction 1
1.1 Introduction of PAR1 2
1.1.1 Identification of thrombin receptors 2
1.1.2 Activation of PAR1 3
1.1.3 Signal transduction of PAR1 . 4
1.1.4 Mechanisms of PAR1 inactivation 7
1.2 Introduction of caveolae 10
1.2.1 Identification of lipid rafts, caveolae and caveolin 10
1.2.2 Signal transduction from caveolae 11
1.2.3 Internalization by caveolae 12
1.2.4 Involvement of caveolae in tumor suppressor gene and oncogene. .......................................................................................................14
1.3 The objective of this work 16
Chapter 2 18
Negative regulation of protease-activated receptor 1-induced Src kinase activity by the association of phosphocaveolin-1 with Csk 18
2.1 Abstract 19
2.2 Introduction 21
2.3 Materials and methods 24
2.3.1 Materials 24
2.3.2 cDNAs and cell lines 25
2.3.3 Transfection methods 26
2.3.4 Detergent-free purification of caveolin-enriched membrane fraction.................. 27
2.3.5 Internalization assays 28
2.3.6 Confocal microscopy 29
2.3.7 Immunoprecipitation 30
2.3.8 Western blot analysis 31
2.4 Results 32
2.4.1 Association of PAR1 and caveolin-1 32
2.4.2 Caveolae-independent and clathrin-dependent internalization of PAR1............. 35
2.4.3 Characterization of PAR1-induced phosphorylation of caveolin-1 on tyrosine 14 in COS7 cells and HEK 293 cells 39
2.4.4 Involvement of p38 MAP kinase in PAR1-induced phosphorylation of caveolin-1 42
2.4.5 Involvement of the Src family kinase in PAR1-induced phosphorylation of caveolin-1 45
2.4.6. Involvement of PTX sensitive G protein in PAR1-induced phosphorylation of caveolin-1 47
2.4.7 Negative regulation of Src kinase activity by the association of phosphocaveolin-1 with Csk after PAR1 activation 49
2.5 Discussion 56
Chapter 3 62
The involvement of caveolae/lipid rafts and phosphorylated ERK1/2 in PAR1-induced cell migration 62
3.1 Abstract 63
3.2 Introduction 64
3.3 Materials and methods 67
3.3.1 Reagents and antibodies 67
3.3.2 Cell culture 68
3.3.3 Detergent-free purification of caveolin-rich membrane fraction... 69
3.3.4 MTS assay 70
3.3.5 Wound-healing assay 70
3.3.6 Western blot analysis 71
3.4 Results 72
3.4.1 Subcellular distribution of PAR1 in caveolae/lipid rafts before and after PAR1 activation in MDA-MB -231 cell 72
3.4.2 Inhibition of PAR1-induced cell migration by disruption of caveolae/lipid rafts 74
3.4.3 Subcellular distribution of Gi protein in caveolae/lipid rafts before and after PAR1 activation 78
3.4.4 Redistribution of phosphorylated ERK1/2 but not PI3K in caveolae/lipid rafts after PAR1 activation 80
3.4.5 PAR1-induced cell migration by activation of Gi protein and ERK1/2 but not by PI3K 82
3.4.6 Involvement of caveolae/lipid rafts in PAR1-induced ERK phosphorylation 866
3.5 Discussion 88
Chapter 4 93
Conclusion 93
Reference 97
Biography........................................................................................................................110

Figures

Chapter 2
Fig. 2.4. 1 Association of PAR1 and caveolin-1 in COS7 cells. 34
Fig. 2.4. 2 Caveolae-independent and clathrin-dependent internalization of PAR1. 38
Fig. 2.4. 3 Characterization of PAR1-induced phosphorylation of caveolin-1 on tyrosine 14 in COS7 and HEK 293 cells. 41
Fig. 2.4. 4 Involvement of p38 MAP kinase in PAR1-induced phosphorylation of caveolin-1. 44
Fig. 2.4. 5 Involvement of Src family kinase in PAR1-induced phosphorylation of caveolin-1. 46
Fig. 2.4. 6 Involvement of PTX-sensitive G protein in PAR1-induced phosphorylation of caveolin-1. 48
Fig. 2.4. 7 Negative regulation of Src kinase activity by the association of phosphocaveolin-1 with Csk after PAR1 activation. 55
Fig. 2.4. 8 Proposed signal transduction mechanism underlying PAR1-induced phosphorylation of caveolin-1 and feedback inhibition of Src kinase by the association of phosphocaveolin-1 and Csk. 61

