La constriction apicale des cellules du mésoderme et l'intercalation des cellules de l'ectoderme sont contrôlées par des réseaux contractiles d'acto-myosine dans l'embryon de Drosophile. Le niveau d'activation et la polarisation du cytosquelette d'acto-myosine détermine la nature des déformations cellulaires observées. Nous montrons que le GPCR Smog et les protéines G (Gα,Gβγ) en aval, activent la signalisation Rho1 et donc la Myosine-II dans les deux tissus. Dans l'ectoderme, Gα12/13 active Rho1 à la membrane apicale (aussi appelé compartiment médio-apical) tandis que les sous-unités Gβ13F-Gγ1 activent Rho1 en médio-apical et aux jonctions cellulaires. Les mécanismes contrôlant l’activation polarisée de Rho1 dans ce tissu demeurent incompris. Nous montrons ici que deux RhoGEFs, RhoGEF2 et une nouvelle RhoGEF Wireless/p114RhoGEF, activent Rho1 sous le contrôle des protéines G dans l’ectoderme. RhoGEF2 stimule Rho1 en médio-apical sous la dépendance de Gα12/13 alors que Wireless/p114RhoGEF contrôle l’activité de Rho1 aux jonctions avec Gβ13F-Gγ1. RhoGEF2 est présente aux jonctions et en médio-apical tandis que Wireless/p114RhoGEF est uniquement jonctionnelle où elle est recrutée par Gβ13F-Gγ1. Pour finir, Wireless/p114RhoGEF est absente des jonctions dans les cellules du mésoderme. En résumé, des GPCRs contrôlent l’activité spatio-temporelle de Rho1 au moyen de deux modules régulatoires dans l’ectoderme. Les protéines G transduisent le signal en recrutant et en activant deux RhoGEFs complémentaires en médio-apical et aux jonctions. Une variation dans la nature des GPCRs, protéines G ou des RhoGEFs détermine le contrôle tissu-spécifique de Rho1 au cours de la morphogenèse. / Cell apical constriction in the mesoderm and cell intercalation in the ectoderm are controlled by contractile actomyosin networks in the developing Drosophila embryo. The extent of both actomyosin activation and polarization determines the nature of these cell deformations. We find that the GPCR Smog and the downstream G proteins (Gα,Gβγ) activate Rho1 signaling and thereby myosin-II in both tissues. In the ectoderm, Gα12/13 activates Rho1 at the apical membrane (also called medial-apical compartment) while Gβ13F-Gγ1 subunits promote Rho1 activity at the apical membrane and at cell junctions. How such a polarized activation of Rho1 is achieved remains unclear. Here, we show that two RhoGEFs, RhoGEF2 and a previously uncharacterized RhoGEF Wireless/p114RhoGEF, control Rho1 activity downstream of G proteins in the ectoderm. RhoGEF2 activates medial-apical Rho1 under control of Gα12/13 and Wireless/p114RhoGEF is required to mediate Gβ13F-Gγ1-dependent activation of Rho1 at junctions. RhoGEF2 is present both at junctions and at the apical membrane. In contrast, Wireless/p114RhoGEF only localizes at junctions together with Gβ13F-Gγ1 which recruit the GEF. Finally, we show that Wireless/p114RhoGEF is absent from junctions in the mesoderm. Collectively, GPCRs shape Rho1 activity through distinct biochemical modules in the ectoderm. Heterotrimeric G proteins transduce the signal by recruiting and activating two complementary RhoGEFs apically and at junctions. Variation in type of GPCRs, G proteins or RhoGEFs underlie the tissue-specific control of Rho1 signaling during morphogenesis.

