Aktivierte G-Protein-gekoppelte Rezeptoren (GPCR) werden schnell desensitisiert, internalisiert und anschließend entweder lysosomal degradiert oder zur Plasmamembran (PM) recycelt. Zur Resensitisierung der Zellen tragen neben recycelten auch neusynthetisierte Rezeptoren bei. Die Überlagerung beider Prozesse erschwert die Untersuchung des Rezeptorrecyclings. In dieser Arbeit sollte mit Hilfe des photokonvertierbaren Fluoreszenzproteins Kaede eine Technik entwickelt werden, mit der es möglich ist Recycling- von Neusyntheseprozessen zu trennen und das Recycling von GPCR mikroskopisch in Echtzeit zu beobachten. Als Modellproteine wurden der Vasopressin-1a-Rezeptor V1aR (recycelnder Rezeptor), der Vasopressin-2-Rezeptor V2R (degradierter Rezeptor) und der Corticotropin-Releasing Factor-Rezeptor Typ 1 (CRF1R) verwendet, wobei bei Letzterem untersucht werden sollte, ob er nach Stimulation zur PM zurücktransportiert wird. Da Kaede als fluoreszierendes Protein mit den GPCR fusioniert wird, wurde zunächst überprüft, ob es die Eigenschaften der Rezeptoren verändert und generell für Transportstudien geeignet ist. Eventuell könnte die bereits publizierte Tetramerisierung von Kaede seine Anwendung verhindern oder erschweren. Mittels Fluoreszenz-Korrelationsspektroskopie konnte gezeigt werden, dass Kaede nicht tetramerisiert, wenn es an ein Membranprotein fusioniert ist. Außerdem konnte in in vitro- und Zellkulturexperimenten belegt werden, dass die native und die photokonvertierte Form von Kaede gleichermaßen stabil sind. Darüber hinaus zeigten Kaede-fusionierte GPCR sowohl in Kolokalisationsstudien als auch in Agonistbindungs- und Rezeptoraktivierungsexperimenten die gleichen Eigenschaften wie CFP- bzw. die unfusionierte Rezeptoren. Lediglich die Expression der Kaede-fusionierten Rezeptoren war geringer. Parallel wurde anhand der bereits publizierten Kaede-Struktur versucht, die Tetramerisierung des Proteins durch den Austausch interagierender Aminosäuren zu unterbinden. Die eingeführten Mutationen bewirkten aber eine Fehlfaltung des Proteins und damit den Verlust der Fluoreszenz. Da zuvor gezeigt werden konnte, dass Kaede-fusionierte Membranproteine nicht tetramerisieren und nicht die Eigenschaften der fusionierten Proteine verändern, war monomerisiertes Kaede zur Untersuchung des Rezeptorrecyclings nicht notwendig. Im zweiten Teil der Arbeit wurde mit Hilfe von Kaede-Fusionsproteinen und mikroskopischer Testsysteme das noch unbekannte Recyclingverhalten des CRF1R untersucht. Hierfür wurden die Kaede-fusionierten Rezeptoren in eukaryotischen Zellen exprimiert und mit Agonisten internalisiert. Die internalisierten Rezeptoren wurden in Endosomen selektiv mit UV-Strahlung photokonvertiert. Anschließend wurde der Transport der photokonvertierten Form verfolgt. Sowohl beim CRF1R als auch beim V1aR wurden Signale in der PM detektiert, beim V2R hingegen nicht. Dies zeigt, dass es sich beim CRF1R um einen recycelnden Rezeptor handelt. Die als Kontrolle eingesetzten Rezeptoren verhielten sich in diesem Experiment wie erwartet: Der V1aR wurde zur PM zurücktransportiert, der V2R nicht. Diese Ergebnisse konnten mit Hilfe biochemischer und durchflusscytometrischer Experimente bestätigt werden. Die Internalisierung des CRF1R verläuft Clathrin-vermittelt in Anwesenheit von β-Arrestin. Je nach Stabilität der β Arrestin-Interaktion unterscheidet man zwei Klassen von Rezeptoren: Klasse A-Rezeptoren interagieren transient mit β Arrestin und können recyceln. Im Gegensatz dazu gehen Klasse B-Rezeptoren eine stabile Interaktion mit β Arrestin ein und werden nach Internalisierung degradiert. In mikroskopischen Untersuchungen konnte für die aktivierten CRF1R und V1aR eine Rekrutierung von β Arrestin zur PM und eine transiente Interaktion mit β Arrestin gezeigt werden (Klasse A-Rezeptoren). Für den V2R wurde dagegen eine stabile Interaktion mit β Arrestin beobachtet (Klasse B-Rezeptor). Diese Daten stützen die Ergebnisse des Kaede-basierten Recyclingversuchs und zeigen, dass der CRF1R ein recycelnder Rezeptor ist. Ferner wurde untersucht, ob der CRF1R zu den schnell oder langsam recycelnden Rezeptoren zählt. Schnell recycelnde Rezeptoren werden direkt aus frühen Endosomen, langsam recycelnde hingegen über das Trans-Golgi-Netzwerk (TGN) bzw. über Recycling-Endosomen zur PM transportiert. Als Marker für das TGN oder die Recycling-Endosomen wurde Rab11 verwendet. In Kolokalisationsstudien konnte gezeigt werden, dass der CRF1R den langsam recycelnden Rezeptoren zugeordnet werden kann. Zusammenfassend konnte in dieser Arbeit belegt werden, dass Kaede als Fusionspartner für Membranproteine genutzt werden kann um deren Transport in Echtzeit zu studieren. Damit wurde erstmals eine mikroskopische Methode etabliert, die es erlaubt