In a HEK 293 cell line stably expressing seabream GHS-R1a (sbGHS-R1a), we found that a synthetic growth hormone secretagogue (GHS) increased [ 3H]-inositol phosphate production, clearly indicating coupling of this receptor to Gq/11-proteins. Using Western blotting, we found that GHS could also stimulate extracellular signal-regulated kinases 1 and 2 (ERK1/2), and that this response was inhibited by the MEK inhibitor U0126. For both the [3H]-inositol phosphate and ERK1/2 assays, the presence of the GHS-R antagonist D-Lys(3)-GHRP-6 significantly inhibited the GHS-stimulated activities, and in addition inhibited basal activities by 50% and 40%, respectively. These results showed that sbGHS-R1a is a constitutively active receptor and the antagonist D-Lys(3)-GHRP-6 is an inverse agonist. We also proposed that the expression of sbGHS-Rs was involved in the regulation of cell apoptosis. / Oligomerization of the human GHS-Rs (hGHS-Rs) was explored by transient transfection of the hGHS-Rs in HEK 293 cells followed by co-immunoprecipitation of differentially epitope-tagged forms of the receptors and bioluminescence resonance energy transfer 2 (BRET2) studies. (Abstract shortened by UMI.) / The concept that G protein-coupled receptors (GPCRs) exist and potentially function as dimers and/or higher oligomers has progressed from hypothesis to being widely accepted recently. Oligomerization of GPCRs has been increasingly noted in the regulation of the biological activity of the receptors. The growth hormone secretagogue receptor 1a (GHS-R1a) is a GPCR which principally regulates the pulsatile release of growth hormone from the pituitary gland. The GHS-R exists in two forms: GHS-R1a being a constitutively-active GPCR with 7 transmembrane (TM) domains, and GHS-R1b being a truncated version of type 1a but having only 5 TM domains. The endogenous agonist for GHS-R1a is ghrelin which exerts a wide range of physiological actions, but the function of GHS-R1b is still unclear. Since the tissue distribution patterns of the two isoforms of GHS-R are different, the objective of the present study is to explore the mechanisms of cell signalling of GHS-R1a and to determine the extent and importance of interactions between these two receptor isoforms. / Leung Po Ki. / "July 2005." / Adviser: Helen Wise. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3728. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 189-210). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / School code: 1307.