Gegenstand der Arbeit ist eine umfassende Charakterisierung von Struktur und Funktion des G-Protein-gekoppelten Rezeptors GPR34 auf genomischer, mRNA und Proteinebene, die mögliche Rückschlüsse auf physiologische Funktionen oder den natürlichen Agonisten zulassen. Dazu wurde die genomische Organisation des GPR34 in Mensch, Maus und Ratte analysiert und festgestellt, dass neben der intronlosen kodierenden Sequenz auch der 5´-Bereich des GPR34 mit seiner nicht-kodierenden Intron-Exon-Struktur stark konserviert ist. Es wurden in der Ratte und der Maus mindestens zwei, beim Menschen ein putativer Transkriptionsstart identifiziert. Beim Menschen konnte ein kryptisches Intron innerhalb der kodierenden Sequenz des GPR34 gefunden werden, was zu einer Verkürzung des N-Terminus um 47 Aminosäuren führt. Auf der Transkriptionsebene wurde der GPR34 und der GPR34-like Rezeptor im Haushuhn (Gallus gallus) und die GPR34-Expression in der Ratte mittels quantitativer RT-PCR analysiert und die ubiquitäre Gewebeverteilung des Rezeptors bestätigt. Beim menschlichen GPR34 konnte festgestellt werden, dass die fünf putativen Translationsstarts innerhalb der kodierenden Sequenz auch als solche funktionstüchtig sind und der zweite Translationsstart bevorzugt genutzt wird. Die genetische Variabilität des GPR34 in der menschlichen Population ist sehr gering. Es konnte innerhalb einer weltweiten DNA-Probensammlung nur ein einziges Mal eine Mutation in der kodierenden Sequenz lokalisiert werden. Mithilfe des während dieser Arbeit entstandenen Mausmodelles ist eine weitere Charakterisierung der physiologischen Relevanz des GPR34 möglich.:Bibliographische Beschreibung 4 1 Einleitung 5 1.1 Das GPCR-Repertoire in Vertebraten 5 1.1.1 Signaltransduktion 5 1.1.2 GPCR - Klassifikation 6 1.1.3 „Orphan“ G-Protein-gekoppelte Rezeptoren 7 1.2 Die Familie der Purin- und Pyrimidinrezeptoren 8 1.2.1 Strukturelle Besonderheiten der P2Y-Rezeptoren 8 1.2.2 Rolle der Nukleotidrezeptoren im kardiovaskulären System 9 1.3 GPR34 - ein „orphan“ G-Protein-gekoppelter Rezeptor 10 1.3.1 Gewebeexpression des GPR34 10 1.3.2 Genomische Organisation und Proteinstruktur des GPR34 11 1.3.3 Lysophosphatidyl-L-Serin – möglicher Agonist des GPR34 12 1.4 GPR34–like ein verwandter „orphan“ G-Protein-gekoppelter Rezeptor 15 2 Zielstellung 16 3 Originalarbeit 17 4 Ergänzungen zur vorgenannten Originalarbeit 29 4.1 Vergleich der Expression von GPR34 und GPR34-like Rezeptoren im Huhn 29 4.2 Populationsgenetische Untersuchungen des humanen GPR34 30 5 Zusammenfassung der Arbeit 31 5.1 Spezifische Promotoraktivität in verschiedenen Organen der Maus 31 5.2 Alternative Translationsstarts im hGPR34 32 5.3 Geringe allelische Variabilität des GPR34-Gens in der menschlichen Population 33 5.4 Ubiquitäre Expression von GPR34 und GPR34-like Rezeptoren 34 5.5 Etabliertes Mausmodell ist vital und fortpflanzungsfähig 34 6 Referenzen 36 Anlagen 38 Erklärung über die eigenständige Abfassung der Arbeit 38 Eigene Publikationen 39 Danksagung 42

Au cours de la gastrulation de l’embryon de Drosophile, l’activation apicale du cytosquelette d’acto-myosine orchestre la constriction apicale dans le mésoderme en invagination ainsi que l’intercalation cellulaire dans l’ectoderme en extension. Un contrôle quantitatif de l’activité des GPCRs et, par conséquent, de l’activation de Rho1 est à l’origine des différences de déformation des cellules du mésoderme et de l’ectoderme mais ces mécanismes demeurent incompris. L’activité du GPCR Smog se concentre respectivement en deux compartiments distincts à