recycelnde von neusynthetisierten Rezeptoren zu unterscheiden. Mit Hilfe dieser Methode war es möglich zu zeigen, dass der CRF1R ein recycelnder Rezeptor ist. / Upon ligand binding and receptor activation, G protein-coupled receptors (GPCR) are rapidly desensitized, internalized and subsequently degraded in lysosomes or recycled back to the plasma membrane. Resensitization of the cell is enabled by both recycling receptors and newly synthesized receptors. The overlap of recycling and synthesis processes largely complicates the study of GPCR recycling mechanisms. One aim of this thesis was to develop a new microscopic technique for real-time visualization of GPCR recycling using the photoconvertible Kaede protein allowing to differentiate newly synthesized from recycling receptors. As model proteins, the V1aR (recycling receptor), the V2R (degraded receptor) and the CRF1R were used. In the case of the CRF1R, it was unknown whether this receptor recycles to the plasma membrane following agonist-promoted internalization. The study of the CRF1R recycling behaviour was another objective of this work. As the Kaede protein is fused C-terminally to the GPCRs, an influence on the pharmacological and trafficking properties of the receptors must be excluded. The previously published tetramerization of Kaede, for example, might hinder or even prevent its usability. To assess for the applicability of Kaede, fluorescence correlation spectroscopy experiments were performed and it was demonstrated that Kaede fused to membrane proteins cannot form tetramers in contrast to the soluble form. In vitro studies and experiments in cell culture revealed that both the native and the photoconverted Kaede are equally stable. Moreover Kaede-fused GPCR displayed the same pharmacological and trafficking properties as the untagged or CFP-tagged receptors. Only the expression levels of the Kaede fusion proteins were reduced, yet this did not affect the microscopic experiments. In parallel to these experiments, the interacting amino acids of the tetrameric Kaede were substituted according to the previously published crystal structure of the protein. Unfortunately, these mutations induced protein misfolding thereby causing loss of fluorescence functions. However, since it could be shown that membrane protein-fused Kaede cannot tetramerize, the monomerized Kaede was no more essential for the microscopic study of receptor recycling. In the second part of this work, Kaede-fusions were used to study the recycling behaviour of the CRF1R and the V1aR and V2R control proteins by the novel real-time recycling assay at the laser scanning microscope. To this end, HEK 293 cells expressing the Kaede-fused receptors were treated with agonist to induce receptor internalization. Internalized receptors were selectively photoconverted in endosomes using UV-irradiation and the subcellular fate of the new fluorescence signals was studied. In the case of the CRF1R, signals of the photoconverted receptors could be detected in the plasma membrane indicating that the CRF1R belongs to the family of recycling receptors. The control receptors showed the expected results: The V1aR recycled back to the plasma membrane whereas the V2R did not. These results were confirmed with biochemical and flow cytometry measurements. The CRF1R internalizes in a clathrin-dependent way via the adaptor protein AP2, dynamin and β arrestin. Depending on the stability of the resulting receptor-β-arrestin-complex, two classes of receptors can be differentiated. Class A receptors are recycling receptors undergoing a more transient β-arrestin interaction. In contrast, class B receptors stably interact with β-arrestin and are degraded after internalization. In the case of the CRF1R and V1aR, microscopic analyzes demonstrated that β arrestin transiently interacts with the stimulated CRF1R and V1aR indicating again that these receptors are recycling GPCRs (class A receptors). The V2R, in contrast, revealed a stable interaction (class B receptor). Moreover, it was studied whether the CRF1R recycles rapidly or more slowly to the plasma membrane. Rapidly recycling receptors are recruited out of early endosomes whereas slowly recycling receptors pass the trans-golgi-network or recycling endosomes before reaching the cell surface. Rab11 colocalization studies demonstrated that the CRF1R belongs to the family of slowly recycling receptors. In conclusion, a novel microscopic technique was established allowing to study GPCR recycling in real-time and to differentiate recycling and synthesis processes. Moreover, it was shown that the CRF1R belongs to the family of recycling receptors. The Kaede technique seems to be very well suited to study membrane protein trafficking in general.

