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title page
ABSTRACT
Contents
Abbreviations 14
Chapter 1. G Protein-coupled Receptor Signaling and Cross-talk : Achieving Rapidity and Specificity. 15
1-1. Introduction. 15
1-2. The established paradigm of GPCR signaling 17
1-3. New paradigms of GPCR signaling 19
1-3-1. Concept of microdomain as a functional signaling platform 19
1-3-2. Cross-talk of GPCRs 22
1-3-2-1. Cross-talk between GPCRs 23
1-3-2-2. Cross-talk between GPCR and other signaling pathways 28
1-4. Concluding remarks 35
1-5. References 38
Chapter 2. Materials and Methods. 44
2-1. Antibodies. 44
2-2. Plasmids. 45
2-3. RNA interference and RT-PCR. 45
2-4. In situ hybridization. 46
2-5. Northern blot. 47
2-6. Western blot and immunoprecipitation. 47
2-7. Preparation of adrenal medullary chromaffin cells. 48
2-8. PC12 cell culture. 49
2-9. Measurement of [Ca2+]i and its imaging.(이미지참조) 49
2-10. Measurement of cAMP contents. 50
2-11. Measurement of IP₃. 51
2-12. Measurement of [³H]NE secretion. 52
2-13. Measurement of membrane potential. 53
2-14. Mn2+ quenching of fura-2 fluorescence.(이미지참조) 53
2-15. Amperometry. 53
2-16. HPLC. 54
2-17. Confocal microscopy and image analysis. 54
2-18. Immunogold electron microscopy. 55
2-19. Detergent solubilization and sucrose gradient fractionation. 56
2-20. Statistical analysis. 57
Chapter 3. Sensitization of Epidermal Growth Factor (EGF)-induced Signaling by Bradykinin Is Mediated by c-Src : Implications for a Role of Lipid Microdomains. 58
3-1. Introduction. 58
3-2. Results. 60
3-2-1. Sensitization of EGF-induced [Ca2+]i increase by BK.(이미지참조) 60
3-2-2. Involvement of EGFR. 61
3-2-3. Role of c-Src in the BK-induced sensitization. 62
3-2-4. Induction of EGF-mediated exocytosis by BK. 64
3-2-5. Signaling events in the detergent-resistant membrane (DRM) fraction. 64
3-2-6. Involvement of Src-dependent EGFR phosphorylation. 65
3-2-7. Effects of cellular cholesterol depletion and add-back. 66
3-3. Discussion. 67
3-4. References. 88
Chapter 4. Junctional Membrane Inositol 1,4,5-trisphosphate Receptor (IP₃R) Complex Coordinates Sensitization of the Silent EGF-induced Ca2+ Signaling.(이미지참조) 91
4-1. Introduction. 91
4-2. Results. 94
4-2-1. Involvement of PKA is involved in sensitization of the silent EGF-induced Ca2+ signaling and exocytosis.(이미지참조) 94
4-2-2. IP₃R1 is required for the sensitization. 96
4-2-3. EGFR, PKA, and yotiao (AKAP9) comprise a macromolecular signaling complex with IP₃R1. 98
4-2-4. IP₃R1 is phosphorylated in response to BK stimulation in a PKA-dependent manner. 99
4-2-5. AKAP signaling complex is required for the PKA-dependent phosphorylation of IP₃R1 by BK and promotes the sensitization process. 100
4-3. Discussion. 101
4-4. References. 126
Chapter 5. Coupling of L-type Voltage-Sensitive Calcium Channels (VSCCL) to P2X₂ Purinoceptors in PC12 Cells.(이미지참조) 129
5-1. Introduction. 129
5-2. Results. 130
5-2-1. Effects of Ca2+ channel inhibitors on P2X-mediated [Ca2+]i increase and secretion.(이미지참조) 130
5-2-2. P2X-mediated [Ca2+]i rise in depolarized cells.(이미지참조) 132
5-2-3. Inhibition of P2X-mediated cation entry by Ca2+ channel blockers.(이미지참조) 132
5-2-4. P2X receptor subtypes are coupled to VSCCL.(이미지참조) 134
5-2-5. Activation of VSCCs by membrane depolarization via Na+ influx through P2X₂ purinoceptors.(이미지참조) 135
