G蛋白偶聯雌激素受體（GPER，又名GPR30）乃最近於各種動物包括小鼠、大鼠、人類及斑馬魚中發現之新型跨膜雌激素受體。 GPER表達於脂肪組織及多種器官之中，其已被證明能與雌激素結合並介導各式快速反應及基因轉錄。針對GPER於成脂作用中角色之研究將達致對雌激素作用之更全面了解，且GPER亦有望成為治療肥胖症之一種新型標靶。 / 脂肪發育調控乃一複雜且精妙之排程，而雌激素已被證明能抑制脂肪形成，是故雌激素替代療法可舒減絶經後婦女之脂肪代謝問題。此項研究發現GPER於小鼠腹部脂肪組織及小鼠前脂肪細胞系3T3-L1中均有表達，且其信使RNA量於受誘導之3T3-L1成脂作用中錄得上調。 / 3T3-L1細胞分化作用會被名為G1之特異性GPER激動劑阻撓於克隆擴增階段（MCE），此即表明GPER有參與成脂調控之可能。通過油紅O染色發現，受G1處理之3T3-L1細胞於分化後所產生之油滴量實比其對照組為低，但此一效果能被特異性GPER小干擾RNA預處理抹除。另外，本研究以流式細胞儀及西方墨點法對細胞週期及細胞週期因子進行分析後，認為激活GPER能觸發對G1期細胞週期停滯之抑制。另一方面，受G1處理並分化中之3T3-L1細胞出現蛋白激酶B磷酸化效應，意味雌激素與GPER結合對成脂作用有雙向調節之可能性。 / 總而言之，本研究結果斷定GPER能介導雌激素對脂肪組織發育之影響，並為成脂作用之負調節因子，故此，一系列成果將有助肥胖症藥物之研發。 / A novel transmembrane estrogen receptor, G-protein coupled estrogen receptor (GPER, also known as GPR30), is recently identified in various animals including mouse, rat, human and zebrafish. GPER is expressed in many organs including fatty tissues, and has been demonstrated to mediate various rapid responses and transcriptional events upon estrogen binding. The study on the role of GPER in adipogenesis would lead to a more comprehensive understanding of estrogenic actions, with the view of identifying novel therapeutic targets for the treatment of obesity. / Regulation of adipose development is a complex and subtly orchestrated process. Estrogen has been shown to inhibit adipogenesis. Estrogen replacement therapy therefore affects fat metabolism in post-menopausal women. In this study, GPER is identified in mouse abdominal fatty tissues; and there is an up-regulation of GPER in the mouse preadipocyte cell line 3T3-L1 during induced adipogenesis. / Differentiation of 3T3-L1 cells is perturbed by the selective GPER agonist G1 at mitotic clonal expansion (MCE), indicating a possible involvement of GPER in the regulation of adipogenesis. By means of Oil-Red-O staining, the production of oil droplets in the G1-treated, differentiated 3T3-L1 cells is shown to be lower than the untreated control; and such effect is reversed by a specific siRNA knockdown of GPER. FACS analysis and Western blot analysis of cell cycle factors during MCE suggest that GPER activation triggers an inhibition of cell cycle arrest at the G1 stage. On the other hand, phosphorylation of Akt in G1-treated differentiating cells implies a possibility of bi-directional estrogenic regulation of adipogenesis via GPER. / To conclude, it is postulated that GPER mediates estrogenic actions in adipose tissues as a negative regulator of adipogenesis. These results provide insights into the development of therapeutic agents for the treatment of human obesity. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yuen, Man Leuk. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 144-166). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract (English version) --- p.I / Abstract (Chinese version) --- p.III / Acknowledgement --- p.V / Table of Contents --- p.VII / List of Abbreviations --- p.XI / List of Tables --- p.XII / List of Figures --- p.XIII / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1. --- Obesity and adipose tissue --- p.1 / Chapter 1.1.1. --- Obesity --- p.1 / Chapter 1.1.2. --- Fat deposition --- p.3 / Chapter 1.1.3. --- Origin and development of white adipose tissue --- p.5 / Chapter 1.2. --- Adipogenesis --- p.7 / Chapter 1.2.1. --- Origins of white adipocytes --- p.7 / Chapter 1.2.2. --- Signals for adipogenesis --- p.10 / Chapter 1.2.3. --- Regulation of gene expression during adipogenesis --- p.12 / Chapter 1.2.4. --- Common adipose cell lines --- p.16 / Chapter 1.2.5. --- Mechanism of in vitro adipogenesis --- p.21 / Chapter 1.2.5.1. --- Growth arrest --- p.23 / Chapter 1.2.5.2. --- Mitotic clonal expansion --- p.23 / Chapter 1.2.5.3. --- Early and terminal differentiation --- p.24 / Chapter 1.3. --- Estrogen and adipogenesis --- p.28 / Chapter 1.4. --- G-protein coupled estrogen receptor-1 --- p.33 / Chapter 1.4.1. --- General introduction of GPER --- p.33 / Chapter 1.4.2. --- Ligands of GPER --- p.36 / Chapter 