Les récepteurs de chimiokines sont des régulateurs essentiels de la migration cellulaire dans le cadre de la surveillance immunitaire, et le développement. Les récepteurs CCR5 et CXCR4 sont de plus spécifiquement impliqués dans les métastases cancéreuses et l'infection par le VIH. Nous avons développé un système permettant de sur-exprimer ces deux RCPGs. Afin de s'affranchir des problèmes de toxicité inhérents à l'expression des protéines membranaires en bactérie notre approche de production consiste à adresser les protéines vers les corps d'inclusion d'E. coli grâce à une fusion protéique N-terminale permettant de hauts niveaux d'expression. Après purification en conditions dénaturantes, les protéines sont alors repliées en présence de surfactants originaux, les amphipoles. La validation de cette nouvelle approche pour les récepteurs des chimiokines représente un des objectifs principaux de ce travail. Afin de tester la fonctionnalité des protéines repliées, une série d'outils a été développée : des versions modifiées des chimiokines ont été produites (RANTES pour CCR5 et SDF 1a pour CXCR4). La fonctionnalité des chimiokines a été évaluée au niveau moléculaire et cellulaire. L'interaction entre le récepteur replié en amphipole et son ligand a été testé par résonance de plasmons de surface (SPR). Différents types de surfaces fonctionalisées avec le récepteur de chimiokine replié en amphipole ont été explorés au cours de ce travail. A la fin de ce projet, la production des chimiokines et de leur récepteur a été mise au point. L'accès à ces outils ouvre la voie à de futures études moléculaires telles que la compréhension de la dimérisation du récepteur ou la détermination de la stoechiométrie du complexe. / Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation and development. The GPCRs (G protein-coupled receptors) CCR5 and CXCR4 are specifically implicated in cancer metastasis and HIV-1 infection. An expression system to over-express these two GPCRs was developed. To overcome the toxicity problem of membrane protein expression in bacterial system, the production approach consists in targeting the proteins towards E. coli inclusion bodies thanks to a N-terminal fusion allowing a high yield expression. After purification under denaturing conditions, these GPCRs were then folded using original polymeric surfactants: the amphipols. The validation of this new approach for the chemokine receptor production is one of the goals of this work. In order to assess the functionality of the folded proteins, series of tools have been developed: engineered chemokine ligands (RANTES for CCR5 and SDF1a for CXCR4) were produced. The functionality of chemokines was evaluated at cellular and molecular levels. Interaction between the receptor folded in amphipols and its ligand was evaluated using Surface Plasmon Resonance (SPR) technique. Several types of surfaces, functionalized with the chemokine receptor/amphipol complex have been explored in this work. At the end of this project the productions of chemokines and their receptors has been set up. These established tools open the way to future studies, at the molecular level, in order to, for instance, investigate receptor dimerization and complex stoichiometry.

Forces requises pour les changements de forme au cours de la morphogenèse des tissus sont générés par d’actine et de myosine. Durant ma thèse, je étudié le rôle de la réglementation MyoII par la voie Rho1-Rok durant l’élongation de l’ectoderme ventro-latéral par intercalation cellulaire. Les pulsations de MyoII médio-apicale se déplacent de manière anisotrope vers les jonctions parallèles avec l’axe dorso-ventral (ou jonctions verticales). Ceci provoque le rétrécissement graduel des jonctions qui sont stabilisées par une population de MyoII polarisée dans le plan du tissu et enrichie au niveau de ces jonctions. Les mécanismes cellulaires qui régulent la pulsatilité, la stabilité et la polarité de la myosine II restent à élucider. J’ai identifié deux propriétés cruciales de la dynamique de la myosine II régie par phospho- à savoir la cinétique d’échange gouvernée par les cycles de phosphorylation-déphosphorylation des chaines légères régulatrices de la MyoII (RLC) et l’advection due à la contraction des moteurs sur le réseau de F-actine. Contrôle spatial sur le chiffre