Chapter 3
Fig. 3.4. 1 Subcellular distribution of PAR1 in caveolae/lipid rafts before and after PAR1 activation in MDA-MB -231 cell. 73
Fig. 3.4. 2 Inhibition of PAR1-induced cell migration by disruption of caveolae/lipid rafts. 77
Fig. 3.4. 3 Subcellular distribution of Gi protein in caveolae/lipid rafts before and after PAR1 activation. 79
Fig. 3.4. 4 Redistribution of phosphorylated ERK1/2 but not PI3K in caveolae/lipid rafts after PAR1 activation. 81
Fig. 3.4. 5 PAR1-induced cell migration by activation of Gi and ERK1/2 but not by PI3K. 85
Fig. 3.4. 6 Involvement of caveolae/lipid rafts in PAR1-induced ERK1/2 phosphorylation. 87

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Biography

Name: Lu, Te-Ling陸德齡
Sex: Female
Date of Birth: Dec 15, 1969
Place of Birth: Tainan City , Taiwan, R.O.C.
Domicile: No.5, Lane 200, Mei-Chih-Cheng Community, Hsinchu City 300, Taiwan
新竹市美之城200巷5號
Telephone: (Office) 03-5715131-3476
(Home) 03-5191272
Mobil: 0982635430
E-mail: d888205@oz.nthu.edu.tw

Education:
09/2000-~Present
Ph.D., Department of Life Science, National Tsing Hua University
國立清華大學生命科學系博士
09/1995~06/1997
Master of Science, Department of Biochemistry National Cheng Kung University
國立成功大學生化所碩士
09/1988~06/1993
Bachelor of Medicine, Department of Pharmacy, China Medical College
私立中國醫藥學院藥學系學士

Professional Experiences:
10/2005~10/2005
Attendance at the Fourth Annual Convention of Cellular and Molecular Biology in France
參加法國舉辦第四屆國際細胞與分子年會
09/1997~07/2000
Lecturer, Department of Medical Technology, Yuan-Pei University of Science and Technology, Hsinchu, Taiwan, R.O.C.
新竹元培科技大學醫技系講師

09/1993~07/1995
Pharmacist, Taipei Veterans General Hospital, Taipei, Taiwan, R.O.C.
台北榮民總醫院藥師

Awards:
10/2005
Honored with Scholarship from “Convention of the Foundation for the Advancement of Outstanding Students”
榮獲李遠哲財團法人傑出人才發展基金會優秀學生出國開會獎 學金
07/1993
National Advanced Pharmacist Qualification Test
國家藥師高等考試及格

Publications:
1. T.L. Lu, F.T. Kuo, T.J. Lu, C.Y. Hsu, and H.W. Fu, Negative regulation of protease-activated receptor 1-induced Src kinase activity by the association of phosphocaveolin-1 with Csk. Cellular Signalling, (2006) (In press).

2. F.T. Kuo, T.L. Lu, and H.W. Fu, Opposing effects of β-arrestin1 and β-arrestin2 on activation and degradation of Src induced by protease-activated receptor 1. Cellular Signalling, (2006) (In press).

3. T.J. Lu, W.Y. Lai, C-Y.F. Huang, W.J. Hsieh, J.S. Yu, Y.J. Hsieh, W.T. Chang,. T.H. Leu, W.C. Chang, W.J. Chuang, M.J. Tang, T.Y. Chen, T.L. Lu, M.D. Lai, Inhibition of cell migration by mammalian sterile 20-like kinase 3 (Mst3) involves autophosphorylation and metal on cofactor. J. Biol. Chem (2006) (Revised).

4. T.J. Lu, C.Y. F. Huang, C.J. Yuan, Y.C. Lee, T.H. Leu, W.C. Chang, T.L. Lu, W.Y. Jeng, and M.D. Lai, Zinc ion acts as a cofactor for serine/threonine kinase MST3 and has a distinct role in autophosphorylation of MST3. J. Inorg. Biochem., 99(6), 1306-13 (2005).

5. T.J. Lu, T.L. Lu, I.J. Su, and M.D. Lai, Tyrosine kinase expression profile in bladder cancer. Anticancer Res. 17(4A), 2635-7 (1997).