Yeung, Barry Ho Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-154). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vii / Publications based on work in this thesis. --- p.ix / List of abbreviations --- p.x / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Dorsal root ganglion cells --- p.1 / Chapter 1.1.1 --- Primary sensory neurons --- p.1 / Chapter 1.1.2 --- Non-neuronal cells --- p.3 / Chapter 1.1.2.1 --- Satellite glial cells --- p.3 / Chapter 1.1.2.2 --- Schwann cells --- p.6 / Chapter 1.2 --- Peripheral sensitization --- p.8 / Chapter 1.3 --- Neuron-glia interactions --- p.9 / Chapter 1.4 --- Aim of Thesis --- p.11 / Chapter Chapter 2 --- "Materials, media, buffers and solutions" --- p.13 / Chapter 2.1 --- Materials --- p.13 / Chapter 2.2 --- "Culture media, buffer and solutions" --- p.19 / Chapter 2.2.1 --- Culture media --- p.19 / Chapter 2.2.2 --- General culture buffers and culture plate coating reagents --- p.19 / Chapter 2.3 --- Antibodies used for identifying DRG cells --- p.23 / Chapter 2.3.1 --- Primary antibodies --- p.23 / Chapter 2.3.2 --- Secondary antibodies --- p.23 / Chapter Chapter 3 --- Methods --- p.24 / Chapter 3.1 --- Preparation of DRG cell cultures --- p.24 / Chapter 3.2 --- Preparation of neuron-enriched and glial cell cultures --- p.25 / Chapter 3.3 --- Immunocytochemistry --- p.26 / Chapter 3.4 --- Immunohistochemistry --- p.27 / Chapter 3.4 --- Determination of [3H]cAMP production in DRG cells --- p.28 / Chapter 3.4.1 --- Principle of assay --- p.28 / Chapter 3.4.2 --- Loading DRG cells with [3H]adenine --- p.28 / Chapter 3.4.3 --- Column preparation --- p.28 / Chapter 3.4.4 --- Measurement of [3H]cAMP production in DRG cells --- p.29 / Chapter 3.4.5 --- Data analysis --- p.30 / Chapter Chapter 4 --- Identification of DRG cells in dissociated cultures --- p.31 / Chapter 4.1 --- Introduction --- p.31 / Chapter 4.2 --- Aim of study --- p.34 / Chapter 4.3 --- Results --- p.35 / Chapter 4.3.1 --- Identification of DRG cells in isolated cultures --- p.35 / Chapter 4.3.2 --- Activation and proliferation of glial cells in isolated cell cultures --- p.36 / Chapter 4.3.3 --- Identification of glial cells in cultures --- p.38 / Chapter 4.3.4 --- Modification of staining methods --- p.40 / Chapter 4.3.5 --- Immunohistochemistry to identify DRG cells in DRG slices --- p.42 / Chapter 4.3.6 --- Comparison of antibody staining in whole DRG and isolated DRG cells --- p.44 / Chapter 4.4 --- Discussion --- p.44 / Chapter 4.5 --- Summary --- p.53 / Chapter Chapter 5 --- Characterization of GPCRs in isolated DRG cultures --- p.69 / Chapter 5.1 --- Introduction --- p.69 / Chapter 5.1.1 --- G-protein coupled receptors --- p.69 / Chapter 5.1.2 --- Pharmacological characterization of prostanoid receptors on DRG cells --- p.73 / Chapter 5.1.3 --- Gs- and Gi/o-coupled GPCRs in DRG cells --- p.75 / Chapter 5.1.3.1 --- Gs-coupled GPCR: β-adrenoceptors --- p.76 / Chapter 5.1.3.2 --- Gs-coupled GPCR: CGRP receptors --- p.79 / Chapter 5.1.3.3 --- Gi/o-coupled GPCR: α2-adrenoceptors --- p.82 / Chapter 5.1.3.4 --- Gi/o-coupled GPCR: Cannabinoid receptors --- p.85 / Chapter 5.1.3.5 --- Gi/o-coupled GPCR: 5-HT1Areceptors --- p.88 / Chapter 5.1.3.6 --- Gi/o-coupled GPCR: opioid and opioid-receptor-like 1 receptors --- p.90 / Chapter 5.2 --- Aims of study --- p.93 / Chapter 5.3 --- Results --- p.94 / Chapter 5.3.1 --- Characterization of prostanoid receptors in isolated DRG cells --- p.94 / Chapter 5.3.2 --- Characterization of CGRP receptors in isolated DRG cells --- p.96 / Chapter 5.3.3 --- Investigation of the effect of CGRP8.37 on CGRP responses --- p.97 / Chapter 5.3.4 --- Characterization of β1-adrenoceptors in isolated DRG cells --- p.97 / Chapter 5.3.5 --- Characterization of β2-adrenoceptors in isolated DRG cells --- p.98 / Chapter 5.3.6 --- Identification of β-adrenoceptor subtype mediating isoprenaline-stimulated responses.. --- p.99 / Chapter 5.3.7 --- Characterization of α2-adrenceptors in isolated DRG cells --- p.100 / Chapter 5.3.8 --- Characterization of cannabinoid 1 receptors in isolated DRG cells ... --- p.100 / Chapter 5.3.9 --- Characterization of cannabinoid 2 receptors in isolated DRG cells --- p.101 / Chapter 5.3.10 --- Characterization of 5-HT1A receptors in isolated DRG cells --- p.101 / Chapter 5.3.11 --- Characterization of μ-opioid receptors in isolated DRG cells --- p.102 / Chapter 5.3.12 --- Characterization of opioid-receptor-like 1 receptors in isolated DRG cells --- p.102 / Chapter 5.3.13 --- Effect of nerve growth factor on DRG cells --- p.103 / Chapter 5.4 --- Discussion --- p.106 / Chapter 5.5 --- Summary --- p.114 / Chapter Chapter 6 --- Conclusion and further studies --- p.134 / References --- p.137

Regrowth in the lesioned central nervous system is impeded by inhibitory molecules including myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs). Inhibitory molecules engage neuronal cell surface receptors and activate the small GTPase RhoA in injured neurons to mediate neurite outgrowth inhibition through targeted modifications to the cytoskeleton. Inhibition of RhoA with the ribosyltransferase C3 attenuates neurite outgrowth inhibition in vitro and in vivo but the ubiquitous expression and multifunctionality of RhoA may limit the specificity of therapeutic RhoA antagonists. The hypothesis of the thesis is that molecules that functionally interact with RhoA to mediate myelin-dependent inhibition may represent more specific targets for therapeutic intervention. We have explored the contribution of two RhoA interacting proteins to the neurite outgrowth inhibitory effects of MAIs. In Chapter 2 we describe the contribution of the rho effector, Rho kinase (ROCK) to MAI responses in neurons. In Chapter 3 we identify the cytosolic phosphoprotein CRMP4b (Collapsin Response Mediator Protein 4b) as a novel RhoA binding partner that mediates neuronal responses to CNS inhibitors. By structure function analysis we have developed a molecular antagonist of CRMP4b-RhoA binding that promotes neurite outgrowth on inhibitory substrates in vitro and has the potential to be a potent and specific molecular therapeutic for spinal cord injury. In Chapter 4 we identify glycogen sythase kinase 3b (GSK3b) as an important kinase in the MAI pathway that regulates protein interactions with RhoA. This thesis provides insights into the signal transduction machinery that is engaged in response to CNS inhibitors and suggests several novel therapeutic targets to promote axon regeneration following CNS injury.

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