Teng, Man Kuen. / Thesis submitted in: November 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 160-169). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Declaration --- p.ii / Acknowledgments --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / List of Abbreviation --- p.viii / Table of Contents --- p.x / List of Figures --- p.xiv / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Biochemistry of the Renin-Angiotensin System --- p.2 / Chapter 1.2 --- Physiological Roles of Angiotensin II --- p.7 / Chapter 1.3 --- Physiological Roles of Angiotensin Receptors --- p.9 / Chapter 1.4 --- Characterization of Type 2 Angiotensin Receptor --- p.12 / Chapter 1.5 --- Trafficking of Type 2 Angiotensin Receptor --- p.16 / Chapter 1.6 --- SUMO and protein SUMOylation --- p.19 / Chapter 1.7 --- Aims of Study --- p.21 / Chapter Chapter 2 --- Preparation of EGFP-and FLAG tagged wild-type AT2 and K343R-AT2 mutant constructs / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials --- p.27 / Chapter 2.2.1 --- Chemicals --- p.27 / Chapter 2.2.2 --- Enzymes --- p.27 / Chapter 2.2.3 --- DNA Purification Kit --- p.27 / Chapter 2.3 --- Methods --- p.28 / Chapter 2.3.1 --- Preparation of pEGFP/AT2 Construct --- p.28 / Chapter 2.3.1.1 --- PCR amplification --- p.30 / Chapter 2.3.1.2 --- Agarose gel electrophoresis --- p.30 / Chapter 2.3.1.3 --- Restriction enzyme digestion --- p.31 / Chapter 2.3.1.4 --- Purification of DNA fragment by ethanol precipitation --- p.31 / Chapter 2.3.1.5 --- Ligation --- p.32 / Chapter 2.3.1.6 --- Preparation of competent cells --- p.33 / Chapter 2.3.1.7 --- Bacterial transformation --- p.33 / Chapter 2.3.1.8 --- Minipreparation of　plasmid DNA --- p.34 / Chapter 2.3.1.9 --- Quantitation of DNA --- p.35 / Chapter 2.3.1.10 --- DNA sequencing --- p.36 / Chapter 2.3.2 --- Preparation of pEGFP/AT2-01igo Construct --- p.36 / Chapter 2.3.2.1 --- PCR amplification of AT2-oligo --- p.39 / Chapter 2.3.2.2 --- PCR amplification of oligo-GFP --- p.39 / Chapter 2.3.2.3 --- Overlapping PCR amplification --- p.40 / Chapter 2.3.2.4 --- Gel extraction of DNA fragment --- p.41 / Chapter 2.3.2.5 --- Restriction enzyme digestion --- p.41 / Chapter 2.3.2.6 --- Ligation and transformation --- p.42 / Chapter 2.3.2.7 --- Construction of pEGFP/oligo --- p.42 / Chapter 2.3.3 --- Preparation of pCMV/AT2 Construct --- p.43 / Chapter 2.3.3.1 --- PCR amplification --- p.45 / Chapter 2.3.3.2 --- Restriction enzyme digestion --- p.45 / Chapter 2.3.3.3 --- Ligation and transformation --- p.45 / Chapter 2.3.4 --- Preparation of mutants --- p.46 / Chapter 2.3.4.1 --- Site directed mutagenesis at SUMOylation site --- p.46 / Chapter 2.3.4.2 --- Transformation of mutants --- p.47 / Chapter 2.4 --- Results --- p.48 / Chapter 2.4.1 --- Preparation of pEGFP/AT2 Construct --- p.48 / Chapter 2.4.2 --- Preparation of pEGFP/AT2-oligo Construct --- p.50 / Chapter 2.4.3 --- Preparation of pCMV/AT2 Construct --- p.53 / Chapter 2.4.4 --- Preparation of Mutants --- p.55 / Chapter 2.5 --- Discussion --- p.57 / Chapter Chapter 3 --- Transient Expression of AT2 and K343R mutants in CHO-K1 and HEK-293 cells / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Materials --- p.64 / Chapter 3.2.1 --- Chemicals --- p.64 / Chapter 3.2.2 --- Antibodies --- p.64 / Chapter 3.2.3 --- Protein Concentration Measurement Kit --- p.65 / Chapter 3.3 --- Methods --- p.66 / Chapter 3.3.1 --- Expression of AT2 in Mammalian Cells --- p.66 / Chapter 3.3.1.1 --- Cell culture --- p.66 / Chapter 3.3.1.2 --- Counting cells --- p.67 / Chapter 3.3.1.3 --- Transient transfection --- p.67 / Chapter 3.3.2 --- Western Blot Analysis --- p.68 / Chapter 3.3.2.1 --- Preparation of protein sample from total lysate --- p.68 / Chapter 3.3.2.2 --- Protein sample derived from immunoprecipitation --- p.69 / Chapter 3.3.2.3 --- SDS PAGE and Western blot analysis --- p.70 / Chapter 3.3.3 --- Confocal microscopy --- p.71 / Chapter 3.4 --- Results --- p.73 / Chapter 3.4.1 --- Expression Analysis of GFP-tagged AT2 --- p.73 / Chapter 3.4.1.1 --- Western blot analysis with anti-GFP antibody --- p.73 / Chapter 3.4.1.2 --- Western blot analysis with anti-AT2 antibody --- p.79 / Chapter 3.4.1.3 --- Confocal microscopy --- p.81 / Chapter 3.4.2 --- Western Blot Analysis of FLAG-tagged AT2 --- p.90 / Chapter 3.5 --- Discussion --- p.92 / Chapter Chapter 4 --- Stable Expression of AT2 and K343R mutants in CHO-K1 cells / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.2 --- Materials --- p.99 / Chapter 4.2.1 --- Chemicals --- p.99 / Chapter 4.2.2 --- Enzymes --- p.99 / Chapter 4.2.3 --- Antibodies --- p.99 / Chapter 4.2.4 --- Protein Concentration Measurement Kit --- p.100 / Chapter 4.3 --- Methods --- p.101 / Chapter 4.3.1 --- Linearization of Vector --- p.101 / Chapter 4.3.2 --- Transfection by Lipofectamine 2000 --- p.101 / Chapter 4.3.3 --- Screening for the Stably Transfected Cells --- p.101 / Chapter 4.3.4 --- Western Blot Analysis --- p.103 / Chapter 4.3.5 --- Confocal Microscopy --- p.103 / Chapter 4.4 --- Results --- p.104 / Chapter 4.4.1 --- Stable expression of wild type and mutant AT2-GFP in CHO-K1 --- p.104 / Chapter 4.4.2 --- Stable expression of wild type and mutant AT2-Gly10Ser5-GFP in CHO-K1 --- p.115 / Chapter 4.4.3 --- Stable expression of wild type and mutant AT2-FL AG in CHO-K1 --- p.123 / Chapter 4.5 --- Discussion --- p.125 / Chapter Chapter 5 --- Co-immunoprecipitation Analysis of CHO-K1 stably expressing wild type and mutant AT2-Gly10Ser5-GFP / Chapter 5.1 --- Introduction --- p.129 / Chapter 5.2 --- Materials --- p.129 / Chapter 5.2.1 --- Chemicals --- p.130 / Chapter 5.2.2 --- Antibodies --- p.130 / Chapter 5.2.3 --- Protein Concentration Measurement Kit --- p.130 / Chapter 5.3 --- Methods --- p.131 / Chapter 5.3.1 --- Transfection by Lipofectaime 2000 --- p.131 / Chapter 5.3.2 --- Western Blot Analysis --- p.131 / Chapter 5.4 --- Results --- p.132 / Chapter 5.4.1 --- Western blot analysis of SUMO 1 transfected stable cell lines --- p.132 / Chapter 5.4.2 --- Western blot analysis of SUM03 transfected stable cell lines --- p.136 / Chapter 5.5 --- Discussion --- p.143 / Chapter Chapter 6 --- General Discussion / Chapter 6.1 --- Investigation of AT2 trafficking in mammalian cells --- p.147 / Chapter 6.2 --- Future Aspects --- p.153 / Appendix I Buffer composition --- p.155 / Appendix II Sequence of Primers --- p.156 / Appendix III Sequencing Results --- p.157 / References --- p.160

Transforming growth factor beta (TGFbeta) and Angiotensin II (AngII) signaling occurs through two distinct receptor superfamilies, the serine/threonine kinase and G protein-coupled receptors (GPCRs). Through diametric actions, TGFbeta and AngII regulate various biological responses, including cell proliferation and migration. Previously, we identified the G protein-coupled receptor kinase 2 (GRK2), which acts through a negative feedback loop mechanism to terminate Smad signaling. To investigate the impact of TGFbeta-induced GRK2 expression on GPCR signaling, we examined its effect on AngII signaling in vascular smooth muscle cells (VSMCs). We show that activation of the TGFbeta signaling cascade results in increased GRK2 expression levels, consequently inhibiting AngII-induced ERK phosphorylation and antagonizing AngII-induced VSMC proliferation and migration. The inhibitory effect of TGFbeta on AngII signaling occurs at the MEK-ERK interface and is abrogated when an anti-sense oligonucleotide directed against GRK2 is used. Thus, we conclude that TGFbeta signaling antagonizes AngII-induced VSMC proliferation and migration through the inhibition of ERK phosphorylation. GRK2 is a key factor in mediating this crosstalk.

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Jiang, Lili. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 215-235). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.