1.4.3. --- Cellular signaling of GPER --- p.38 / Chapter 1.4.4. --- Metabolic actions of GPER: A brief introduction --- p.43 / Chapter 1.4.5. --- Metabolic actions of GPER on obesity and glucose metabolism --- p.48 / Chapter 1.4.6. --- Study objectives --- p.53 / Chapter Chapter 2: --- Expression profiles and cellular localization of Gper/GPER in mouse adipose, 3T3-L1 preadipocytes and 3T3-L1 mature adipocytes --- p.54 / Chapter 2.1. --- Introduction --- p.54 / Chapter 2.1.1. --- Expression and functional roles of GPER in adipose. --- p.55 / Chapter 2.1.2. --- Swiss mouse preadipocytes 3T3-L1 --- p.57 / Chapter 2.1.3. --- Study objectives --- p.57 / Chapter 2.2. --- Materials and Methods --- p.59 / Chapter 2.2.1. --- Reagents --- p.59 / Chapter 2.2.2. --- Animal tissues --- p.59 / Chapter 2.2.3. --- Cell culture --- p.60 / Chapter 2.2.4. --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.62 / Chapter 2.2.5. --- Quantitative real-time RT-PCR (qRT-PCR) --- p.66 / Chapter 2.2.6. --- SDS-PAGE and Western blot analysis --- p.68 / Chapter 2.2.7. --- Immunofluorescence assay --- p.69 / Chapter 2.2.8. --- Statistical analysis --- p.70 / Chapter 2.3. --- Results --- p.71 / Chapter 2.3.1. --- Expression of Gper/GPER in mouse visceral adipose tissues --- p.72 / Chapter 2.3.2. --- Expression profiles of Gper/GPER in undifferentiated 3T3-L1 preadipocytes and differentiated 3T3-L1 adipocytes --- p.73 / Chapter 2.3.3. --- Cellular localization of GPER in undifferentiated 3T3-L1 preadipocytes and differentiated 3T3-L1 adipocytes --- p.75 / Chapter 2.4. --- Discussion --- p.76 / Chapter Chapter 3: --- Rapid cellular responses induced by GPER activation in 3T3-L1 preadipocytes --- p.78 / Chapter 3.1. --- Introduction --- p.78 / Chapter 3.1.1. --- Rapid cellular response of estrogen via GPER --- p.79 / Chapter 3.1.2. --- Study objectives --- p.81 / Chapter 3.2. --- Materials and Methods --- p.82 / Chapter 3.2.1. --- Reagents --- p.82 / Chapter 3.2.2. --- Cell culture --- p.82 / Chapter 3.2.3. --- SDS-PAGE and Western blot analysis --- p.83 / Chapter 3.2.4. --- Statistical analysis --- p.84 / Chapter 3.3. --- Results --- p.86 / Chapter 3.3.1. --- Phosphorylation of p44/42 MAPK after time-dependent activation of GPER by ICI182,780 and G1 --- p.87 / Chapter 3.3.2. --- Phosphorylation of p44/42 MAPK after dose-dependent activation of GPER by a combination of chemical agents --- p.88 / Chapter 3.4. --- Discussion --- p.89 / Chapter Chapter 4: --- GPER activation on cell viability of 3T3-L1 preadipocytes --- p.90 / Chapter 4.1. --- Introduction --- p.90 / Chapter 4.1.1. --- Cell proliferation mediated by GPER --- p.90 / Chapter 4.1.2. --- Study objectives --- p.92 / Chapter 4.2. --- Materials and Methods --- p.93 / Chapter 4.2.1. --- Reagents --- p.93 / Chapter 4.2.2. --- Cell culture --- p.93 / Chapter 4.2.3. --- MTT assay for cell viability --- p.94 / Chapter 4.2.4. --- Statistical analysis --- p.95 / Chapter 4.3. --- Results --- p.96 / Chapter 4.3.1. --- Cell viability of 3T3-L1 after dose-dependent activation of GPER by 17β-estradiol, ICI182,780 and G1 --- p.97 / Chapter 4.4. --- Discussion --- p.99 / Chapter Chapter 5: --- GPER-mediated estrogenic action on lipid accumulation in the mature 3T3-L1 adipocytes --- p.101 / Chapter 5.1. --- Introduction --- p.101 / Chapter 5.1.1. --- Induction of differentiation in Swiss mouse preadipocyte 3T3-L1 --- p.101 / Chapter 5.1.2. --- Study objectives --- p.102 / Chapter 5.2. --- Materials and Methods --- p.103 / Chapter 5.2.1. --- Reagents --- p.103 / Chapter 5.2.2. --- Cell culture --- p.103 / Chapter 5.2.3. --- Oil-Red-O staining and measurement of absorbance --- p.105 / Chapter 5.2.4. --- Knockdown of Gper/GPER by siRNA --- p.107 / Chapter 5.2.5. --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.110 / Chapter 5.2.6. --- SDS-PAGE and Western blot analysis --- p.110 / Chapter 5.2.7. --- Statistical analysis --- p.110 / Chapter 5.3. --- Results --- p.112 / Chapter 5.3.1. --- GPER