The hypothalamic peptide arginine vasopressin (AVP) is an important regulator of adrenocorticotropin (ACTH) release from the anterior pituitary. AVP stimulates ACTH secretion from corticotroph cells by activating the pituitary vasopressin receptor (V1b-R), a member of the G protein-coupled receptor (GPCR) family. In vitro, repeated stimulus of anterior pituitary cells with AVP results in rapid desensitisation. The aim of this research was to develop methods needed to use RNA interference (RNAi) to investigate the role of G protein-coupled receptor kinase 5 (GRK5) in this desensitisation process. This required the development of a model system using human embryonic kidney (HEK) 293 cells transfected with the pituitary vasopressin receptor, V1b-R. AVP binding to the V1bR activates the phosphoinositide signalling pathway, leading to production of inositol phosphates (IPs), which can be measured following radiolabelling of cells with myo-[³H]inositol. Stimulation of V1b-R-transfected cells for 15 min with AVP (100nM) increased IP production to 235.5 ± 23.4 % (n=3, p<0.02) of that seen in un-stimulated control cells. Following a 5 minute pre-treatment with 5nM VP, the IP response to stimulation with 100nM VP for 15 min was reduced to 62.8 ± 9.1 % (n=4, p<0.02) of that seen in control cells that were not pre-treated. These data indicate that AVP-desensitisation can be induced and measured in V1bR-transfected HEK293 cells following a brief pre-treatment with a physiological concentration of AVP. This model system will enable RNAi to be used to investigate the role of GRK5 in AVP-desensitisation. When using RNAi, it is essential to establish that the effects observed are the result of small interfering RNA (siRNA) specific degradation of the target mRNA. Quantitative reverse transcription PCR (qRT-PCR) was used to measure the expression of GRK5 at the mRNA level in HEK293 cells. Human GRK5 mRNA was amplified using qRT-PCR with GRK5 specific primers, providing confirmation that GRK5 is expressed endogenously in HEK293 cells. GRK5 expression studies were carried out to evaluate whether the qRT-PCR methods developed would be suitable to measure knockdown of GRK5 mRNA using RNAi. These experiments were also designed to assess the impact of HEK293 cell culture methods on expression of GRK5. Expression of GRK5 did not vary with passage number (2-26 passages). The GRK5 expression in HEK293 cells that were maintained in culture for 5 days (grown to a confluence of approximately 100%) was 7.4 ± 0.9 fold greater (n=2, p<0.05) than for cells cultured for 3 days (grown to a confluence of approximately 65%). These data indicate that GRK5 expression is affected by HEK293 culture conditions. Furthermore, the results demonstrated that a significant difference in GRK5 expression could be measured in HEK293 cells using qRT-PCR. Therefore the results reported in this thesis provide the basis for future studies utilising RNAi to investigate mechanisms underlying V1b-R desensitisation.

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The role of NMDAR subtypes in synaptic plasticity is very controversial, partially caused by the lack of specific GluN2A containing NMDA receptor (GluN2AR) antagonists. Here we took a novel approach to selectively modulate NMDAR subtype activity and investigated its role in the induction of plasticity. Whole cell recording in both acutely isolated CA1 cells and hippocampal slices demonstrated that pituitary adenylate cyclase activating peptide 1 receptors (PAC1 receptors), which are Gαq coupled receptors, selectively recruited Src kinase and enhanced currents mediated by GluN2ARs. In addition, biochemical experiments showed that the activation of PAC1 receptors phosphorylated GluN2ARs specifically. In contrast, vasoactive intestinal peptide receptors (VPAC receptors), which are Gαs coupled receptors, selectively stimulated Fyn kinase, potentiated currents mediated by GluN2B containing NMDARs (GluN2BRs). Furthermore, dopamine D1 receptor activation (another Gαs coupled receptor) specifically phosphorylated GluN2BRs. Interestingly, field recording experiments showed that PAC1 receptor activation lowered the threshold for LTP whilst LTD was enhanced by dopamine D1 receptor activation. In conclusion, the activity of GPCRs can signal through different pathways to selectively modulate absolute contribution of GluN2ARs versus GluN2BRs in CA1 neurons via Src family kinases. Furthurmore, Epac, activated by some Gαs coupled receptors, also modulated NMDAR currents via a PKC/Src dependent pathway, but whether it selectively modulates NMDAR subtypes, and has capacity to change the induction of plasticity, requires further study. By this means, we can investigate the role of NMDAR subtypes in the direction of synaptic plasticity by selectively modulating the activity of GluN2ARs or GluN2BRs. In addition, based on my work, some interfering peptides and drugs can be designed and used to selectively inhibit the activity of GluN2BRs and GluN2ARs by interrupting Fyn- and Src - mediated signaling cascade respectively. It will provide new candidate drugs for the treatment of some neurological diseases such as Alzheimer disease (AD) and schizophrenia.