d'affaires MyoII établit 2 régimes stables des taux élevés et faibles dissociation résultant dans MyoII polarité. Pulsatilité est un comportement auto-organisé qui émerge à taux de dissociation intermédiaires permettant d'advection MyoII et les régulateurs en amont. Dans la deuxième partie de ma thèse, je l'ai montré que la protéine GPCR- GRsmog et la brume, et la voie G-protéines en aval permettent l'activation progressive des MyoII, établissant pulsatilité et de la stabilité pour produire des déformations de forme polarisées cours de la morphogenèse. / Forces required to power shape changes during tissue morphogenesis are generated by non-muscle MyosinII (MyoII) pulling filamentous actin. During my PhD, I investigated the role of MyoII regulation through the conserved Rho1-Rok pathway during Drosophila germband extension. The morphogenetic process is powered by cell intercalation involving shrinkage of junctions in the dorsal-ventral axis (‘vertical junctions’) followed by junction extension in the anterior-posterior axis. Advances in light microscopy revealed that the actomyosin networks exhibit pulsed contractions to power junction shrinkage, and alternate with steps of stabilization by MyoII enriched on vertical junctions (planar-polarity) to result in irreversible shape changes. Although described in many different contexts, the underlying mechanisms of this ratchet-like behavior remained unclear. Using genetic and biophysical tools, quantitative imaging and subtle perturbations, I identified 2 critical properties underlying MyoII dynamics- turnover governed by phospho-cycling of the MyoII Regulatory Light Chain, and advection due to contraction of the motors on actin networks. Spatial control over MyoII turnover establishes 2 stable regimes of high and low dissociation rates resulting in MyoII planar polarity. Pulsatility is a self-organized behavior that emerges at intermediate dissociation rates enabling advection of MyoII and upstream regulators. In the second part of my thesis, I showed that G protein coupled receptors- GRsmog and Mist, and the downstream G-protein pathway allow step-wise activation of MyoII, establishing pulsatility and stability, to drive polarized shape deformations during morphogenesis.

Die Familie der G-Protein-gekoppelten Rezeptoren (GPCRs) bildet die größte Gruppe von Membranrezeptoren im menschlichen Organismus. Für viele GPCRs sind bisher die physiologischen Funktionen nicht bekannt. Das biologische Verständnis der Funktionen im menschlichen Organismus dieser sogenannten „orphan“ GPCRs (oGPCRs) hat, aufgrund möglicher kausaler Beteiligung an der Pathogenese von Erkrankungen sowie deren therapeutische Beeinflussbarkeit, hohe medizinische Relevanz. Die GPCRs der P2Y12-ähnliche Rezeptorgruppe besitzen eine große physiologische Bedeutung bei der Thrombozytenaggregation und der Induktion der Migration von immunokompetenten Zellen in Schädigungsgebiete. Der ADP-Rezeptor P2Y12 kann durch verschiedene pharmakologische Wirkstoffe beeinflusst werden, was bereits klinisch-therapeutisch genutzt wird. Diese Gruppe von GPCRs enthält jedoch auch Mitglieder, deren Funktionen völlig unbekannt sind. Einer dieser oGPCRs ist der GPR34. Ziel dieser Arbeit war es, mittels verschiedener in-vitro-Methoden und anhand eines GPR34-defizienten Mausstamms die physiologische Relevanz dieses P2Y12-ähnlichen Rezeptors zu analysieren. Dazu wurde ein GPR34-Knockout-Mausmodell etabliert. Die GPR34-Defizienz hatte keinen wesentlichen Einfluss auf die Entwicklung, Morphologie, das Wachstum oder die Fertilität bei Mäusen. Die Ergebnisse aus Immunisierungs– und Infektionsstudien zeigten jedoch, dass dieser evolutionär hoch konservierte Rezeptor eine wichtige Funktion in der Feinkontrolle der zellulären Immunabwehr ausübt. Neben einer verstärkten Antwort im Delayed-type Hypersensitivity (DTH)-Test war die Abwehr einer Cryptococcus-Infektion in diesem GPR34-defizienten Tiermodell beeinträchtigt. Signifikant erhöhte Zytokinspiegel nach Antigen- bzw. Pathogenexposition deuteten auf eine gestörte Immunregulation in GPR34-defizienten Mäusen hin. Weiterführende Untersuchungen sollten sich der Identifizierung des endogenen Agonisten und der Funktion des GPR34 bei der Koordinierung der zellulären Immunreaktion widmen.

Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes.