activation on 3T3-L1 differentiation --- p.114 / Chapter 5.3.2. --- Knockdown of Gper/GPER in Swiss mouse preadipocyte 3T3-L1 --- p.114 / Chapter 5.3.3. --- Phosphorylation of p44/42 MAPK in Gper/GPER-knockdown 3T3-L1 after time-dependent activation of GPER by G1 --- p.117 / Chapter 5.3.4. --- Action of drugs on differentiation of Gper/GPER-knockdown 3T3-L1 --- p.117 / Chapter 5.4. --- Discussion --- p.118 / Chapter Chapter 6: --- Role of GPER in regulating cell cycle progression during mitotic clonal expansion (MCE) stage in adipogenesis of 3T3-L1 --- p.120 / Chapter 6.1. --- Introduction --- p.120 / Chapter 6.1.1. --- Differentiation stages of Swiss mouse preadipocyte 3T3-L1 --- p.121 / Chapter 6.1.2. --- Apoptosis and cell cycle progression --- p.122 / Chapter 6.1.3. --- Study objectives --- p.126 / Chapter 6.2. --- Materials and Methods --- p.127 / Chapter 6.2.1. --- Reagents --- p.127 / Chapter 6.2.2. --- Cell culture --- p.127 / Chapter 6.2.3. --- Oil-Red-O staining and measurement of absorbance --- p.129 / Chapter 6.2.4. --- Trypan blue exclusion assay for cell viability determination --- p.129 / Chapter 6.2.5. --- SDS-PAGE and Western blot analysis --- p.131 / Chapter 6.2.6. --- Flow cytometry for analysis of cell cycle progression --- p.132 / Chapter 6.2.7. --- Statistical analysis --- p.133 / Chapter 6.3. --- Results --- p.134 / Chapter 6.3.1. --- Temporal effect of GPER activation on differentiation progress of Swiss mouse preadipocyte 3T3-L1 --- p.137 / Chapter 6.3.2. --- Effect of GPER activation on cell viability during adipogenesis --- p.139 / Chapter 6.3.3. --- Effect of GPER activation on apoptosis during adipogenesis --- p.139 / Chapter 6.3.4. --- Effect of GPER activation on cell cycle distribution during induced adipogenesis --- p.140 / Chapter 6.3.5. --- Effect of GPER activation on expression of cell cycle markers during induced adipogenesis --- p.142 / Chapter 6.3.6. --- Activation of PI3K/Akt pathway by GPER stimulation during induced adipogenesis --- p.143 / Chapter 6.4. --- Discussion --- p.144 / Chapter Chapter 7: --- Conclusions and Future Perspectives --- p.148 / References --- p.155

Ectopic and over-expression of G protein-coupled receptor (GPCR) have been reported to induce tumor formation. Mas protein is a member of GPCR family and was originally isolated from human epidermoid carcinoma. It was demonstrated that mas mRNA was abundantly expressed in human and rat brains by in situ hybridization and RNase protection assays. However, cellular mechanism that leads to such tumorigenic transformation is still an open question. / In order to identify the cellular mechanism of mas-induced tumor formation, a full-length mas cDNA was cloned into a mammalian expression vector pFRSV with dihydrofolate reductase gene as a selection marker. Detailed analyses of mas-transfected cell lines by Southern blot, Northern blot and tumorigenicity assay indicated that tumorigenicity of mas-transfected cells depended on the sites of chromosomal integration and the levels of mas expression. These results suggest that overexpression of mas is not sufficient to induce tumor formation. In line with the ability of mas-transfected cells Mc0M80 to form solid tumor in nude mice, MTT cell proliferation assay indicated that the mas-transfected cells Mc0M80 proliferated faster than vector-transfected cells. Moreover, mas-transfected cells Mc0M80 exhibited significantly increased anchorage-independent growth. Furthermore, mas-transfected cells Mc0M80 showed higher percentage cells in G2/M phase but lower in S-phase in comparison with vector-transfected cells. / Interestingly, Southern blot analysis of individual xenografted tumor tissue indicated that tumor was composed of cells not only derived from injected mas-transfected CHO cells but also cells from mouse tissues. The presence of mouse stromal cells in the tumor was confirmed by immunohistochemistry and in situ hybridization. Previously our laboratory had identified