Osmoregulation is critical to life and is tightly regulated by integrated physiological and behavioral responses to maintain the osmolality of body fluid. In particular, this involves recovery from dehydration both at the intracellular and extracellular levels. To achieve appropriate body fluid balance, three major hormones namely secretin (SCT), angiotensin II (ANGII) and vasopressin (VP) are responsible. Of note, SCT and ANGII share overlapping physiological roles including similar expression pattern within the brain, dipsogenic actions and activation of VP expression and/or release in mice. However, it remains unclear how their receptor pathways may cross-interact to aid osmoregulation. In recent years, G protein-coupled receptor (GPCR) oligomerization has been implicated to play roles in regulating processes such as expression, pharmacological diversity, signal transduction and internalization. Though not as extensively studied, class B GPCRs are also gaining merit in their oligomerization abilities, within which the wealth of available information is focused on SCT receptor (SCTR) homomers and heteromers. Moreover, there is also evidence indicating the ability for ANGII receptors to oligomerize. On the basis of this information, this project predominantly aims to explore the molecular association between SCTR and ANGII receptors via in vitro experiments and provide insights into its physiological relevance. In this study, bioluminescence resonance energy transfer (BRET) assays revealed SCTR and ANGII type 1a receptor (AT1aR) to form hetero-complexes. This oligomerization event was found by BRET competition to be contributed predominantly by transmembrane (TM) domain regions 2 and 4 in SCTR, and TM1 and TM4 in AT1aR. Within which, combinational use of mutant TM peptides and SCTR chimeras revealed the importance of lipid-exposed residues, particularly Leu204 and Ser205 in SCTR TM2 as key contact points for formation of the SCTR/AT1aR complex. Morphologically, the heteromers were visualized by confocal FRET imaging at the cell surface and found have a role in modulating AT1aR trafficking. It was also found that the SCTR/AT1aR complex affected Gαs signaling specifically, reducing maximal response values by 24.3 ± 2.8 % compared to CHO-K1 cells transfected with only SCTR. While, this negative effect could be abolished by co-application of SCT and ANGII peptides, use of constitutively active AT1aR mutants or disruption of the hetero-complex using SCTR mutants. Taken together, the SCTR/AT1aR complex was proposed to impose conformational restraints on the SCTR that could be overcome upon activation of the AT1aR. Physiologically, hyperosmolality isovolemic induced drinking could be attenuated by central administration of TM peptides and the protein kinase A pathway blocker H-89, indicating receptor oligomerization to have a role in neural osmoregulation via a Gαs dependent pathway. This study presents novel findings regarding the receptor oligomerization of SCTR and AT1aR, which may be the molecular basis to the overlapping roles of SCT and ANGII in water homeostasis. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

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