la surface de la membrane plasmique (PM) et dans ses invaginations (PMI). Au moyen de la FCS, nous avons étudié la surface de la PM et pu montrer que Smog oligomérise en homo-clusters en réponse à son activation par le ligand Fog. L’endocytose de Smog est facilitée par la kinase Gprk2 et sa protéine adaptatrice la β-Arrestine-2 qui retire Smog actif de la PM. Lorsque que la concentration de Fog est élevée ou que l’endocytose est réduite, Smog s’organise en homo-clusters et s’accumule au niveau des PMI qui agissent comme des centres d’activation de Rho1. Une concentration plus importante d’homo-clusters de Smog et un nombre plus important PMI dans le mésoderme par comparaison avec l’ectoderme. Répartition dynamique de Smog actif à la surface de la PM ou dans ses invaginations impacte directement sur la signalisation Rho1. Les PMI accumulent de hauts niveaux de Rho1-GTP suggérant qu’elles forment des centres de signalisation. La concentration de Fog et l’endocytose de Smog sont des processus régulateurs couplés qui contrôlent la différence quantitative d’activation de Rho1 dans le mésoderme et l’ectoderme de la Drosophile. / During Drosophila gastrulation, apical activation of the actomyosin networks drives apical constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Here, we show that cell-surface G-protein coupled receptor, Smog activates G-proteins, Rho1 and Rho-kinase that is required for apical constriction and cell-cell intercalation. Quantitative control over GPCR activity and thereby Rho1 activation underlies differences in deformation of the mesoderm and ectoderm cells but the mechanisms remain elusive. We show that GPCR-Smog activity is concentrated on two different apical plasma membrane compartments i.e. the surface and the plasma membrane invaginations. Using FCS, we probe the surface of the plasma membrane (PM) and show that Smog homo-clusters in response to its activating ligand Fog. Endocytosis of Smog is facilitated by the kinase Gprk2 and the adaptor protein β-Arrestin-2 that clears active Smog from the surface of PM. When Fog concentration is high or endocytosis is low, Smog arranges in homo-clusters and accumulates in plasma membrane invaginations (PMI), that are hubs for Rho1 activation. Lastly, we find high Smog homo-cluster concentrations and numerous apical PMIs in the mesoderm compared to the ectoderm. We identify that dynamic partitioning of active Smog on the surface of the PM or PMI directly impact on Rho1 signaling. PMIs accumulate high Rho1-GTP suggesting they form signaling centers. Fog concentration and Smog endocytosis form coupled regulatory processes that regulate quantitative differential Rho1/MyoII activation in the Drosophila mesoderm and ectoderm.

G protein-coupled receptors (GPCRs) represent the largest membrane protein family and a significant target class for therapeutics. Receptors from GPCRs’ largest class, class A, influence virtually every aspect of human physiology. About 45% of the members of this family endogenously bind flexible peptides or peptides segments within larger protein ligands. While many of these peptides have been structurally characterized in their solution state, the few studies of peptides in their receptor-bound state suggest that these peptides interact with a shared set of residues and undergo significant conformational changes. For the purpose of understanding binding dynamics and the development of peptidomimetic drug compounds, further studies should investigate the peptide ligands that are complexed to their cognate receptor.

Serving as key players in cell signaling, nearly all cells in the human body contain GPCRs. As the largest and most diverse superfamily of proteins in the human body, GPCRs are linked to some of the most prevalent current disease states including cardiovascular disease, type II diabetes, and various types of cancers. The development of new biosensors capable of simple, specific, sensitive, high-throughput screenings of the ligand-binding events of GPCRs are crucial to the diagnosis and maintenance of such diseases. To this end, this research is focused on the development of a novel biosensor platform incorporating ICCRs reconstituted into BLMs. Although ICCRs have been expressed previously in oocytes and HEK293 cells, no occurrence of cell-free expression has yet been performed. The advantages of such a platform include the specificity and real-time measurement capabilities of GPCRs, the innate sensitivity of electrophysiological ion channel flux measurements, and the simplified cellular mimicking of the BLM and cell-free expression. The majority of the presented research was based in the molecular cloning of M2Kir6.2, an ICCR incorporating a muscarinic acetylcholine receptor (M2), from Xenopus oocyte vector pGH2 into cell-free expression vector pT7CFE1-CGST-HA-His. Much optimization of the cloning procedure (PCR, digestion, and ligation) was necessary involving studies into polymerase fidelity, inclusion of DpnI for degradation of methylated DNA, and ligation parameter alterations in time, temperature, and insert:vector ratios. It was discovered that Deep VentR polymerase was beneficial to preventing mutations within the sequence of M2Kir6.2 during PCR, DpnI was capable of degrading unwanted residual M2Kir6.2 pGH2, and ligation performance was optimal using a 1:1 (insert:vector) ratio and reaction time and temperature of 18 h and 4 °C, respectively. With the successful ligation of M2Kir6.2 into cell-free expression vector pT7, expression of the ICCR via cell-free expression lysate kit was performed with direct reconstitution into a micropipette suspended BLM attempted. Five reconstitution trials were performed with electrophysiological single-channel recording results suggesting ICCR insertion based on ion channel currents of ~ 3 pA and mean open-times of ~ 3 ms observed corresponding to literature values for native Kir6.2 channels. Additionally, a Western blot analysis of the cell-free expression mixture contained products with molecular weights corresponding to monomer (~ 100 kDa) and tetramer (~ 400 kDa) constructs of M2Kir6.2. With the successful optimization of the M2Kir6.2 pT7 cloning procedure, this procedure can be used in future cloning attempts with similar ICCR constructs, such as D2Kir6.2. Although preliminary electrophysiological results suggest ICCR expression and BLM reconstitution, further work needs to be done in controlling the amount of ICCR insertion and optimizing BLM stability. Additionally, in order to confirm the functionality of both M2 and Kir6.2 ligand dose response curves must be performed. The evidence supporting ICCR expression and direct reconstitution into suspended BLMs via cell-free protein expression is both exciting and promising. Not only has this research involved the first cell-free expression of M2Kir6.2 but also has great benefits to the further development of such novel ligand-binding biosensor platforms.

Die meisten membranständigen Rezeptoren in Säugetieren sind G-Protein-gekoppelte Rezeptoren (GPCR). Sie sind entscheidend beteiligt an der Verarbeitung von sensorischen Reizen, an der Wirkung von Signalmolekülen, an Zellwachstum, Zellreifung und Entzündungsprozessen. Dennoch sind für viele GPCR die molekularen und auch physiologischen Funktionen nicht bekannt. Die Erforschung dieser Rezeptorproteine ist von großer medizinischer Relevanz, da deren Fehlfunktion eine mögliche Ursache für Krankheiten ist und sich GPCR häufig als pharmakologische Zielmoleküle eignen. Mit 33 Mitgliedern bilden die Adhesion GPCR die zweitgrößte Klasse von GPCR im Menschen, die bisher allerdings am wenigsten erforscht wurde. Sie sind bedeutsam für das Immunsystem, die zelluläre Organisation während der Embryonalentwicklung, die Angiogenese und die Entstehung von Tumoren. Adhesion GPCR besitzen im Transmembranbereich die typische Struktur von GPCR, unterscheiden sich jedoch von klassischen GPCR aufgrund ihrer sehr großen und komplexen N Termini. Die Frage, ob diese Rezeptorfamilie funktionell an G-Proteine koppelt, blieb lange unbeantwortet. Für einige wenige Rezeptoren der Subfamilie wurde die Bindung von Liganden beschrieben, dennoch fehlt für diese bisher der Beweis einer Rezeptoraktivierung. Unsere Arbeitsgruppe konnte zum ersten Mal eine Gαs vermittelte Adenylylcyclase (AC) Aktivierung durch den Adhesion GPCR GPR133 zeigen. Aufbauend auf diesen Ergebnissen wurden in dieser Arbeit die Signalkopplungswege der drei Adhesion GPCR GPR126, GPR56 und CD97 mittels verschiedener in vitro Methoden untersucht, um eine G-Protein-vermittelte Kopplung zu beweisen und weiter zu spezifizieren. Eine Grundlage bildeten die erfolgreiche Entwicklung einer Klonierungsstrategie und der Nachweis einer suffizienten Expression der Adhesion GPCR sowohl intrazellulär, als auch an der Membran. Durch die N-terminale Integration eines Rhodopsin Epitops gelang eine Steigerung der Zellmembranexpression für GPR126 und GPR56. Weiterhin wurden eine funktionelle Kopplung von GPR126 und GPR56 an das Gs-Protein sowie von GPR126 an das Gi-Protein bewiesen. Da eine G-Protein-gekoppelte Signaltransduktion somit für zwei weitere Adhesion GPCR gezeigt werden konnte, kann diese als etabliert für die gesamte Rezeptorklasse gelten.