G protein-coupled receptors (GPCR) are interesting drug targets. In this degree project the focus is on GPR183 and GPR61 which play a role in immune cell trafficking and metabolic regulation respectively. This project aims to test specific mutations from both blood cancer and high obesity samples, with the goal of seeing any different behaviors compared to wild type receptors. Behavior in this case pertains to differences in expression and activation, both constitutively and in the presence of ligands. The project employs a plethora of different physical instruments to obtain these results. The use of Bioluminescence Resonance Energy Transfer (BRET), Fluorescence Resonance Energy Transfer (FRET) and a combination of multiple different assays together form a robust way of measuring both expression and activation for different mutations. The main takeaways for GPR183 were that the mutation, L125P, found in Non-Hodgkin lymphoma samples, differ significantly in both expression and activation compared to wild type. Specifically at the native membrane domain. There were also results that speculated that this mutation might induce misfold, hindering its transportation to the membrane from the inner compartments of the cell.For GPR61, a majority of the mutations investigated showed lower expression compared to the wild type counterpart, and only a few saw a significant reduction in activation. Ligand experiments on GPR61 did not yield any increase in constitutive activity, prompting further assessment in the future. Overall, the project gained interesting results that prompts further investigation of GPR183 mutations, and signaling, specifically in the realm of G_α signaling. The project also narrows down the large magnitude of GPR61 mutations interesting for further research. This provides a solid foundation into GPCR signaling in said diseases. / G-proteinkopplade receptorer (GPCR) är intressanta läkemedelsmål. I detta examensarbete ligger huvudfokus på GPR183 och GPR61 som spelar varsin roll i trafikering av immunceller respektive metabolisk reglering.Detta projekt syftar till att testa specifika mutationer från prover kopplade till blodcancer och hög fetma med målet att se olika beteenden jämfört med vildtyp. Beteende i detta fall hänför sig till skillnader i uttryck och aktivering, både konstitutivt och med närvaro av ligander.Projektet använder en uppsjö av olika fysiska instrument för att få dessa resultat. Användningen av Bioluminescensresonasenergiöverföring (BRET), fluorescensenergiöverföring (FRET) och en kombination av flera andra metoder, bildar de tillsammans ett robust sätt att mäta både uttryck och aktivering för olika mutationer.De viktigaste för GPR183 var att mutationen, L125P, som hittades i non-Hodgkin-lymfom prover, skiljer sig signifikant i både uttryck och aktivering jämfört med vildtyp. Specifikt vid den naturliga membrandomänen. Det fanns också resultat som spekulerade i att denna mutation kan inducera felveckning, vilket hindrar dess transport till membranet från cellens inre avdelningar.För GPR61 visade en majoritet av de undersökta mutanterna lägre uttryck jämfört med vildtypens motsvarighet och endast ett fåtal såg en signifikant minskning av aktivering. Ligandexperiment på GPR61 gav ingen ökning av konstitutiv aktivitet, vilket föranleder till ytterligare bedömningar i framtida experiment.Sammantaget fick projektet intressanta resultat som föranleder ytterligare undersökning av GPR183-mutationer och deras beteenden, särskilt kopplat till G_α signalering. Projektet begränsar också den stora mängden GPR61-mutanter som är intressanta för vidare forskning. Detta ger en solid grund för GPCR-signalering i nämnda sjukdomar.

Níveis elevados de hormônios tireoidianos (HTs) são comumente associados à ativação do sistema renina angiotensina local e ao desenvolvimento da hipertrofia cardíaca. O envolvimento do receptor de angiotensina II tipo 1 (AT1R) nos efeitos hipertróficos dos HTs fora descrito previamente. No entanto, os mecanismos subjacentes a essa interação ainda são desconhecidos. O AT1R pertence à família dos receptores acoplados à proteína G e, portanto, promove a transdução de sinal por mecanismos dependentes e independentes de proteína G. Recentemente, a sinalização dependente de beta-arrestinas (independente de proteína G) tem sido descrita por contribuir com a resposta hipertrófica em diferentes modelos experimentais. Assim, no presente estudo investigou-se o envolvimento da sinalização dependente de beta-arrestinas nos efeitos hipertróficos dos HTs, mediados pelo AT1R, bem como a participação de ERK½ nesse processo. Culturas primárias de cardiomiócitos foram estimuladas com T3 (triiodotironina; 15nM) para indução da hipertrofia. O tratamento dos cardiomiócitos com T3 por tempos rápidos (5-30 min) resultou na ativação transiente de ERK½, a qual foi parcialmente atenuada quando da administração de Losartan (1µM), antagonista do AT1R. A contribuição de ERK½ na hipertrofia dos cardiomiócitos foi verificada através do uso de PD98059 (20µM), inibidor de MEK½, o qual preveniu a transcrição de marcadores hipertróficos. Ensaios de imunoprecipitação revelaram o aumento da interação entre AT1R e beta-arrestina 2 sob estímulo do T3, sugerindo o recrutamento de beta-arrestina 2 e, possível, internalização do AT1R. Através de ensaios de imunofluorescência e fracionamento subcelular, foi demonstrado que o T3 estimula a translocação do AT1R, amentando sua expressão no núcleo dos cardiomiócitos. Além disso, tanto a ativação de ERK½ quanto a hipertrofia cardiomiocítica mostraram-se sensíveis à inibição da endocitose, a qual foi avaliada através de Concanavalina A (0,5µg/ml). Ensaios de silenciamento gênico por RNA de interferência foram eficientes em demonstrar o envolvimento de beta-arrestina 2 na ativação de ERK½ e na hipertrofia cardiomiocítica induzida por T3. Desta forma, os resultados evidenciam o envolvimento da sinalização dependente de beta-arrestina 2 na ativação de ERK½, através do AT1R, a qual contribui com a hipertrofia cardiomiocítica promovida pelo T3. / Elevated levels of thyroid hormones (THs) are commonly associated with activation of the local renin angiotensin system and the development of cardiac hypertrophy. The involvement of the angiotensin II receptor type 1 (AT1R) in the hypertrophic effects of the THs was previously described. However, the mechanisms underlying this interaction are still unknown. AT1R belongs to the G-protein coupled receptor family and promotes its signal transduction by G-protein dependent and independent mechanisms. Recently, beta-arrestin signaling (G-protein independent) has been described as contributing to the hypertrophic response in different experimental models. Thus, the present study investigated the involvement of beta-arrestin signaling in the hypertrophic effects of THs mediated by AT1R, as well as the participation of ERK½ in this process. Primary cardiomyocytes cultures were stimulated with T3 (triiodothyronine; 15nM) for the induction of hypertrophy. Cardiomyocytes acutely treated with T3 (5-30 min) resulted in transient activation of ERK½, which was partially attenuated upon Losartan (1µM) administration, an AT1R antagonist. The contribution of ERK½ to cardiomyocyte hypertrophy was verified by using PD98059 (20µM), a MEK½ inhibitor, which prevented the transcription of hypertrophic markers. Immunoprecipitation assays revealed increased interaction between AT1R and beta-arrestin 2 under T3 stimulation, suggesting the recruitment of beta-arrestin 2 and, possibly, the internalization of AT1R. Through immunofluorescence and subcellular fractionation assays, T3 has been shown to stimulate AT1R translocation, enhancing its expression in the cardiomyocyte nucleus. In addition, both ERK½ activation and cardiomyocyte hypertrophy were sensitive to the inhibition of endocytosis, which was assessed by Concanavalin A (0.5µg/ml). Interfering RNA assays were efficient in demonstrating the involvement of beta-arrestin 2 in ERK½ activation and in T3-induced cardiomyocyte hypertrophy. Therefore, the results evidenced the involvement of beta-arrestin-2-dependent signaling in the activation of ERK½, through the AT1R, which contributes to the cardiomyocyte hypertrophy promoted by T3.

Rhodopsin, der Rezeptor der visuellen Kaskade, gehört zu größten Klasse A der G-Protein-koppelnden Rezeptoren (GPCRs) und gilt als Modell-Rezeptor in der GPCR-Forschung. Über 3 % des humanen Genoms kodieren für GPCRs, doch trotz der physiologischen Bedeutung dieser Proteinfamilie sind die fundamentalen Mechanismen, mit denen diese Rezeptoren extrazelluläre Signale in das Zellinnere weiterleiten noch nicht verstanden. In der vorliegenden Dissertation werden Aspekte des Aktivierungsmechanismus von Rhodopsin sowie der Kopplung und Aktivierung des G-Proteins Transduzin untersucht. Die Arbeit ist in drei Schwerpunkte unterteilt: I. Es wurde ein in GPCR’s hochkonserviertes NPxxYx(5,6)F Motiv (Aminosäuresequenz Asn-Pro-x-x-Tyr-x(5,6)-Phe) in der siebten und achten Helix charakterisiert. In diesem konservierten Motiv sind mehrere für die Ausbildung der aktiven Rezeptorkonformation wichtige Funktionen vereint: Verknüpfung zu einem Wasserstoffbrückennetzwerk, Helixflexibilität sowie die exakte Positionierung der achten Helix. Letzteres hat nicht nur bei der Rezeptoraktivierung sondern auch bei der nachfolgenden Interaktion mit dem G-Protein eine Bedeutung. II. Anhand von chimären Rezeptoren, bei denen Teile der achten Helix durch homologe Sequenzen des beta2-adrenergen Rezeptors ausgetauscht wurden, wurde die Rolle der achten Helix bei der Rezeptor-Aktivierung und Bindung des G-Proteins untersucht. Auch bei dieser Studie wurde gezeigt, dass die exakte Positionierung der achten Helix essentiell für die Interaktion mit dem G-Protein ist. Zudem wurde ein bezüglich der G-Protein-Aktivierung funktionsfähiger chimärer Rezeptor gefunden, was auf einen übergeordneten Mechanismus bei der Aktivierung von G-Proteinen durch GPCRs hindeutet. III. Die Funktion des ß-Ionon-Rings des Retinals beim Aktivierungsmechanismus von