The role of NMDAR subtypes in synaptic plasticity is very controversial, partially caused by the lack of specific GluN2A containing NMDA receptor (GluN2AR) antagonists. Here we took a novel approach to selectively modulate NMDAR subtype activity and investigated its role in the induction of plasticity. Whole cell recording in both acutely isolated CA1 cells and hippocampal slices demonstrated that pituitary adenylate cyclase activating peptide 1 receptors (PAC1 receptors), which are Gαq coupled receptors, selectively recruited Src kinase and enhanced currents mediated by GluN2ARs. In addition, biochemical experiments showed that the activation of PAC1 receptors phosphorylated GluN2ARs specifically. In contrast, vasoactive intestinal peptide receptors (VPAC receptors), which are Gαs coupled receptors, selectively stimulated Fyn kinase, potentiated currents mediated by GluN2B containing NMDARs (GluN2BRs). Furthermore, dopamine D1 receptor activation (another Gαs coupled receptor) specifically phosphorylated GluN2BRs. Interestingly, field recording experiments showed that PAC1 receptor activation lowered the threshold for LTP whilst LTD was enhanced by dopamine D1 receptor activation. In conclusion, the activity of GPCRs can signal through different pathways to selectively modulate absolute contribution of GluN2ARs versus GluN2BRs in CA1 neurons via Src family kinases. Furthurmore, Epac, activated by some Gαs coupled receptors, also modulated NMDAR currents via a PKC/Src dependent pathway, but whether it selectively modulates NMDAR subtypes, and has capacity to change the induction of plasticity, requires further study. By this means, we can investigate the role of NMDAR subtypes in the direction of synaptic plasticity by selectively modulating the activity of GluN2ARs or GluN2BRs. In addition, based on my work, some interfering peptides and drugs can be designed and used to selectively inhibit the activity of GluN2BRs and GluN2ARs by interrupting Fyn- and Src - mediated signaling cascade respectively. It will provide new candidate drugs for the treatment of some neurological diseases such as Alzheimer disease (AD) and schizophrenia.

The hypothalamic peptide arginine vasopressin (AVP) is an important regulator of adrenocorticotropin (ACTH) release from the anterior pituitary. AVP stimulates ACTH secretion from corticotroph cells by activating the pituitary vasopressin receptor (V1b-R), a member of the G protein-coupled receptor (GPCR) family. In vitro, repeated stimulus of anterior pituitary cells with AVP results in rapid desensitisation. The aim of this research was to develop methods needed to use RNA interference (RNAi) to investigate the role of G protein-coupled receptor kinase 5 (GRK5) in this desensitisation process. This required the development of a model system using human embryonic kidney (HEK) 293 cells transfected with the pituitary vasopressin receptor, V1b-R. AVP binding to the V1bR activates the phosphoinositide signalling pathway, leading to production of inositol phosphates (IPs), which can be measured following radiolabelling of cells with myo-[³H]inositol. Stimulation of V1b-R-transfected cells for 15 min with AVP (100nM) increased IP production to 235.5 ± 23.4 % (n=3, p<0.02) of that seen in un-stimulated control cells. Following a 5 minute pre-treatment with 5nM VP, the IP response to stimulation with 100nM VP for 15 min was reduced to 62.8 ± 9.1 % (n=4, p<0.02) of that seen in control cells that were not pre-treated. These data indicate that AVP-desensitisation can be induced and measured in V1bR-transfected HEK293 cells following a brief pre-treatment with a physiological concentration of AVP. This model system will enable RNAi to be used to investigate the role of GRK5 in AVP-desensitisation. When using RNAi, it is essential to establish that the effects observed are the result of small interfering RNA (siRNA) specific degradation of the target mRNA. Quantitative reverse transcription PCR (qRT-PCR) was used to measure the expression of GRK5 at the mRNA level in HEK293 cells. Human GRK5 mRNA was amplified using qRT-PCR with GRK5 specific primers, providing confirmation that GRK5 is expressed endogenously in HEK293 cells. GRK5 expression studies were carried out to evaluate whether the qRT-PCR methods developed would be suitable to measure knockdown of GRK5 mRNA using RNAi. These experiments were also designed to assess the impact of HEK293 cell culture methods on expression of GRK5. Expression of GRK5 did not vary with passage number (2-26 passages). The GRK5 expression in HEK293 cells that were maintained in culture for 5 days (grown to a confluence of approximately 100%) was 7.4 ± 0.9 fold greater (n=2, p<0.05) than for cells cultured for 3 days (grown to a confluence of approximately 65%). These data indicate that GRK5 expression is affected by HEK293 culture conditions. Furthermore, the results demonstrated that a significant difference in GRK5 expression could be measured in HEK293 cells using qRT-PCR. Therefore the results reported in this thesis provide the basis for future studies utilising RNAi to investigate mechanisms underlying V1b-R desensitisation.