Les récepteurs couplés aux protéines G (RCPG) constituent la plus grande famille de protéines transmembranaires et régulent une panoplie de processus physiologiques. La densité de récepteurs exprimés à la surface d'une cellule dicte l’intensité de la réponse cellulaire suite à l’activation par un ligand. Un débalancement dans les niveaux d’expressions protéiques est la cause de plusieurs pathologies. C’est pourquoi il est d’une importance capitale de comprendre les mécanismes qui régissent les niveaux d’expressions totaux et membranaires des RCPG. Dans un premier temps, nos travaux ont permis de caractériser la dynamique d’internalisation et de recyclage du récepteur CRTH2. Nous avons identifié les domaines qui sont impliqués dans la régulation du trafic du récepteur. Des essais d’expression de surface ont révélé les cibles majeures des GRK2, GRK5, de la PKC et de l’arrestine-3 pour l’internalisation de CRTH2 suite à une stimulation du récepteur. De plus, nous rapportons la présence d’un motif de recyclage interne de type PDZ dans la queue cytoplasmique de CRTH2. Avant de se rendre à la membrane plasmique, les RCPG doivent être synthétisés au réticulum endoplasmique (RE), subir des étapes de maturation au Golgi et être transportés à la membrane plasmique. Les évènements coordonnant ces processus sont encore très peu étudiés. La dernière partie de nos travaux a permis d’élucider différents mécanismes régulant le contrôle qualité des RCPG à un stade précoce de synthèse. Tout d’abord, une nouvelle stratégie de purification protéomique a été développée et validée dans le but d’identifier des nouveaux partenaires d’interaction des RCPG dans un contexte cellulaire. Les résultats des criblages ont montré une interaction entre le récepteur [beta indice inférieur 2]AR et l’ubiqutine ligase RNF5. Ceci a conduit à la découverte d’un nouveau mécanisme de contrôle qualité des RCPG faisant intervenir les protéines RNF5 et JAMP comme étant des composantes importantes d’un système de régulation qui module la stabilité des RCPG. En dernier lieu, une nouvelle protéine, ANKRD13A, régulerait l’expression des RCPG par un mécanisme encore inconnu. Des évidences nous permettent de croire que les protéines RNF5, JAMP et ANKRD13A feraient partie d’un complexe protéique régulant le contrôle qualité des RCPG au RE.

GPCR accessory proteins regulate the strength, efficiency and specificity of signal transfer upon receptor activation. Due to the inherent difficulties of studying membrane proteins in vitro and in vivo, little is known about the structure and topology of these small accessory proteins. Two examples of GPCR accessory proteins are the Melanocortin-2 receptor accessory protein (MRAP) and the Receptor-activity-modifying protein (RAMP) family. MRAP and RAMP1 are the main focus of this thesis in which they are thoroughly characterised by solution-state NMR and further biophysical techniques. The single-pass transmembrane domain protein MRAP regulates the class A GPCR melanocortin receptors. It is specifically required for trafficking the melanocortin-2-receptor from the endoplasmic reticulum to the cell surface and subsequent receptor activation. A remarkable characteristic of MRAP is its proposed native dual-topology, which leads to an antiparallel homodimeric conformation. Investigation of the biochemical and biophysical properties of MRAP revealed an α-helical transmembrane domain, and an α-helical N-terminal LD(Y/I)L-motif. Further efforts concentrated on establishing the homodimeric conformation of MRAP in vitro. RAMP1 facilitates receptor trafficking and alters the ligand specificity of the GPCR Class B receptors calcitonin receptors and calcitonin receptor-like receptors. Moreover, RAMP1 is required to act as a Calcitonin-gene-related peptide (CGRP) receptor (RAMP1). RAMP1 has been shown to form stable parallel homodimers in the absence of its cognate receptor. Its dimerisation and the possible dimerisation motif PxxxxP-motif were studied extensively. With the goal of understanding the mechanism of dimerisation and the role of GPCR accessory proteins I have used solution-state NMR in detergent micelles as my main technique. NMR provides unique possibilities for understanding the structure and dynamics of such small membrane proteins.