Rhodopsin wurde an einem Retinal studiert, bei welchem Teile des Retinal-Rings fehlten (azyklisches Retinal). Auch diesem azyklischen Retinal können Eigenschaften eines partiellen Agonisten zugeschrieben werden. Beim Vergleich zu Pigmenten mit dem nativen 11-cis-Retinal wurden starke Analogien bei der initialen Energieaufnahme durch die Retinal-Isomerisierung sowie bei der Weiterleitung der Lichtenergie ins Protein gefunden. Allerdings wird die Energie schlechter auf das Protein übertragen, wodurch wesentlich weniger der aktiven G-Protein bindenden Rezeptorkonformation gebildet wird. Als wichtigste Funktion des Retinal-Rings wurde die Aufrechterhaltung der aktiven Meta-II-Konformation identifiziert. / Rhodopsin, the receptor of the visual cascade, belongs to the largest group A of G-protein coupled receptors (GPCRs) and can be seen as a model receptor in GPCR research. More than 3 % of the human genome code for GPCRs. But despite their physiological relevance, the detailed mechanism of signal transduction from extra cellular signal to different cellular pathways remains to be fully understood. Different aspects of receptor activation and the coupling and activation of the G-protein transducin are investigated in this dissertation. The thesis focuses on the following three subjects: I. A NPxxYx(5,6)F motif (amino acid sequence Asn-Pro-x-x-Tyr-x(5,6)-Phe) has been characterized for rhodopsin. It is localized in helix VII and VIII and is highly conserved throughout the GPCR family. Various roles for rhodopsin activation are combined in this motif: linkage to a hydrogen-bond network, helix flexibility and the exact positioning of helix VIII. The latter is not only relevant for the activation of the receptor but also for interaction with its G-protein. II. The role of helix VIII for receptor activation and G-protein coupling was studied on chimeric receptors, in which parts of helix VIII were exchanged against homologous sequences of the beta2 adrenergic receptor. This study confirmed the importance of helix VIII’s position for G-protein coupling. Furthermore, a chimeric receptor was found, which was fully functional concerning G-protein activation. This indicates that GPCRs might use a single, generic mechanism for G-protein activation. III. The role of the ß-ionone-ring for the activation mechanism of rhodopsin was studied by means of an acyclic retinal, which lacks four carbon atoms of the ß-ionone-ring. This modified retinal could be classified as a partial agonist for rhodopsin. Energy input by retinal isomerization and formation of the G-protein binding Meta-II conformation were found to be very similar to rhodopsin when bound to its native 11-cis-retinal. However, the lack of the ring structure resulted in a lower amount of Meta-II and a fast decay of activity. It was concluded that the main role of the ring structure is to maintain the active state of rhodopsin.

O sistema olfatório de mamíferos é capaz de detectar milhares de substâncias químicas diferentes, mesmo em baixas concentrações. Um odorante disperso no ar pode se ligar a um receptor olfatório (OR) iniciando o processo de detecção. Os ORs são membros da super família de receptores acoplados a proteína G (GPCRs). Apesar de a via de transdução de sinal de odorantes estar bem descrita, pouco se sabe sobre os seus moduladores. Em 2005, nosso laboratório identificou RIC-8B como um possível fator de troca de nucleotídeos de guanina (GEF) que poderia amplificar a atividade da proteína G olfatória (Golf). No presente trabalho mostramos que RIC-8B é capaz de interagir com Gγ13. Procurando os outros componentes desse complexo identificamos Gβ1 como sendo a subunidade Gβ mais expressa no epitélio olfatório. Além disso, RIC-8B, Gαolf, Gβ1 e Gγ13 encontram-se concentrados nos cílios dos neurônios olfatórios e se co-localizam nesse compartimento celular. Nossos experimentos de co-imunoprecipitação mostram que RIC-8B interage mais fortemente com Gαolf, na presença de GDP do que na presença de GTP, como esperado para uma GEF. Curiosamente quando Gβ1 e Gβ13 estão presentes, RIC-8B e Gαolf co-imunoprecipitam igual, independente do nucleotídeo de guanina utilizado. Apesar de, na presença de Gβ1 e Gγ13, não observarmos dissociação física de RIC-8B e Gαolf com a mudança do estado de ativação, o efeito de RIC-8B na produção de AMPc não é afetada. Também mostramos que a quantidade de Gαolf, Gβ1 e Gγ13 presentes na membrana celular aumenta quando estas são co-transfectadas com RIC-8B em células de mamíferos. Nossos resultados apontam para um papel duplo da RIC-8B. Primeiro, RIC-8B poderia funcionar como uma chaperona auxiliando na formação e transporte do complexo heterotrimérico, Golf, em neurônios olfatórios. Em segundo lugar, RIC-8B também atuaria como uma GEF sobre Gαolf, aumentando a produção de AMPc e portanto amplificando a via de transdução de sinal de odorantes. Por fim, acreditamos que um possível papel para Gβ1 e Gγ13 nesse complexo seria funcionar como um andaime molecular, aproximando RIC-8B de seu alvo, Gαolf, potencializando