The prostaglandins comprise a group of bioactive lipids generated from arachidonic acid by cyclooxygenases and cell type-specific prostaglandin and thromboxane synthases. Prostaglandins mediate local cell signaling interactions by activation of G-protein coupled prostanoid receptors. Because the prostaglandins and their receptors are active in all tissues, they have an extraordinarily broad spectrum of physiological and pathophysiological functions that have hampered the development of safe prostanoid-based medications. This situation has emphasized the importance of understanding the functional properties of the prostanoid receptors and developing selective ligands capable of being used in patient care.The aims of this project were to identify novel regulatory functions of endogenous EP and FP prostanoid receptors in cultured human cells. Our results show that activation of EP₂ receptors in human microglia and astrocytes led to increased secretion of BDNF, a growth factor that regulates the survival of neurons. In the same cell lines, FP receptors regulate the induction of TNF-&alpha; gene expression through a classic G_q-PKC pathway. In microglia these FP receptors also stimulate a novel signaling crosstalk mechanism involving the up-regulation of TCF transcriptional function by Raf kinases, which culminates in the expression of the angiogenic inducer Cyr61. FP receptors also regulate the induction of angiogenic immediate early genes in cultured ciliary muscle cells, which may constitute the early steps in a mechanism by which commercial FP agonists reduce intraocular pressure in glaucoma therapy.The up-regulation of BDNF through glial EP₂ receptors constitutes a mechanism by which elevated PGE₂ in the inflamed brain might elicit either healing processes in the brain or neuronal apoptosis. On the other hand, induction of TNF-&alpha; and Cyr61 by glial FP receptors may mediate neuroinflammation and may also contribute to glioma tumor growth. Stimulation of FP receptors in the ciliary muscle leads to the induction of immediate early genes capable of coordinating tissue remodeling processes that have been previously documented. The results of these studies suggest novel regulatory functions of the prostanoid receptors in the brain and eye. Furthermore, these findings provide insight on how the selective modulation of the EP₂ and FP receptors might be therapeutically advantageous.