<p>The neuropeptides orexin-A and -B act as endogenous ligands for G-protein-coupled receptors (GPCRs) called OX₁ and OX₂ receptors. Previous observations have established that orexin receptors have an ability to couple to different G-proteins and signaling pathways and induce Ca²⁺ elevations via both receptor-operated Ca²⁺ channels (ROCs) and store-operated Ca²⁺ channels (SOCs). This thesis further elucidates the intracellular signaling mechanisms of orexin receptors.</p><p>Orexin receptors were shown to activate ERK (extracellular signal-regulated kinase) via Ras, protein kinase C, phosphatidylinositol-3 kinase and Src. Ca²⁺ influx was shown to be obligatory for the activation of ERK and adenylyl cyclase, wherewith a hypothesis was formed that submembrane Ca²⁺ elevation is of central importance for the regulation of orexin receptors' coupling to different signaling pathways. This was further investigated with respect to OX₁R-mediated activation of phospholipase C (PLC) showing that ROC influx was of more central importance for the OX₁R signaling, but also SOCs amplified PLC activity. A technique to block OX₁R-induced IP₃increase and subsequent Ca²⁺ release was devised, leaving ROC influx as the only source of Ca²⁺ elevation upon OX₁R activation. This block had no effect on OX₁R-mediated activation of ERK, showing that ROC-dependent influx is the most central Ca²⁺ elevating process in OX₁R signaling. OX₁Rs' coupling to PLC was further investigated by measuring the metabolites generated, inositol phosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities, which results in, at low orexin-A concentrations, DAG production without concomitant production of IP₃. At even lower orexin-A concentrations, OX₁Rs generate DAG by activating phospholipase D. In conclusion, the results strengthen the hypothesis that ROCs have a central role in orexin receptor signaling and DAG may be the signal of preference.</p>

Cancer is lacking early detection methods and treatment specificity. In order to increase the sensitivity and specificity towards cancer cells, we propose the use of multivalent interactions targeting specific receptor combinations at the cancer cell surface. In this thesis, we explored the design of multimers, which could provide such interactions. The design was investigated and revisited based on specific parameters, essential for the creation of multivalent interactions such as thermodynamics. The synthesis was designed so that libraries of homo- and hetero-multimers of different valencies can be obtained efficiently with good yields. The established synthetic scheme is empowered by its modularity, necessary to investigate different essential factors. Trimers composed of micromolar affinity MSH(4) targeting the MC1-R, overexpressed in melanoma, were investigated on a model cell line and resulted in the creation of nanomolar affinity constructs with up to 350 fold increase in affinity. Different multimers such as hexavalent and nonavalent dendrimers were synthesized and studied for their properties. All constructs had nanomolar affinity and showed to be non-toxic up to micromolar concentrations and imaging studies also confirmed their internalization, which overall demonstrate the potential for these compounds to be used as markers for cancer cells and as delivery agents. Trimers targeting the CCK2-R were similarly investigated for their potential as pancreatic cancer markers. However, those constructs did not seem to result in the expected enhancements in affinity, but the affinity of the initial monovalent agonist was in the 10-50 nanomolar range. As we were unable to design micromolar affinity agonist we investigated the use of antagonists. This study, revealed the importance of thermodynamics in the creation of multivalent interaction. Heterotrivalent ligands (CCK and MSH) were investigated for their potential in cross-linking different receptors and the study demonstrated the subtility to detect cross-linking. Finally, the different attempts toward the efficient synthesis of a tetra-orthogonal scaffold, a key feature needed to generate multimers that could target up to 3 different receptors was investigated and showed promising results. It is our hypothesis that such an approach will ultimately lead to specific markers of tumor cells, which could be used as diagnosis agents when modified with an imaging moiety and as a therapeutic agent when modified with a drug.