ainda mais a atividade de GEF. / The mammalian olfactory system detects small amounts of thousands of different chemical compounds. Odorant perception starts when an odorant in the air binds to an olfactory receptor (OR). ORs belong to the super family of G-protein coupled receptors (GPCRs). Even though the odorant signaling pathway is well known, little is known about its modulators. In 2005, our lab identified RIC-8B as a putative guanine nucleotide exchange factor (GEF) that is able to interact with the olfactory G-protein (Golf) and amplify its activity. Here we show that RIC-8B also interacts with Gγ13. We also found that Gβ1 is the Gβ subunit that is predominantly expressed in the olfactory epithelium. Furthermore, RIC-8B, Gαolf, Gβ1 and Gγ13 are highly concentrated in the cilia of olfactory neurons and co-localize in this cellular compartment. We also show that RIC-8B interaction with G&#945olf is stronger in the presence of GDP than GTP, as expected for a GEF. Curiously, in the presence of Gβ1 and Gγ13, RIC-8B and Gαolf remain associated, in the presence of both GDP or GTP, probably through an indirect interaction via Gβ1/Gγ13. We also showed that the amounts of Gαolf, Gβ1 and Gγ13 in the cell membrane increase if RIC-8B is cotransfected in the same cell. Our results suggest that RIC-8B plays two roles. First, it may act as a chaperone which assists in the assembly and trafficking of the G protein complex. Second, RIC-8B would also act as a GEF to increase Gαolf dependent cAMP production and thereby amplify odorant signal transduction. Lastly, we believe that Gβ1 and Gγ13 may act as a scaffold to position RIC-8B close to its target, Gαolf, further enhancing the GEF activity.

[Truncated abstract] G-protein coupled receptors (GPCRs) form one of the largest superfamilies of cell-surface receptors and respond to a vast range of stimuli including light, hormones and neurotransmitters. Although structurally similar, GPCRs are regulated by many diverse proteins, which allow the specific functions of each receptor to be carried out. This thesis focussed on two well-documented GPCRs, the thyrotropin releasing hormone receptor (TRHR) and gonadotrophin-releasing hormone receptor (GnRHR), which control the thyroid and reproductive endocrine pathways respectively. Although each of these anterior pituitary receptors is responsible for distinct physiological responses, both are integral to normal development and homeostasis. This thesis focused on three areas of GPCR regulation: ?-arrestin recruitment, transcription factor regulation and receptor up-regulation. The role of the cytoplasmic protein, ?-arrestin, has perhaps been previously underestimated in GPCR regulation, but it is now increasingly apparent that ?-arrestins not only inhibit further G-protein activation and assist in GPCR internalisation but also act as complex scaffolding platforms to mediate and amplify downstream signalling networks for hours after initial GPCR activation. It is therefore becoming increasingly important to be able to monitor such complexes in live cells over longer time-frames. ... Members of the E2F transcription family have been previously identified by this laboratory as potential GnRHR interacting proteins, via a yeast-2-hybrid screen and BRET. This thesis further investigated the role of E2F family members and demonstrates that a range of GPCRs are able to activate E2F transcriptional activity when stimulated by agonist. However, despite GnRHR displaying robust E2F transcriptional activation upon agonist stimulation, this did not result in any conclusive evidence for functional regulation, although it is possible E2F may modulate and assist in GnRHR trafficking. Furthermore it is apparent that E2F family members are highly redundant, as small effects in GnRHR binding and cell growth were only observed when protein levels of both E2F4 and E2F5 were altered. During the course of the investigation into the effect of E2F transcription on GPCR function, it was evident that long-term agonist stimulation of GnRHR had a profound effect on its expression. As this was explored further, it became clear that this agonist-induced up-regulation was both dose- and time-dependent. Furthermore, altering levels of intracellular calcium and receptor recycling/synthesis could modulate GnRHR up-regulation. In addition, an extremely sensitive CCD camera has been used for the first time to visualise the luciferase activity attributed to GnRHR up-regulation. Overall, this thesis demonstrates the complex nature of GPCR regulation. For the first time, long-term BRET analysis on ?-arrestin interactions with both classes of GPCRs has been examined in a variety of cellular formats. This has given valuable insights into the roles of phosphorylation and internalisation on ?-arrestin interaction. Additionally, this thesis has revealed that prolonged agonist exposure increases receptor expression levels, which has major implications for drug therapy regimes in the treatment of endocrine-related disorders and tumours.