Regulation of the human delta opioid receptor (hDOR) is implicated in the development of tolerance to chronic morphine (Zhu et al., 1999). In addition, DORs are promising analgesic targets for the management of chronic pain states such as inflammatory or neuropathic pain (Cahill et al., 2007). Therefore, in this study, we investigated multiple aspects of hDOR regulation, including receptor phosphorylation, beta-arrestin binding, receptor internalization, down-regulation and desensitization, using recombinant Chinese hamster ovary (CHO) cells expressing the wild-type or various mutant hDOR constructs. We found that structurally diverse delta opioid agonists regulate the hDOR by different mechanisms. We demonstrate that morphine is able to activate the initial step of the regulatory events, phosphorylation of S363, but due to requirements for simultaneous activation of multiple sites, morphine fails to promote beta-arrestin binding, receptor internalization and down-regulation. We also report that peptide delta opioid receptor agonists and a non-peptide agonist SNC80 differ in their ability to down-regulate the hDOR. Further differences in receptor phosphorylation, desensitization and beta-arrestin translocation between these two classes of full DOR agonists are reveled by truncation of the receptor's C-terminus or by mutation of the primary phosphorylation site, S363. Studies using the mutant receptors identify the C-terminus as the important domain for hDOR phosphorylation, beta-arrestin binding and down-regulation by both peptide and non-peptide agonists. S363 within the C-terminus is critically involved in receptor phosphorylation, desensitization and down-regulation, but not in beta-arrestin binding and receptor internalization. In contrast to peptide agonists, SNC80 is able to phosphorylate and activate secondary intracellular domain(s), in addition to the C-terminus, which participate in beta-arrestin recruitment and receptor desensitization and down-regulation. Therefore, agonist-specific differences were detected for multiple regulatory events between morphine, peptide agonists and SNC80. Differential agonist-mediated regulation of the human delta opioid receptor may be used to design pain therapy drugs with improved analgesic properties and minimal side effects.

Little is known about the outward trafficking of receptor dimers from the endoplasmic reticulum to the plasma membrane, or the role that trafficking plays in assembly, targeting and specificity of receptor signalling. Bimolecular fluorescence complementation was used to follow prescribed receptor homo/heterodimers in Jurkat cells and clarify the trafficking itineraries those receptors follow to reach the plasma membrane. Chemokine receptors CXCR4 and CCR5 were chosen due to their implication in numerous pathologies including, HIV and cancer, and their ability to form homo and hetero-oligomers. This study demonstrates that although the individual receptors composing heterodimeric complexes are the same as in homodimeric complexes, the heterodimer traffics and signals independently of its constituent homodimers. The presence of CD4 affects the trafficking of CCR5 containing dimers but not the CXCR4 homodimer. These observations demonstrate the importance of considering receptor heterodimers as distinct signalling entities that should be more carefully and individually characterized.

Apelin, the endogenous ligand to the apelin receptor, is a small peptide involved with cardiovascular regulation. Using nuclear magnetic resonance (NMR) spectroscopy, I demonstrate that at low temperature, residues R6-L9 and G13-F17 of apelin are more structured than the rest of the peptide. I also study the interactions of apelin with sodium dodecylsulphate (SDS), dodecylphosphocholine (DPC) and 1-palmitoyl-2-hydroxy-sn- glycero-3-[phospho-RAC-(1-glycerol)] (LPPG) micelles. Apelin binds to SDS micelles through residues R6-L9, with structure being induced in this region as well as the C- terminus of the peptide. The binding to micelles along with the corresponding change in structure make it likely that apelin binds to the apelin receptor following the membrane catalysis hypothesis. NMR spectroscopy was used to determine the structure of the N- terminal tail and first transmembrane segment of the apelin receptor (AR55) in DPC micelles. AR55 has two disrupted helices from D14-K25 and from A29-K57. The second helix is the membrane spanning region of AR55 and has a significant kink located at N46. Mutagenesis of the apelin receptor and functional assays indicate that G42, G45 and N46 are essential for the proper trafficking and function of AR. In the N-terminal tail, the functionally critical residues E20 and D23 form an anionic face that could take part in initial binding of apelin to AR. The structure of AR55 was also determined in SDS micelles, LPPG micelles and a 1:1 water: 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) solution. Overall, the micelle spanning region of AR55 has a consistent structure with a kink near N46. The N-terminal tail of AR55 is more variable, having similar structures in the micelle conditions but being largely helical in 50% HFIP. NMR relaxation experiments indicate that the N-terminal tail of AR55 undergoes much more motion in LPPG micelles compared to SDS and DPC micelles. Finally, I created a program named MC-HELAN that characterizes the kinks that occur in protein helices. I used MC- HELAN to analyze all non-redundant membrane protein structures as of March 2010. Membrane protein helix kinks are remarkably common and diverse. Initial attempts to predict membrane protein kinks using only the protein sequence were unsuccessful.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18675号 / 医博第3947号 / 新制||医||1007(附属図書館) / 31608 / 京都大学大学院医学研究科医学専攻 / (主査)教授 清水 章, 教授 渡邉 大, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM