Die Bedeutung der β-Arrestine als multifunktionelle Adapterproteine GPCR-vermittelter Signaltransduktion hat in den letzten Jahren immer mehr zugenommen. In der vorliegenden Arbeit lag der Schwerpunkt auf der Untersuchung der molekularen Basis und der Ligandenabhängigkeit sowohl der β-Arrestin/Rezeptor-Interaktion als auch β-Arrestin- (un-)abhängiger Signaltransduktionsmechanismen. Im ersten Teil wurde der Einfluß potentieller Phosphorylierungsstellen im C-Terminus des β2AR bzw. im C-Terminus und der TM3 des P2Y1R auf die agonisteninduzierte β-Arrestin/Rezeptor-Interaktion, Internalisierung und Desensibilisierung untersucht. Durch Mutationsanalysen konnten Ser 352/Thr 358 im distalen C-Terminus des P2Y1R als Schlüsselstellen der β-Arrestin-Translokation und Internalisierung identifiziert werden, während ein oder mehrere Phosphorylierungsstellen im proximalen P2Y1R C-Terminus die molekulare Grundlage der Rezeptordesensibilisierung darstellen. Darüber hinaus machte die Anwendung verschiedener PKC- oder CaMK-Inhibitoren sowie der Einsatz des PKC-Aktivators PMA deutlich, dass die P2Y1R-Desensibilisierung und β-Arrestin-Translokation durch unterschiedliche Kinasen kontrolliert werden. Zudem konnte mit Hilfe der FRET-Technik gezeigt werden, dass die Phosphorylierungsstellen zwischen den Positionen 355 und 364 im proximalen β2AR C-Terminus essentielle Bereiche der β-Arrestin-Translokation darstellen. Im zweiten Teil der vorliegenden Arbeit wurden Agonisten am β2-adrenergen Rezeptor bzw. dem P2Y2R auf ihre Fähigkeit hin untersucht verschiedene mit dem jeweiligen Rezeptor verknüpfte G-Protein- bzw. β-Arrestin-Funktionen in unterschiedlichem Ausmaß zu aktivieren („biased agonism“). Da eine solche ligandenselektive Aktivierung rezeptorvermittelter Signalwege bis dato nur mit synthetischen Liganden detailliert untersucht wurde, galt das besondere Interesse der Analyse der durch die endogenen Substanzen induzierten Signalmuster. Die Betrachtung der Noradrenalin- bzw. Adrenalin-induzierten β-Arrestin/Rezeptor-Interaktion, β-Arrestin2-Translokation, Rezeptorinternalisierung, G-Protein-Aktivierung sowie cAMP-Produktion am β2AR machte deutlich, dass es sich beim Phänomen des „biased agonism“ um einen endogenen Mechanismus handelt. Darüber hinaus konnte gezeigt werden, dass auch zur Tokolyse eingesetzte β2AR-Agonisten spezifische Signalmuster induzieren. Die Beobachtung, dass UTP und ATP sowohl unterschiedliche β-Arrestin1/2-Translokationsals auch ERK-Aktivierungsmuster am P2Y2R induzieren bestärkte das Konzept des „biased agonism“ als endogenes Phänomen. Das ligandenabhängige β-Arrestin-Translokationsverhalten des P2Y2R ließ zudem die agonistenbedingte Zuteilung des Rezeptors zu den „Klasse A“ oder „Klasse B“ Rezeptoren zu. Die detaillierte Untersuchung agonisteninduzierter Rezeptor/Effektor-Interaktionen und Signalmuster dürfte helfen die Anwendung klinisch relevanter Substanzen zu optimieren. / In recent years, the significance of β-arrestins as multifunctional adapter proteins of GPCR mediated signal transduction has steadily been increasing. In this thesis the main focus is to research the molecular basis and the ligand dependence of the β-arrestin recruitment as well as β-arrestin-(in-)dependent signal transduction mechanisms. In the first part, the influence of potential phosphorylation sites in the C-terminus of the β2AR or the C-terminus and the TM3 of the P2Y1R, respectively, on the agonist-induced β-arrestin2/receptor-interaction, receptor internalization and desensitization was examined. Using mutation analysis, Ser 352 and Thr 358 were identified as key points of the β-arrestin2 translocation and receptor internalization in the distal C-terminus of the P2Y1R. In contrast, one or more phosphorylation sites in the proximal P2Y1R C-terminus represent the molecular basis of receptor desensitization. In addition, the use of different PKC- or CaMK inhibitors and the application of the PKC activator PMA made it clear that the P2Y1R desensitization and β-arrestin translocation are controlled by different kinases. Using the FRET technique we were able to show that the phosphorylation sites between position 355 and 364 in the proximal C-terminus of the β2AR represent essential areas of the β-arrestin2 translocation. In the second part of the study at hand, agonists of the β2AR or the P2Y2R were examined with respect to their ability to activate distinct receptor associated G-protein or β-arrestin functions to varying degrees (“biased agonism”). Since this kind of ligandselective activation of receptor-mediated signaling pathways has only been studied in detail with synthetic ligands to this day, special interest in the analysis of the signaling pattern induced by the endogenous substances was taken. The analysis of norepinephrine- or epinephrine-induced β-arrestin/receptor interaction, β-arrestin translocation, receptor internalization, G-protein activation and cAMP production at the β2AR made clear that “biased agonism” is an endogenous phenomenon. Moreover, it has also been shown that β2AR agonists used for tocolysis induced a specific signaling pattern. The observation that UTP and ATP both induce different β-arrestin translocation as well as ERK activation patterns at the P2Y2R confirmed the concept of “biased agonism” as an endogenous phenomenon. The ligand dependent β-arrestin behavior of the P2Y2R also allowed the allocation of the receptor to the “class A” or “class B” receptors depending on the agonist used for stimulation. The detailed testing of agonist induced receptor/effector interactions and signaling pattern could help to optimize the application of clinically relevant substances.

Die vorliegende Arbeit befasst sich mit der Synthese von Liganden der Melatonin-Rezeptoren (MR). Die zwei humanen MR-Subtypen, MT1 und MT2, gehören zur Familie der G-Protein-gekoppelten Rezeptoren. Als „Schlafhormon“ wirkt es schlafinduzierend und vermittelt den circadianen Rhythmus. Zum genauen Verständnis der physiologischen Funktionen der MT1- und MT2-Rezeptoren ist die Verfügbarkeit von subtypselektiven MR-Liganden unentbehrlich. Zum Design von MT2-selektiven MR-Liganden modifizierte man die Melatonin-Grundstruktur durch formale Substitution in 2-Stellung, z.B. mit dem 2-Methylen-N-methyl-anilin- oder 2-Methylen-1´-indol-Rest. Weiterhin wurden trizyklische Derivate mit 1,2,3,4-Tetrahydro-pyrazino[1,2-a]indol- oder 2,3,4,5-Tetrahydro-1H-[1,4]diazepino[1,2-a]indol-Grundgerüst hergestellt. Das Synthesekonzept für dieses Teilprojekt basierte auf dem Synthesebaustein 3-Cyanomethyl-5-methoxy-1H-indol-2-carbonsäure. Da bislang nur wenige MT1-selektive MR-Liganden bekannt sind, wurde zur Untersuchung der Voraussetzung für MT1-Selektivität, die 5-Methoxygruppe von Melatonin formal durch Phenylalkyloxy-Reste verschiedener Kettenlängen substituiert. Die Synthese der Derivate erfolgte ausgehend von N-Acetylserotonin. Als Referenzverbindung wurde der bis heute MT1-selektivste MR-Antagonist (Descamps-Francois et al. 2003) hergestellt. Zu dessen Synthese benötigte man Agomelatin als Ausgangsverbindung. Eine neuartige vierstufige Route zu Agomelatin wurde daher entwickelt. Die Testung der Referenzverbindung ergab eine drastische Abweichung vom Literaturwert, da diese als nahezu unselektiv getestet wurde. Unter den O-Phenylalkyl-N-Acetylserotonin-Derivaten wurden zwei Verbindungen mit einer 11-fachen MT1-Selektivität getestet. Zur Absicherung der Reinheit wurden die Verbindungen mit RP-HPLC untersucht. Schließlich wurden noch melatoninerge Dimere mit einem 1-1´, 1-2´ und 5-5´ Verknüpfungsmuster hergestellt. / The present work is focused on the synthesis of ligands of melatonin receptors (MR). The two human MR subtypes, MT1 and MT2, belong to the family of G-protein-coupled receptors. As a “sleep hormone”, it induces sleep and also moderates the circadian rhythm. An accurate characterization of melatonin receptor-mediated functions requires MT1 and MT2 selective ligands. In order to design MT2-selective MR-ligands the melatonin scaffold was formally substituted in 2-position, for example with methylen-N-methyl-aniline- or methylen-1´-indole. Furthermore, tricyclic derivatives with the 1,2,3,4-tetrahydro-pyrazino[1,2-a]indole- or 2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a]indole-scaffold were synthesized. The synthetic concept based on the useful building block 3-cyanomethyl-5-methoxy-1H-indole-2-carboxylic acid. While many series of MT2-selective agents are known, the design of MT1-selective agents still is a challenge. A common structural feature of MT1-selective ligands is the presence of a bulky hydrophobic substituent linked to an alkyl chain in a position topologically equivalent to the MeO-group of melatonin. In order to probe the melatonin receptors for MT1-selectivity, a series of melatonin analogues obtained by the replacement of the ether methyl group with phenylalkyl substituents was prepared. The derivates were synthesized from N-acetylserotonine as a starting compound. The most MT1-selective MR-antagonist (Descamps-Francois et al. 2003) was synthesized additionally as a reference compound. Therefore we need agomelatine as starting material, which could be received from our newly developed four-step route. Surprisingly, the reference compound displayed a much lower affinity for the MT1 receptor than reported earlier. In the homologous series of melatonin analogs, the compound with Ph(CH2)3- and PhO(CH2)3-groups were the most MT1-selective agents, revealing that a C3-spacer is optimal to generate MT1-selectivity. Finally, three series of dimeric melatonin analogues, with a 1-1´-, 1-2´- and 5-5´-junction patter were prepared.

Lau Pui Ngan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 171-181). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / Abbreviations --- p.viii / Publications Based on work in this thesis --- p.xii / Chapter Chapter 1 --- Introduction and project overview --- p.1 / Chapter 1.1 --- Ghrelin structure and its synthesis --- p.3 / Chapter 1.2 --- Types of growth hormone secretagogues (GHSs) --- p.6 / Chapter 1.3 --- Characterization of GHS-R --- p.7 / Chapter 1.3.1 --- Cloning of GHS-Rla and GHS-Rlb --- p.7 / Chapter 1.3.1.1 --- GHS-R subtypes --- p.7 / Chapter 1.3.1.2 --- Properties of GHS-R subtypes --- p.7 / Chapter 1.3.1.3 --- Evidence of non-GHS-Rla stimulated by ghrelin and GHSs --- p.9 / Chapter 1.3.1.4 --- Distribution of GHS-R --- p.10 / Chapter 1.3.2 --- Signal transduction pathways of GHS-R --- p.11 / Chapter 1.3.3 --- Comparison between human and seabream GHS-R --- p.12 / Chapter 1.4 --- Is adenosine a partial agonist at GHS-Rla? --- p.15 / Chapter 1.5 --- Physiological effects of ghrelin --- p.17 / Chapter 1.6 --- Apoptosis --- p.19 / Chapter 1.6.1 --- Introduction --- p.19 / Chapter 1.6.2 --- Apoptosis versus necrosis --- p.19 / Chapter 1.6.3 --- Mechanisms of apoptosis --- p.20 / Chapter 1.6.4 --- Methods to study apoptosis --- p.23 / Chapter 1.6.5 --- Different types of apoptotic inducers --- p.24 / Chapter 1.7 --- Apoptotic and anti-apoptotic pathways regulated by GPCRs --- p.27 / Chapter 1.7.1 --- Bcl-2 family pathway --- p.27 / Chapter 1.7.2 --- Caspase pathway --- p.27 / Chapter 1.7.3 --- ERK pathway --- p.28 / Chapter 1.7.4 --- PI3K/Akt pathway --- p.29 / Chapter Chapter 2 --- Materials and solutions --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.2 --- "Culture medium, buffer and solutions" --- p.37 / Chapter 2.2.1 --- Culture medium --- p.37 / Chapter 2.2.2 --- Buffers --- p.37 / Chapter 2.2.3 --- Solutions --- p.38 / Chapter Chapter 3 --- Methods --- p.41 / Chapter 3.1 --- Maintenance of cell lines --- p.41 / Chapter 3.1.1 --- Human Embryonic kidney (HEK293) cells --- p.41 / Chapter 3.1.2 --- HEK293 cells stably expressing black seabream growth hormone secretagogues receptors (HEK-sbGHS-Rla and HEK-sbGHS-Rlb) --- p.41 / Chapter 3.2 --- Preparation of plasmid DNA --- p.42 / Chapter 3.2.1 --- Preparation of competent E. coli --- p.42 / Chapter 3.2.2 --- Transformation of DNA into competent cells --- p.42 / Chapter 3.2.3 --- Small-scale and large-scale plasmid DNA preparation --- p.43 / Chapter 3.2.4 --- Confirmation of the purity and the identity of the plasmid DNA --- p.43 / Chapter 3.3 --- Transient transfection of mammalian cells --- p.45 / Chapter 3.4 --- Development of stable cell lines --- p.46 / Chapter 3.4.1 --- Determination of the optimum concentration of each antibiotic used in selection of clones --- p.46 / Chapter 3.4.2 --- Development of monoclonal stable cell line --- p.46 / Chapter 3.4.3 --- Confirmation the expression of 2myc-hGHS-Rla and myc-hGHS-Rlb --- p.48 / Chapter 3.5 --- Measurement of phospbolipase C activity --- p.49 / Chapter 3.5.1 --- Introduction --- p.49 / Chapter 3.5.2 --- Preparation of columns --- p.49 / Chapter 3.5.3 --- [3 H]-inositol phosphate assay --- p.49 / Chapter 3.5.4 --- Measurement of [3H]-inositol phosphates production --- p.50 / Chapter 3.5.5 --- Data analysis --- p.50 / Chapter 3.6 --- Determination of transient transfection efficiency --- p.51 / Chapter 3.7 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.52 / Chapter 3.7.1 --- RNA extraction and first strand cDNA production --- p.52 / Chapter 3.7.2 --- PCR and visualization of amplicons --- p.52 / Chapter 3.7.3 --- Real-time PCR --- p.59 / Chapter 3.7.3.1 --- Construction of standard curve --- p.60 / Chapter 3.7.3.2 --- Data analysis --- p.60 / Chapter 3.8 --- Measurement of caspase-3 activity --- p.65 / Chapter 3.8.1 --- Determination of caspase-3 activity using colorimetric assay --- p.65 / Chapter 3.8.1.1 --- Introduction --- p.65 / Chapter 3.8.1.2 --- Induction of apoptosis --- p.65 / Chapter 3.8.1.3 --- Preparation of cell lysates --- p.65 / Chapter 3.8.1.4 --- Quantification of caspase-3 activity by measuring pNA absorbance --- p.66 / Chapter 3.8.1.5 --- Data analysis --- p.67 / Chapter 3.8.2 --- Determination of caspase-3 activity using bioluminescence resonance energy transfer (BRET2) assay --- p.67 / Chapter 3.8.2.1 --- Introduction --- p.67 / Chapter 3.8.2.2 --- Quantification of caspase-3 activity using BRET2 assay --- p.68 / Chapter 3.8.2.3 --- Data analysis --- p.69 / Chapter 3.8.3 --- Determination of caspase-3 activity using fluorescence resonance energy transfer (FERT) assay --- p.70 / Chapter 3.8.3.1 --- Introduction --- p.70 / Chapter 3.8.3.2 --- Quantification of caspase-3 activity using FRET assay --- p.70 / Chapter 3.8.3.3 --- Data analysis --- p.71 / Chapter Chapter 4 --- Results --- p.72 / Chapter 4.1 --- Characterization of GHS-R --- p.72 / Chapter 4.1.1 --- Properties of GHS-Rla --- p.72 / Chapter 4.1.1.1 --- Constitutively active receptor --- p.72 / Chapter 4.1.1.2 --- Characterization of epitope-tagged hGHS-Rla --- p.73 / Chapter 4.1.2 --- Properties of GHS-Rlb --- p.75 / Chapter 4.1.3 --- Conclusions --- p.75 / Chapter 4.2 --- Effect of co-transfection of HEK293 cells --- p.85 / Chapter 4.2.1 --- Effect of balancing DNA concentrations transfected into HEK293 cells --- p.85 / Chapter 4.2.2 --- Effect of balancing DNA concentration using another Gq-coupled receptor --- p.87 / Chapter 4.2.3 --- Effect of Gi- and Gs-coupled receptor on GHS-Rla signaling --- p.88 / Chapter 4.2.4 --- Potentiating effect of co-transfection appeared using different transfection reagents --- p.88 / Chapter 4.2.5 --- Co-transfection improves transfection efficiency --- p.89 / Chapter 4.2.6 --- Discussions --- p.91 / Chapter 4.3 --- Development of cell lines stably expressing hGHS-Rla or hGHS-Rlb --- p.102 / Chapter 4.3.1 --- Advantages of using a monoclonal cell line --- p.102 / Chapter 4.3.2 --- Sensitivity of HEK293 cells to antibiotics --- p.102 / Chapter 4.3.3 --- Production of polyclonal stable cell line --- p.103 / Chapter 4.3.4 --- Monoclonal stable cell line selection --- p.104 / Chapter 4.3.5 --- Discussions --- p.105 / Chapter 4.4 --- Effect of adenosine on GHS-Rla signaling --- p.111 / Chapter 4.4.1 --- Adenosine acts as partial agonist --- p.111 / Chapter 4.4.2 --- Effect of substance P analog on adenosine-mediated GHS-Rla signaling --- p.112 / Chapter 4.4.3 --- Effect of adenosine deaminase (ADA) on adenosine- and ghrelin-stimulated GHS-Rla signaling --- p.113 / Chapter 4.4.4 --- Specificity of ADA --- p.115 / Chapter 4.4.5 --- Conclusions --- p.116 / Chapter 4.5 --- Role of GHS-R in apoptosis --- p.124 / Chapter 4.5.1 --- Different methods to measure caspase-3 activity --- p.124 / Chapter 4.5.1.1 --- Colorimetric assay --- p.124 / Chapter 4.5.1.1.1 --- Time course for staurosporine and etoposide in HEK293 cells --- p.125 / Chapter 4.5.1.1.2 --- Effect of 2myc-hGHS-Rla on staurosporine- and etoposide-induced caspase-3 activity --- p.127 / Chapter 4.5.1.1.3 --- Time course for staurosporine and etoposide in sbGHS-R monoclonal stable cell line --- p.128 / Chapter 4.5.1.1.4 --- Effect of sbGHS-Rs on staurosporine- and etoposide- induced caspase-3 activityin HEK 293 cells --- p.129 / Chapter 4.5.1.1.5 --- Effect of sbGHS-Rs on staurosporine- induced caspase-3 activity in sbGHS-R monoclonal stable cell line --- p.130 / Chapter 4.5.1.1.6 --- Differences between epitope-tagged and non-tagged sbGHS-Rs in staurosporine- induced caspase-3 activity --- p.131 / Chapter 4.5.1.1.7 --- The role of epitope-tagged sbGHS-Rlbin staurosporine-induced caspase-3 activity --- p.132 / Chapter 4.5.1.1.8 --- Effect of staurosporine and etoposide on GHS-Rla signaling --- p.133 / Chapter 4.5.1.2 --- BRET2 assay --- p.135 / Chapter 4.5.1.3 --- FRET assay --- p.136 / Chapter 4.5.1.4 --- Conclusions --- p.136 / Chapter 4.6 --- Determination of GHS-R amount in terms of mRNA --- p.155 / Chapter 4.6.1 --- Determination of GHS-R amount in stable cell lines --- p.155 / Chapter 4.6.2 --- Transfected DNA amount match with stable cell lines --- p.155 / Chapter Chapter 5 --- "Discussion, Conclusions and Future Plan" --- p.159 / Chapter 5.1 --- General Discussion and Conclusions --- p.159 / Chapter 5.2 --- Future Plan and Experimental Design --- p.168 / References --- p.171

In a phage binding assay, phage clone (3p5A190) expressing a surrogate mas ligand displayed punctate binding and were internalized in cell expressing native mas and GFP-tagged variants. However, the number of bound and internalized phages in cells expressing mas-GFP was substantially less than the cells expressing mas-(Gly10Ser5)GFP and native mas. In parallel, biotinylation experiment quantitatively showed that the extent of mas-(Gly10Ser 5)-GFP translocation was higher than that of mas-GFP. Consistently, cells expressing mas-(Gly10Ser5)-GFP and native mas showed a rapid and sustained increase of intracellular calcium levels upon MBP7 stimulation. By contrast, cells expressing mas-GFP only response to higher concentration of MBP7 challenge and showed a delayed increase of intracellular calcium level. Moreover, cells expressing native mas had a higher proportion (80%) of cells responsive to MBP7 stimulation; in contrast to only 10∼20% of cells expressing mas fusion proteins. / MBP7-like motif was identified in human facilitative GLUT1 and GLUT7 indicating that mas might interact with glucose transporter (GLUT) and regulate cellular glucose uptake. GLUT4 was found to be expressed endogenously in the CHO cell by RT-PCR, but expression of insulin receptor was not detectable. Although no statistical difference was detected in basal glucose uptake among control cells Vc0M80 and cells with different levels of mas expression, cells expressing mas-(Gly10Ser5)-GFP showed a high glucose uptake in response to insulin. Furthermore, basal 2-DOG uptake in Mc0M80 cells was not affected by pretreatment with various kinase inhibitors or transient expression of Rho variants. By contrast, MBP7 was found to induce a significant elevation of glucose uptake specifically in Mc0M80 cells transiently transfected with GLUT1. / Orphan G protein-coupled receptor (GPCR) mas was initially isolated from a human epidermal carcinoma. Previous study from our lab identified a surrogate ligand---MBP7 (mas binding peptide 7) for mas, and suggested that GFP tagging might affect the receptor activity of mas. In this project, three stable CHO cell lines expressing native mas, mas-GFP and mas-(Gly10Ser 5)-GFP were used to characterize receptor activity of mas. / To summarize, direct GFP tagging at the C-terminus of mas decreased its interactions with ligand and downstream signaling molecules. Partial recovery of mas receptor activity by adding a peptide linker was confirmed by phage binding, membrane fusion protein translocation and calcium response. In addition, mas was possibily coupled with GLUT1 to affect cellular glucose uptake via signaling pathways yet to be fully characterized. / Sun, Jingxin. / Adviser: Cheung Wing Tai. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0104. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 150-170). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Animals rely on the integration of a variety of external cues to understand and respond appropriately to their environment. The relative amounts of food and constitutively secreted pheromone detected by the nematode C. elegans determines how it will develop and grow. Starvation conditions cause the animal to enter a protective stage, termed dauer. Dauer animals are non-eating, long-lived and stress resistant. Yet, when these animals are introduced to food replete conditions they will recover from dauer and proceed into normal development. Furthermore, food restriction has been demonstrated to extend the lifespan of a wide-range of species including C. elegans. However, the exact mechanism by which food signals are detected and transduced by C. elegans to influence development and longevity remains unknown. Here, we identify a G protein-coupled receptor (GPCR) DCAR-1 that acts in two chemosensory neurons to mediate food signaling in an autophagy-related manner. The DCAR-1 ligand Dihydrocaffeic acid (DHCA) competes with dauer-inducing pheromone to promote growth. DHCA is a key intermediate in the shikimate pathway, which is required to synthesize folate and aromatic amino acids. We report that dcar-1 mutations influence dauer formation and extend wildtype lifespan via a mechanism of dietary restriction. Moreover, we show that the lifespan extension of dcar-1 mutants is completely dependent on autophagy gene atg- 18. Furthermore, our data suggests metabolites derived from shikimate are food signals that control aging and dauer development through GPCR signaling in C. elegans. These studies will contribute to the delineation of mechanisms behind the beneficial effects of dietary restriction in eukaryotic organisms. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

The Rho family of GTPases plays a crucial role in the regulation of diverse cellular processes, including proliferation and actin cytoskeletal rearrangement to promote cell migration. However, dysregulation of RhoGTPases has been associated with disease, particularly cancers such as leukemia. Despite this, RhoGTPases are rarely mutated in cancer. Rather, dysregulation of their regulatory proteins through mutation or overexpression contributes to disease pathogenesis. RhoGTPases are activated through Rho guanine nucleotide exchange factors (GEFs). Although over eighty RhoGEFs have been identified that activate the 25 RhoGTPases, the pathological role of the majority of these proteins remains unclear. Further, whereas the majority of RhoGEFs are activated through tyrosine phosphorylation, a small subset can be activated through heterotrimeric G proteins, including through GΒ;Γ; subunits. However, the mechanism by which GΒ;Γ; induces RhoGEF activation remains unclear. PLEKHG2 is a Dbl family RhoGEF that was originally identified as a gene upregulated in a leukemia mouse model, and later shown to be activated by heterotrimeric G protein Β;Γ; subunits. However, its function and activation mechanisms remain elusive. Here we show that, as compared to primary human T cells, the expression of PLEKHG2 is upregulated in leukemia cell lines. Downregulation of PLEKHG2 by siRNAs specifically inhibited GΒ;Γ;-stimulated Rac and Cdc42, but not RhoA activation. Consequently, inhibition of PLEKHG2 blocked actin polymerization, protrusion formation, and leukemia cell migration in response to SDF1alpha;. Additional studies indicate that GΒ;Γ; likely activates PLEKHG2 by binding the N-terminus of PLEKHG2. This interaction results in the release of autoinhibition imposed by the C-terminus within a region encompassing the catalytic DH domain. As a result, overexpressing either the N-terminus of PLEKHG2 that binds GΒ;Γ; or the C-terminus that autoinhibits PLEKHG2 blocked GΒ;Γ;-stimulated Rac and Cdc42 activation and the ability of leukemia cell to form membrane protrusions and to migrate. Together, our results have demonstrated that PLEKHG2 functions as a novel GΒ;Γ; -stimulated RhoGEF that could contribute to chemokine-induced leukemia cell dissemination and leukemia pathogenesis.

RKIP reguliert Proteinkinasen der Signaltransduktionskaskaden von G Protein-gekoppelten Rezeptoren, der Raf/MEK/ERK-MAPK, des Transkriptionsfaktors NFκB und von GSK3β. Unklar war bisher, wie die spezifische Interaktion von RKIP mit seinen mannigfaltigen Interaktionspartnern ermöglicht und reguliert wird. Raf1 und GRK2 sind die einzigen bekannten direkten Interaktionspartner von RKIP und wurden deshalb gewählt, um die zugrundeliegenden molekularen Mechanismen dieser Interaktion genauer zu untersuchen. In dieser Arbeit wurde gezeigt, dass RKIP nach PKC-vermittelter Phosphorylierung von Serin153 dimerisiert und dass diese Dimerisierung für die RKIP/Raf1-Dissoziation und die RKIP/GRK2-Interaktion essentiell ist. Co-Immunpräzipitationsexperimente mit einer phosphorylierungsdefizienten Mutante zeigten, dass für diese Dimerisierung die Phosphorylierung von beiden RKIP-Molekülen notwendig ist. Als Dimerinteraktionsfläche wurden die Aminosäuren 127-146 von RKIP identifiziert, da das Peptid RKIP127-146 die Dimerisierung von RKIP spezifisch und effizient hemmte. Um die Bedeutung dieser phosphorylierungsinduzierten Dimerisierung von RKIP für seine Interaktion mit Raf1 und GRK2 zu untersuchen, wurden eine phosphomimetische Mutante (RKIPSK153/7EE) und eine Mutante von RKIP generiert, welche bereits unphosphoryliert dimerisiert (RKIP∆143-6). Folgende Ergebnisse legen nahe, dass die Dimerisierung von RKIP für die spezifische Interaktion mit Raf1 bzw. GRK2 entscheidend ist: (i) Die Dimerisierung von phosphoryliertem RKIP ging mit der Dissoziation von RKIP und Raf1 und der Assoziation von RKIP und GRK2 einher; (ii) die Mutanten RKIPSK153/7EE und RKIP∆143-6, die bereits in unstimulierten Zellen eine starke Dimerisierung zeigten, hatten eine höhere Affinität zu GRK2 als zu Raf1; (iii) die Hemmung der RKIP-Dimerisierung interferierte nur mit der RKIP/GKR2- aber nicht mit der RKIP/Raf1-Interaktion; (iv) in vitro und in Mausherzen konnte ein RKIP- und GRK2-immunreaktiver Komplex nachgewiesen werden; (v) Untersuchungen zur RKIP-vermittelten Hemmung der Kinaseaktivität von GRK2 und Raf implizierten, dass dimerisiertes RKIP nur die Aktivität von GRK2, nicht aber von Raf hemmt. Diese Arbeit zeigt, dass die phosphorylierungsinduzierte Dimerisierung von RKIP die spezifische Interaktion von RKIP mit Raf1 und GRK2 koordiniert. Die Aufklärung dieses Mechanismus erweitert unser Verständnis der spezifischen Interaktion von Kinasen mit ihren Regulatorproteinen. / RKIP is a regulator of several distinct kinases and modulates diverse signal transduction cascades such as the signaling of G protein coupled receptors, of the Raf/MEK/ERK-cascade, of the transcription factor NFκB, and of GSK3β. Until now, it was not well understood how the specific interaction of RKIP with its diverse targets is achieved and regulated. Raf1 and GRK2 are the only known direct interaction partners of RKIP and were thus chosen to untangle the molecular mechanisms regulating the specific interaction of RKIP with these kinases. In this dissertation it is shown that RKIP dimerizes upon PKC-mediated phosphorylation of serine153 and that this dimerization is essential for RKIP/Raf1-dissociation and RKIP/GRK2-association. Co-immunoprecipitation experiments with a phosphorylation-deficient mutant revealed that the dimerization of RKIP requires the phosphorylation of two RKIP molecules. The amino acids 127-146 of RKIP were identified as dimer-interface, since RKIP-dimerization was efficiently and specifically inhibited by the peptide RKIP127-146. To elucidate the implication of this phosphorylation-induced dimerization on the target specificity of RKIP, a phosphomimetic RKIP mutant (RKIPSK153/7EE) and a dimeric RKIP mutant (RKIP∆143-6) were generated. The following results indicated that dimerization of RKIP controls its specific interaction with Raf1 or GRK2, respectively: (i) dimerization of phosphorylated RKIP occurred concomitantly with the release of RKIP from Raf1 and its association with GRK2; (ii) the RKIP mutants RKIPSK153/7EE and RKIP∆143-6, which had a higher propensity for RKIP-dimerization already under basal conditions, had a higher affinity to GRK2 than to Raf1; (iii) inhibition of RKIP-dimerization prevented only RKIP/GRK2-binding but did not interfere with RKIP/Raf1-binding; (iv) an RKIP- and GRK2-immunoreactive complex was detected in vitro as well as in mouse hearts; (v) analyses of RKIP-mediated inhibition of GRK2 and Raf showed that a higher propensity for RKIP-dimerization translates into efficient GRK2-inhibition but not into Raf-inhibition. The results of this thesis show that phosphorylation-induced dimerization of RKIP regulates its specific interaction with Raf1 and GRK2. The elucidation of this mechanism improves our understanding how specificity in the interaction of kinases and their regulatory proteins can be achieved.