some up- and down-regulated genes in mas-transfected cells by fluorescent differential display (FluoroDD). Northern blot showed that these differential expressed genes were up- or down-regulated in mas-transfected cells and tumor samples, which might play an important role in cancerous growth. / Taken together, these results suggest that over-expression of GPCR mas up-regulated tumor-related genes, resulting in promoting excessive cell growth and tumorigenic transformation. In addition, when the tumor mass formed they secreted some growth factor(s) which altered the migration of mouse stromal cells into tumor mass. The interactions of transformed cells and stromal cells further aggravate the tumorigenicity process. / To complement our fluorescent differential display study and to compare changes of gene expression when transformed cells were exposed to the microenvironment in nude mice, protein expression profiles of mas over-expressing cells as well as tumor tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. The 2D-PAGE analysis showed that a similar but distinct protein expression profiles in mas-transfected cells and in mas-induced tumor. Mass spectrometry analysis identified several cancerous growth-related proteins and they are involved in processes such as cell signaling, energy metabolism, transcription and translation and cytoskeleton organization. / Lin Wenzhen. / "December 2005." / Adviser: Cheung Wing Tai. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6381. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 222-240). / 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. / Abstracts in English and Chinese. / School code: 1307.

Bikkavilli Rama Kamesh. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 212-223) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

The apelin system is an evolving transmitter system consisting of the G protein coupled apelin receptor and two endogenous peptide ligands, apelin and elabela. It is implicated as a potential therapeutic for a number of diseases; however, the endogenous peptides are limited by half-life and bioavailability. This study aims to identify and pharmacologically characterise apelin agonists in vitro and in vivo and to evaluate their therapeutic potential in pulmonary arterial hypertension as a model disease. CMF-019 was identified as the first G protein biased apelin agonist. To date, suitable small molecule apelin agonists as experimental tool compounds have been limited and CMF-019 represents an important advance. CMF-019 was active in vivo, producing an increase in cardiac contractility and vasodilatation, similar to apelin. These effects were achieved without receptor desensitisation, supporting the remarkable G protein bias observed in vitro. Furthermore, it was disease-modifying in vitro in an endothelial cell apoptosis assay but despite this, did not prevent pulmonary arterial hypertension in a monocrotaline rat model of the disease. An apelin mimetic peptide possessing an unnatural amino acid, MM202, conjugated chemically via a polyethylene glycol linker to an anti-serum domain antibody (AlbudAb) was also characterised. The product MM202-AlbudAb represents the first time an AlbudAb has been conjugated chemically to an unnatural peptide mimetic, providing protection from proteolysis and glomerular filtration. Importantly, it retained binding to albumin and demonstrated in vitro and in vivo activity at the apelin receptor. In conclusion, this thesis has identified and pharmacologically characterised two novel apelin agonists that possess significant advantages over the endogenous peptides. CMF-019 is suitable as an experimental tool compound and, as the first G protein biased small molecule, provides a starting point for more suitable therapeutics. In addition, MM202-AlbudAb proves that unnatural peptides can be conjugated to AlbudAb, supporting use of this technology in other small-peptide ligand transmitter systems.

Regulator of G-protein Signaling (RGS) proteins temporally regulate the G protein signaling cascades initiated by GPCR activation. Reports have established dysregulation of RGS expression in a variety of disease states including several cancers. Additionally, use of genetic ablation techniques has implicated RGS proteins in a variety of other disease states through the native action of the RGS i.e. not a consequence of dysregulation of RGS expression. Therefore identification and optimization of small molecule lead compounds that alter RGS protein function has emerged as a promising therapeutic strategy. In this thesis, we use high throughput screening to interrogate small molecule libraries targeting two RGS proteins, RGS6 and RGS17. RGS6 has been reported as an essential mediator of doxorubicin induced cardiotoxicity, alcohol induced cardio and hepatotoxicity, anxiety, depression, and alcohol dependence. RGS17 has largely been implicated in a variety of cancer pathogenesis, with reported over expression in prostate, lung, breast, and hepatocellular carcinomas. Chapter 2 of this work focuses on the screening efforts targeting RGS6. Three separate screening campaigns interrogating over 20K compounds led to the identification of 3 small molecules that inhibit the RGS6: Gαo protein protein interaction with appreciable selectivity over control assays. The development of a cell based protein interaction assay is discussed, and the compounds were investigated using this system. All compounds tested did not appreciably alter signal over control, meaning that the cellular activity of these compounds remains ambiguous. Chapter 3 details the screening and follow up efforts targeting RGS17. The primary screening and/or follow up of four separate screening campaigns interrogating over 110K compounds is discussed. In total, 10 identified leads and a panel of analogs were subjected to significant follow up evaluation. All compounds were found to be cysteine dependent. The second generation RGS17 inhibitors (UI series) were determined to be both cytostatic and cytotoxic against lung and prostate cancer cell lines in culture, although whether this is due to RGS17 dependent mechanisms or due to general promiscuity of the compounds remains to be determined. Lead compounds from a library provided by the NCI were found to have cellular activity and were subjected to an investigation of structure activity relationships via commercially available compounds. The active form of three of these compounds was found to be a degradation product, which is likely due to decomposition of furan or methyl furan moieties that these compounds shared. One compound demonstrated robust SAR which allowed for the generation of schemes detailing putative inhibitory mechanisms. Finally, the role of RGS17 in the transition from epithelial to mesenchymal phenotypes is investigated. RGS17 was found to cause a sub population of PC3 cells to shift to mesenchymal phenotype, indicating that RGS17 may indeed play a role in this transition. Chapter 4 focuses on efforts to investigate variable potencies of published RGS4 inhibitors against a panel of RGS proteins, with the goal of gleaning insight in to structural characteristics that influence the inhibitability of RGS proteins. Most compounds tested were found to be more potent inhibitors of RGS14 rather than RGS4 in biochemical assays. We developed the NanoBit protein complementation assay to assess the interaction of RGS proteins with either Gαi1 or Gαq in a cellular context, and used this system to investigate compound selectivity in a cellular context. The compounds tested showed selectivity for RGS2, RGS4, and RGS14 over the other RGS proteins tested. The structural differences between the RGS proteins is discussed. Chapter 5 focuses on the future directions the lab may take with respect to the projects outlined in the previous chapters. This includes the screening of more targeted libraries or even virtual screening for RGS6, the development of in vivo assessment tools for RGS17, and an expanded structural examination of RGS proteins including NMR and crystal structure analysis. Additionally, the development of the NanoBit system to interrogate RGS protein interactions that are not RGS: Gα interactions is discussed.

Der Fluoreszenz-Resonanz-Energie-Transfer ist ein Phänomen, welches erstmals 1948 von Theodor Förster beschrieben wurde. Mit der Entwicklung von Fluoreszenzproteinen konnten in Kombination mit Mikroskopietechniken Einblicke in zellbiologische Vorgänge gewonnen werden, die durch biochemische oder physiologische Experimente nicht möglich sind. Dabei spielt die hohe zeitliche und räumliche Auflösung eine wichtige Rolle. Auf dem Forschungsgebiet der GPCR, welche die größte Gruppe von Membranproteinen bei den Säugetieren darstellen, wurden insbesondere Erkenntnisse über Konformationsänderungen der Rezeptoren, die Kinetik der Rezeptoraktivierung und die Interaktion mit intrazellulären Signalproteinen gewonnen. Der µ-Opioidrezeptor gehört zur Familie der GPCR und stellt aufgrund seiner analgetischen Wirkungen eine wichtige pharmakologische Zielstruktur dar. Das Ziel dieser Arbeit war sowohl den Rezeptor als auch seine Signalwege mittels FRET-Mikroskopie zu untersuchen. Zunächst sollte ein intramolekularer FRET-Sensor des µ-Opioidrezeptors entwickelt werden, dazu wurden basierend auf den Kenntnissen über die Tertiärstruktur und dem Aufbau bereits bekannter GPCR-Sensoren verschiedene Rezeptorkonstrukte kloniert. Bei den Konstrukten wurden entweder zwei Fluoreszenzproteine oder ein Fluoreszenzprotein und ein Fluorophor-bindendes Tetracysteinmotiv kombiniert. Auch die Positionen der eingefügten Sequenzen wurden in den intrazellulären Domänen variiert, da der Rezeptor auf die Modifikationen mit beeinträchtigter Membranlokalisation reagierte. Durch die Optimierung wurden Rezeptoren konstruiert, die an der Zellmembran lokalisiert waren. Jedoch zeigte keines der Rezeptorkonstrukte Funktionalität im Hinblick auf die Rezeptoraktivierung. Im zweiten Teil wurden die pharmakologischen Effekte der Metabolite von Morphin am humanen µ-Opioidrezeptor systematisch analysiert. Dazu wurde die Fähigkeit der Metabolite, Gi-Proteine zu aktivieren und β-Arrestin2 zu rekrutieren, mittels FRET-basierter Messungen an lebenden Zellen untersucht. Außerdem wurde die Affinität der Metabolite zum humanen µ Opioidrezeptor anhand der Verdrängung eines radioaktiven Liganden analysiert. Meine Experimente identifizierten eine Gruppe mit stark agonistischen und eine mit schwach agonistischen Eigenschaften. Die starken Partialagonisten aktivieren den Rezeptor bereits bei nanomolaren Konzentrationen, während die schwachen Metabolite den Rezeptor erst bei Konzentrationen im mikromolaren Bereich aktivieren. Die Metabolite Normorphin, Morphin-6-Glucuronid und 6-Acetylmorphin zeigen geringere Potenz als Morphin bei der Gi-Aktivierung aber überraschenderweise höhere Potenz und Effizienz für die β-Arrestin-Rekrutierung. Dies deutet auf eine bevorzugte Aktivierung von β-Arrestin2 hin. Die aus diesen Studien gewonnenen Ergebnisse liefern Hinweise darauf, welche Metabolite bei der Signalverarbeitung am µ Opioidrezeptor in vivo beteiligt sind. / Fluorescence resonance energy transfer was first described by Theodor Förster in 1948. The discovery and development of intrinsic fluorescent proteins revolutionized cell and molecular biology. The FRET-technique allows the analysis of protein-protein interactions and intramolecular conformational changes. In this method, its high temporal and spatial resolution plays a crucial role. Especially in the research field of GPCR, which are the largest family of membrane proteins in mammals, insights into receptor conformational changes, kinetics of receptor activation and the interaction with intracellular proteins were obtained. The µ-opioid receptor belongs to the GPCR family and is involved in analgesia. Therefore, the receptor is an important pharmacological target. Its pharmacological properties were extensively analyzed in the current thesis by FRET. Engineering of an intramolecular MOR-biosensor was initially planned. Based on the knowledge about the tertiary receptor structure and earlier GPCR-sensors, different receptor constructs were cloned. For each receptor construct either two fluorescent proteins or one fluorescent protein and one fluorophore binding tetracysteine motif were combined. The insertion of the additional amino acid sequences prevented the membrane localization for some constructs. Hence, the insertion site of the amioacid sequences was varied in the intracellular loops. Ultimately, the optimization resulted in some membrane localized receptor constructs with the tetracysteine motif in the third intracellular loop. Nevertheless, none of the receptor constructs was functional in terms of measurable conformational change upon receptor activation. In the second part of this thesis, the pharmacological effects of morphine and its metabolites were studied. The analgesic effects of morphine are mainly mediated via the activation of the µ opioid receptor. This receptor activates inhibitory G-proteins and induces the recruitment of β-arrestin2. Therefore I analyzed activation of these two pathways induced by morphine metabolites using FRET-microscopy in living cells. Furthermore, radioligand binding studies were used to determine the affinity of each compound to the human µ-opioid receptor. This approach identified two groups of metabolites, which were classified into strong and weak ligands. Strong partial agonists showed efficacies in the nanomalar range. In contrast, weak metabolites activated µ opioid receptor pathways in the micromolar range. Normorphine, morphine-6-glucuronide and 6 acetylmorphine had lower potencies regarding Gi-protein activation but higher potencies and efficacies for β-arrestin2 recruitment than morphine. These findings indicate that these metabolites are biased towards β-arrestin2 pathways.

G protein-coupled receptors (GPCRs) are the largest family of signaling proteins in animals and represent the largest family of druggable targets in the human genome. Therefore, it is of no surprise that the molecular mechanisms of GPCR activation and signal transduction have attracted close attention for the past few decades. Several stabilizing interactions within the GPCR transmembrane (TM) domain helices regulate receptor activation. An example is a salt bridge between 2 highly conserved amino acids at the bottom of TM3 and TM6 that has been characterized for a large number of GPCRs. Through structural modeling and molecular dynamics (MD) simulations, we predicted several electrostatic interactions to be involved in metabotropic glutamate receptor 2 (mGlu2R) activation. To experimentally test these predictions, we employed a charge reversal mutagenesis approach to disrupt predicted receptor electrostatic intramolecular interactions as well as intermolecular interactions between the receptor and G proteins. Using two electrode voltage clamp in Xenopus laevis oocytes expressing mutant receptors and G-proteins, we revealed novel electrostatic interactions, mostly located around intracellular loops 2 and 3 of mGlu2R, that are critical for both receptor and G-protein activation. These studies contribute to elucidating the molecular determinants of mGluRs activation and conformational coupling to G-proteins, and can likely be extended to include other classes of GPCRs.

Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is known about Groups 3 and 4 medulloblastomas. Identification of tumor subgroup using molecular classification is poised to become an important component of the medulloblastoma diagnosis and staging and will likely guide therapeutic options. G-protein coupled receptors (GPCR) possess characteristics that make them ideal targets for molecular imaging and therapeutics. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We have found that clusters of medulloblastoma tumors arise based solely on differential GPCR expression patterns. Further, two of these clusters correspond with high fidelity to the WNT and SHH subgroups. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our results identify GPCRs with potential to act as imaging and therapeutic targets; elucidating tumorigenic mechanisms is a secondary benefit to identifying differential GPCR expression patterns in medulloblastoma tumors. Current imaging for diagnosis, staging, and measuring response to therapy for medulloblastoma patients relies heavily on MRI; single photon emission tomography (SPECT) using 111In-DTPA-Octreotide targeting the somatostatin type 2 receptor (SSTR2) is also available. Positron emission tomography (PET) affords a more sensitive and specific imaging modality than SPECT; however, the most common tracer 18FDG, is of limited usefulness for the delineation of brain tumors. Smoothened (SMO) is a GPCR that is overexpressed in a subset of medulloblastoma; we hypothesized that SMO overexpression could be exploited as a specific PET target in these tumors. Genentech generously provided the synthetically-derived small-molecule SMO ligand, GDC-0449, for use as the lead compound for development of a PET tracer. GDC-0449 has already been demonstrated to localize in brain tumors and has Cl- atoms incorporated in positions that are predicted to readily exchange with fluorine-18 to generate a fluorinated analog of the compound. We have successfully fluorinated GDC-0449, with very high radiochemical purity. Binding assays reveal affinities of the fluorinated analog of GDC-0449 for SMO to be comparable to precursor GDC-0449, and biodistribution experiments demonstrate accumulation of the fluorinated compound in tumors. The fluorinated analog of GDC-0449 holds promise as a novel PET imaging agent in medulloblastoma, providing highly specific and sensitive imaging for use in diagnosis, staging and measurement of response-to-treatment.

G–Protein Coupled Receptors are one of the most important targets in drug development, making up over 60% of drug targets. Recent studies have implicated a role of Regulator of G–Protein Signaling (RGS) proteins in the development and progression of pathologies, including some cancers. RGS17, the most–recently identified family member of the RZ family of RGS proteins, has been implicated in the growth, proliferation, metastasis and migration of prostate tumors as well as small–cell and non–small cell lung cancers. In neoplastic tumor tissues RGS17 is up–regulated 13 fold over patient–matched normal tissues in prostate cancer. Studies have shown that RGS17 RNAi knockdown inhibits colony formation and decreases tumorigenesis in nude mice. Based on these findings, this thesis explores the research undertaken to develop small molecule inhibitors of the RGS17: Gαo protein: protein interaction. In this thesis, we implemented AlphaScreen® technology to develop a high–throughput screening method for interrogating small molecule libraries for inhibitors of RGS17. Chapter 3 focuses on the initial results of the AlphaScreen® in 384–well format. The screen utilizes a measurement of the Gα: RGS17 protein: protein interaction (PPI) and with an excellent Z–score exceeding 0.73, a signal to noise ratio >70 and a screening time of 1,100 compounds per hour. Chapter 3 presents the development, validation and initial high–throughput screening for inhibitors of Gα: RGS17 interaction as well as preliminary characterization of the RL series of hits. In this pilot screen the NCI Diversity Set II was interrogated, yielding 35 initial hits of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 ΜM in dose–response experiments for inhibiting the Gα: RGS17 interaction. Four exhibited IC50 values <6 ΜM while inhibiting the Gα: RGS17 interaction >50% when compared to a biotinylated GST control (TrueHits). Compounds RL–1 and RL–2 were confirmed by flow cytometry protein interaction assay (FCPIA) while RL–3 and RL–4 were unable to disrupt this PPI in FCPIA. All four compounds were tested using the differential scanning fluorimetry (DSF) method, which is based on energetic coupling between ligand binding and protein unfolding and found compounds RL–1 to RL–4 all slightly increased protein stability upon ligand binding. Chapter 4 focuses on the miniaturization and optimization of AlphaScreen® to a 1536–well format and screening of the MicroSource SPECTRUM and NDL3000 small molecule libraries. This increased throughput 11–fold and decreased our working volumes from 45 ΜL to 10 ΜL, which reduced reagent cost. After optimization, we retained in an excellent Z–factor ≥0.70 with S/N>5.77 and increased the screening rate to more than 12,000 compounds per hour. In this format, the initial screening of the SPECTRUM and NDL3000 libraries was completed and filtered the initial hits by counter screening and PAINs filtering as well as developing four powerful orthogonal assays for the characterization of potential lead molecules. Chapter 6 focuses on the future directions, which include the screening the in–house 50,000 compound library in the University of Iowa HTS Core facility as well as the development of cell based assays to determine the activity of these leads in the cellular milieu. These screens are the first step to developing novel pharmacophores for further optimization of structure with the focus on RGS17 activity in enzymatic, whole cell, xenograft and whole animal models as well as providing new avenues for the development of anticancer therapies.