5-3. Discussion. 138
5-4. References. 153
SUMMARY(KOREAN) 155
Appendix 159
Curriculum vitae 167
Acknowledgements 173
Table 4-1. BK increases cAMP contents via activation of B₂ receptors. 109
Table 4-2. Distribution of distances between EGFR at the PM and IP₃R1 at Ca2+ stores in rat adrenal medullary chromaffin cells(이미지참조) 110
Chapter 1. 12
Fig. 1-1. Effector molecules regulated by different Gα- and Gβγ-subunits.(이미지참조) 36
Fig. 1-2. Schematic model of arising paradigms in GPCR signaling. 37
Chapter 3. 12
Fig. 3-1. Proposed model for the possible role of lipid raft in signal transduction. 74
Fig. 3-2. BK sensitizes EGF-induced [Ca2+]i increase.(이미지참조) 75
Fig. 3-3. Sensitization of the EGF-induced [Ca2+]i increase.(이미지참조) 76
Fig. 3-4. PLC-dependent Ca2+ release is induced by EGF after BK treatment.(이미지참조) 78
Fig. 3-5. Involvement of EGFR in the sensitization. 79
Fig. 3-6. Involvement of Src kinase in the sensitization. 80
Fig. 3-7. Src-mediated sensitization of EGF-evoked exocytosis. 82
Fig. 3-8. Phosphorylation and translocation events occur in the DRM fraction. 83
Fig. 3-9. Src-dependent phosphorylation of EGFR on Tyr-845 is critical to the sensitization. 84
Fig. 3-10. Effects of cellular cholesterol depletion and add-back on the Sensitization. 85
Fig. 3-11. EGF-induced ERK phosphorylation is not affected by BK. 87
Chapter 4. 12
Fig. 4-1. IP₃R as a signal integrator. 111
Fig. 4-2. Sensitization of the functionally silent EGF-induced [Ca2+]i increase and exocytosis by BK in rat adrenal chromaffin cells.(이미지참조) 112
Fig. 4-3. Involvement of PKA in the sensitization process. 114
Fig. 4-4. Expression and subcellular localization of IP₃R subtypes. 116
Fig. 4-5. IP₃R1 is required for the sensitization. 118
Fig. 4-6. Yotiao (AKAP9), PKA, and EGFR comprise a macromolecular signaling complex with IP₃R1. 119
Fig. 4-7. Immunogold EM showing colocalization of EGFR and IP₃R1 in adrenal chromaffin cells. 120
Fig. 4-8. Immunoconfocal microscopy images showing colocalization of endogenous EGFR and IP₃R1 in adrenal chromaffin cells. 121
Fig. 4-9. Phosphorylation of IP₃R1 upon application of BK. 123
Fig. 4-10. AKAP signaling complex facilitates BK-induced phosphorylation of IP₃R1 and the sensitization process. 124
Fig. 4-11. Suggested model for the sensitization of EGF-induced Ca2+ release and exocytosis by BK.(이미지참조) 125
Chapter 5 13
Fig. 5-1. P2 purinoceptors in PCl2 cells. 142
Fig. 5-2. VSCCs in PC12 cells. 143
Fig. 5-3. Effect of VSCC blockers on the ATP-, 2-MeSATP-, and UTP-induced responses in PC12 cells. 144
Fig. 5-4. Effect of 70 mM K+ pretreatment on the ATP-, 2-MeSATP-, and UTP-induced [Ca2+]i increase.(이미지참조) 145
Fig. 5-5. Effect of VSCC blockers on ATP-, 2-MeSATP-, and UTP-evoked [Ca2+]i increase after treatment with TG.(이미지참조) 146
Fig. 5-6. Effect of nifedipine on Mn2+ quenching and Ca2+ influx.(이미지참조) 147
Fig. 5-7. P2X receptor subtypes coupled to L-type VSCCs. 148
Fig. 5-8. ATP-, 2-MeSATP-, and UTP-induced depolarization measured with bisoxonol. 149
Fig. 5-9. Effect of nifedipine on the ATP- and 2-MeSATP-induced [Ca2+]i increase under Na+-free conditions.(이미지참조) 150
Fig. 5-10. Effect of 2-MeSATP and K+ on depolarization and [Ca2+]i rise.(이미지참조) 151
Fig. 5-11. Proposed model for the coupling of P2X₂ purinoceptors and VSCCL.(이미지참조) 152
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