The superfamily of G protein-coupled receptors (GPCR) regulates numerous physiological and pathophysiological processes. Hence GPCRs are of significant interest for pharmacological therapy. Embedded into cytoplasmic membranes, GPCRs represent the core of large signaling complexes, which are critical for transduction of exogenous stimuli towards activation of downstream signaling pathways. As a member of the GPCR family B, the parathyroid hormone receptor (PTHR) activates adenylyl cyclases, phospholipases C β as well as mitogen-activated protein kinase-dependent signaling pathways, thereby mediating endocrine and paracrine effects of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), respectively. This regulates, calcium homeostasis, bone metabolism and bone development. Paradoxically, PTH is able to induce both catabolic and anabolic bone metabolism. The anabolic effect of PTH is successfully applied in the therapy of severe osteoporosis. Domination of anabolic or catabolic bone-metabolism is entailed by temporal and cell-type specific determinants. The molecular bases are presumably differential arrangements of adaptor proteins within large signaling complexes that may lead to differential activation of signaling pathways, thereby regulating physiological effects. The molecular mechanisms are largely unclear; thus, there is significant interest in revealing a better understanding of PTHR-related adaptor proteins. To identify novel adaptor proteins which direct PTHR signaling pathways, a proteomic screening approach was developed. In this screening, vav2, a guanine-nucleotide exchange factor (GEF) for small GTPases which regulates cytoskeleton reorganization, was found to interact with intracellular domains of PTHR. Evidence is provided that vav2 impairs PTH-mediated phospholipase C β (PLCβ) signaling pathways by competitive interactions with G protein αq subunits. Vice versa, PTH was shown to regulate phosphorylation and subsequent GEF activity of vav2. These findings may thus shed new light on the molecular mechanisms underlying the effects of PTH on bone metabolism by PLC-signaling, cell migration and cytoskeleton organization. In addition to the understanding of intracellular molecular signaling processes, screening for ligands is a fundamental and demanding prerequisite for modern drug development. To this end, ligand binding assays represent a fundamental technique. As a substitution for expensive and potentially harmful radioligand binding, fluorescence-based ligand-binding assays for PTHR were developed in this work. Based on time-resolved fluorescence, several assay variants were established to facilitate drug development for the PTHR. / Die Superfamilie der G-Protein-gekoppelten Rezeptoren (GPCRs) reguliert eine Vielzahl von physiologischen und pathophysiologischen Prozessen, was sie bedeutend für die Pharmakotherapie macht. Eingebettet in die Zytoplasmamembran sind GPCRs das Zentrum von Signalkomplexen, die eine Transduktion äußerer Stimuli zur Aktivierung von nachgeschalteten Signalwegen ermöglichen. Der zur Familie B der GPCRs gehörige Parathormon-Rezeptor (PTHR) aktiviert Adenylyl-Zyklasen-, Phospholipasen Cβ- und Mitogen-aktivierte Proteinkinase (MAPK)-abhängige Signalwege, wodurch endokrine und parakrine Wirkungen des Parathormons (PTH) und des Parathormon-ähnlichen Peptides (PTHrP) vermittelt werden. Dies ermöglicht die Regulation der Calcium-Homöostase, des Knochenmetabolismus und der Knochenentwicklung. Paradoxerweise kann PTH sowohl katabole als auch anabole Effekte auf den Knochenstoffwechsel induzieren. Den anabolen Effekt von PTH nutzt man erfolgreich in der Therapie der schweren Osteoporose. Ob ein anaboler oder kataboler Knochenmetabolismus überwiegt, wird durch zeitliche und Zelltyp-spezifische Faktoren bestimmt. Dem zugrunde liegt vermutlich unter anderem eine differenzielle Anordnung verschiedener Adapterproteine innerhalb der Signalkomplexe, die zur differenziellen Aktivierung von Signalwegen führen und so eine Steuerung bestimmter physiologischer Effekte ermöglichen. Die molekularen Mechanismen sind jedoch noch weitgehend unklar, weshalb großes Interesse besteht, ein besseres Verständnis über die PTHR-assoziierten Adapterproteine zu entwickeln. Zur Identifizierung neuer Adapterproteine, die PTHR-Signalwege beeinflussen, wurde in dieser Arbeit ein auf dem Proteom-basierender Screening-Ansatz entwickelt. Dieser führte zur Entdeckung einer Interaktion von intrazellulären Domänen des PTHR mit vav2, einem Guanin-Nukleotid Austauschfaktor (GEF) für kleine GTPasen, der die Zytoskelett-Reorganisation steuert. Des Weiteren wurde nachgewiesen, dass vav2 über kompetitive Interaktionen mit G Protein αq Untereinheiten PTH-vermittelte Phospholipase Cβ (PLCβ)-abhängige Signalwege beeinflusst. Umgekehrt wurde gezeigt, dass PTH die Phosphorylierung und damit die GEF Aktivität von vav2 reguliert. Diese Befunde können Aufschluss über molekulare Mechanismen geben, die den Wirkungen von PTH auf den Knochenstoffwechsel durch PLC-Signalwege, Zellmigration und Zytoskelett-Reorganisation zugrunde liegen. Neben dem Verständnis über molekulare Prozesse der intrazellulären Signalgebung ist die Suche nach Liganden eine herausfordernde Grundvoraussetzung für die aktuelle Arzneistoffentwicklung. Liganden-Bindungs-Experimente stellen dafür elementare Techniken dar. Zur Substitution kostenintensiver und potentiell gesundheitsschädlicher Radioliganden-Bindungen, wurden in dieser Arbeit Fluoreszenz-basierte Liganden-Bindungs-Experimente für den PTHR entwickelt. Basierend auf Zeit-aufgelöster Fluoreszenz wurden mehrere Varianten dieser Experimente etabliert, um die Arzneistoffentwicklung am PTHR zu unterstützen.

Phototransduction is a well characterized system for study of G protein coupled receptor (GPCR) signaling. The GPCR rhodopsin couples to the heterotrimeric G protein transducin. Light-stimulated activation of transducin in turn activates phosphodiesterase (PDE), leading to closure to cGMP-gated channels and inhibition of glutamate release. Rod and cone photoreceptors are highly polarized neurons consisting of the outer segment (OS) where phototransduction biochemistry occurs, the inner segment containing mitochondria and other organelles, the nuclear layer, an axon, and a glutamatergic synapse. Upon illumination, activated G protein transducin redistributes from the rod OS (where it is localized in the dark) to the inner compartments of the cell. Interestingly, cone transducin does not translocate in light. Opposite to this, visual arrestin migrates from the inner compartments to the OS, where it binds to rhodopsin. Previous reports from other groups and our lab argue for either an active or passive mechanism for transducin and arrestin redistribution. Our lab has shown that arrestin migration occurs by diffusion which is restricted by molecular sinks (Nair et al, 2005b). The focus of my dissertation was to unravel the molecular mechanism of rod transducin translocation. Specifically, I found energy (ATP) was not required for transducin movement within photoreceptors. Also, I found that the disc membranes of the rod outer segments as well as protein-protein interactions with retinal guanylate cyclase serve to restrict transducin diffusion through the cell. In addition, I used the insights gained from these studies of transducin to re-examine the relationship of other G proteins' subcellular localization and signal transduction. Ultimately, I found that most G proteins do not undergo subunit dissociation under physiological activating conditions.

Cell adhesion and migration play crucial roles in development of multicellular organisms, immune surveillance, wound repair and cancer metastasis. The Gβγ subunits of heterotrimeric G-proteins have been implicated in signalling activities that promote cell adhesion and migration but the molecular mechanisms are unclear. Using a mass-spectrometry based proteomic approach we identified a protein complex between Gβγ and Rap1a that is bridged by a novel Rap1 effector, Radil. Overexpression of constitutively active Rap1a, Gβγ or stimulation of cells with the GPCR ligand fMLP triggers recruitment of Radil to the plasma membrane. Exogenous expression of Radil promotes cell spreading through Rap1-dependent inside-out activation of integrins leading to enhanced cell-matrix adhesion. Structure function experiments demonstrated that the RA and PDZ domains of Radil are required for its ability to promote cell adhesion. Using phage-display and mass-spectrometry we identified the kinesin family protein KIF14 as a novel interacting partner for Radil. Both KIF14 and Radil colocalized on microtubules in a PDZ-dependent manner. Depletion of KIF14 or disruption of microtubules led to accumulation of Radil at the cell membrane. Functionally, KIF14 is a negative regulator of Radil signalling as its depletion increased cell spreading and integrin activation and both phenotypes are rescued by simultaneous knockdown of Radil. Knockdown of KIF14 affects focal adhesion dynamics, which we determined is due to delayed adhesion disassembly. Depletion of either KIF14 or Radil dramatically decreased breast cancer cell migration and invasion in vitro. Additionally, knockdown of Radil compromised the ability of cells to metastasize to the lung and reduced tu-mor growth in xenograft mouse models. Collectively, these studies describe a functional re-quirement for the Gβγ-Rap1a-Radil complex during GPCR signalling for the control of integrin-mediated cell adhesion, cell motility and cancer progression.