The endogenous neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts various neuromodulatory functions in mammalian brain. Enhancement of synaptic activity, mediation of chronic inflammatory and neuropathic pain, and neuroprotection in cerebral ischemia reperfusion injury constitute some of the exemplary functions of PACAP. However, it remains unclear whether PACAP signaling can directly influence the function of critical voltage-gated ion channels, which could profoundly alter the excitability of neurons. Voltage-gated K+ (Kv) channels are critical regulators of neuronal excitability. The major Kv channel in the dendrites of mammalian neurons, Kv4.2, contributes most of the fast-activating and rapidly-inactivating K+ currents (IA), and is a key regulator of dendritic excitability, as well as modulation of synaptic inputs. In addition, the major somatic Kv channel Kv2.1 that contributes the bulk of slow-activating and non-inactivating K+ currents (IK), acts as an integrator of neuronal inputs and limits high frequency firing in neurons. As such, it provides homeostatic control of excitability under hyperexcitable and ischemic conditions. Both these Kv channels are known to undergo extensive post-translational modifications mainly by phosphorylation that alters their localization and biophysical properties. PACAP can activate its specific receptor PAC1 that could result in downstream activation of various kinases including protein kinase A (PKA), protein kinase C (PKC), extracellular signal-regulated kinase (ERK1/2). Therefore, I hypothesize that PACAP activation of PAC1 receptor can cause phosphorylation-dependent modulation of somatodendritic Kv4.2 and Kv2.1 channels, resulting in altered neuronal excitability. First, I identified the various PAC1 receptor isoforms expressed in rat and mouse brain and elucidated that their activation by PACAP caused downstream PKA- and PKC-dependent signaling pathways, ultimately converging on ERK1/2 activation. Further, PACAP caused reduction in IA that was mediated by phosphorylation-dependent internalization of the channel protein from the plasma membrane. These effects were mediated by direct phosphorylation of the channel by ERK1/2 at the cytoplasmic C-terminus of the channel. Although PACAP did not significantly alter the voltage-dependence of Kv4.2 channel activation/inactivation, I observed distinct ERK1/2- and PKA-dependent changes in the extent and kinetics of channel inactivation. Next, I observed that PACAP induced dephosphorylation of the Kv2.1 channel in CHN that was mediated by protein phosphatase 2A (PP2A), and was dependent on PKC activation but was independent of the effects of PACAP on Kv4.2 currents. Rapid but reversible dephosphorylation of Kv2.1 was also observed following induction of ischemia in neurons by oxygen-glucose deprivation (OGD). PACAP prolonged the dephosphorylation of Kv2.1 following in vitro ischemia-reperfusion and also reduced neuronal death. My results therefore suggest a novel PACAP/PAC1-PKC-PP2A-Kv2.1 signaling axis that provides neuroprotection during ischemia reperfusion injury. In summary, my results suggest that PACAP can induce direct phosphorylation-dependent modulation of the Kv4.2 and Kv2.1 channel localization and function in mammalian brain neurons. The effect of PACAP on these two critical somatodendritic ion channels occurs via distinct signaling - convergent PKA-PKC-ERK-mediated phosphorylation of Kv4.2 channel, and PKC-PP2A-mediated dephosphorylation of the Kv2.1 channel. Such distinct modulations of these ion channels are presumably responsible for the multifarious roles of PACAP in the central nervous system.