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No description available.

Over the past two decades, a vast body of research has demonstrated the importance of G protein-coupled receptor kinase 2 (GRK2) in the physiology and pathophysiology of the heart. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 following cardiac insult exacerbates injury and speeds progression to heart failure. In this dissertation we turned our attention towards two novel aspects of GRK2 biology. Firstly, despite the importance of this GRK2 activity in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In the first study of this dissertation, we generated a novel skeletal muscle specific GRK2 knockout (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle, yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared to wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β2-adrenergic receptor agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, this study provides the first insights into the role of GRK2 in skeletal muscle physiology, and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β2-adrenergic receptor-induced hypertrophy. In the second part of this dissertation, we report surprising novel metabolic phenotypes that arise from modulating GRK2 activity exclusively in the heart. We show that transgenic βARKct (TgβARKct) mice (cardiac specific expression of a GRK2 inhibitory peptide) are more susceptible to high-fat diet (HFD) induced obesity. TgβARKct mice exhibit marked increase in adiposity on HFD relative to control animals. Conversely transgenic GRK2 mice (TgGRK2) mice (cardiac specific overexpression of GRK2) show resistance to weight gain on a HFD and decrease in adipose tissue mass relative to control animals. Furthermore, conditioned media from βARKct expressing neonatal rat ventricular myocytes enhances adipocyte differentiation in vitro. These results suggest that the heart produces a secreted factor to control whole body metabolism, and that GRK2 is a regulator of this mechanism. / Biomedical Sciences

Sphingosine-1-phosphate receptor type 1 (S1PR1), one of the five S1PRs that signals in response to bioactive lysosphingolipid S1P, regulates several fundamental processes in distinct cell types and is implicated in atherosclerosis. Using the cre-lox recombination system, previous studies identified that knocking out S1PR1 in myeloid and endothelial cells promotes plaque development in atherogenic mouse models. In the process of generating S1pr1lox/lox; ApoEKO/KO control mice, we unexpectedly noticed that S1pr1lox/lox mutation alone, in the absence of cre recombinase, reduces high-fat (HF) diet-induced atherosclerosis in S1pr1lox/lox; ApoEKO/KO mice compared to S1pr1WT/WT; ApoEKO/KO mice. Although S1pr1lox/lox allele partially suppressed S1pr1 levels in macrophages and vascular smooth muscle cells (VSMC), the presence of this mutation in a non-BM derived cell type was responsible for this reduced atherosclerosis in S1pr1lox/lox; ApoEKO/KO mice. We speculated that it could be VSMCs due to their abundance in the vascular wall and their role in foam cell formation. In this thesis, we directly tested the effects of inactivating S1PR1 in smooth muscle cells (Tagln-creTG; S1pr1lox/lox; ApoEKO/KO mice) on atherosclerosis. Our results demonstrated that deleting S1PR1 in smooth muscle cells drastically reduces atherosclerosis in apoE-deficient mice. The aortic SMCs isolated from these mice also exhibited reduced cell proliferation and lipid droplet formation in response to S1PR1 agonist SEW2871 compared to S1PR1-WT VSMCs. Furthermore, we also tested the effects of directly inhibiting S1PR1 with S1PR1 selective antagonist Ex26 at a dosage of 0.1 mg/kg/hr in S1pr1WT/WT; ApoEKO/KO mice and Tagln-creTG; S1pr1lox/lox; ApoEKO/KO mice. The prolonged exposure to Ex26 substantially reduced atherosclerotic plaque development in apoE KO mice on an HFD compared to DMSO-treated apoE KO mice. However, this protection was completely lost in mice that lack the S1pr1 gene in VSMCs. Overall, our results suggest that knocking out S1PR1 in VSMCs results in atheroprotection that surpasses the effects of inactivating S1PR1 in macrophages and endothelial cells which are known to promote atherosclerosis. / Dissertation / Doctor of Philosophy (PhD)

Os receptores acoplados à proteína G (GPCRs), também chamados de receptores 7TM, são conhecidos por regular virtualmente todos os processos fisiológicos em mamíferos e cerca de 40% de todas as drogas comerciais agem através destes receptores. A sinalização mediada por eles é classicamente atribuída à proteína G, que é ativada pela troca de GDP por GTP, promovendo a separação das subunidades G e G, e leva à produção de mensageiros secundários como cAMP, Ca2+ e DAG. Após a resposta os GPCRs são fosforilados pelas quinases de GPCRs (GRKs), sinalizando para recrutamento das -arrestinas citoplasmáticas, que por sua vez desencadeiam a formação de endossomos internalizando e dessensibilizando o receptor. Entretanto, estudos mostram que este endossomo, contendo o complexo ligante-receptor--arrestina, pode interagir com proteínas sinalizadoras no citoplasma desencadeando vias de sinalização independentes de proteína G. Recentemente foram descritos para diferentes receptores, ligantes capazes de ativar seletivamente uma das duas vias, proteína G ou -arrestina, chamados agonistas seletivos. O receptor AT1 é um GPCR particularmente interessante no estudo do agonismo seletivo, tanto por sua vasta expressão em tecidos quanto pelo conhecimento de agonistas seletivos já estabelecidos, tais como os ligantes SII e TRV120027. O objetivo deste trabalho foi analisar comparativamente os perfis de sinalização decorrente da ativação de AT1 por SII ou TRV120027 através do uso de arranjos de quinases e da modulação de genes relacionados a sinalização de GPCRs. Ang II que é ligante natural e total (ativa via dependente de proteína G e de -arrestina) neste receptor foi usada como controle para fins de comparação. Nossos dados mostraram que o perfil da sinalização mediada pelo receptor AT1 varia não só entre AngII e os agonistas seletivos, mas também entre os dois ligantes seletivos SII e TRV120027, mostrando que a interação receptor-ligante pode influenciar a sinalização em um grau mais refinado, além da ativação dependente de -arrestina ou proteína G. Estes dados mostram que existem perspectivas para o desenvolvimento futuro de ligantes com ainda maior grau de seletividade. / G protein coupled receptors (GPCRs), also known as 7TM receptors, are known to regulate virtually all physiological processes in mammals and approximately 40% of all current clinical drugs act by modulating such receptors. The signaling mediated by them is classically by coupling to G protein, which is activated by exchanging bound GDP for GTP, dissociation of G and G subunits, then leading to production of second messengers such as cAMP, Ca2+, and DAG. After the signal transduction, GPCR are phosphorylated by GPCR kinases (GRKs), followed by recruitment of cytoplasmic -arrestins, which initiate the endosome formation with consequent internalization and desensitization of the receptor. However, is has been demonstrated that the endosome assembling the ligand-receptor--arrestin complex can interact with cytoplasmic signaling proteins, therefore activating signaling pathways independently of G protein coupling. Recently, for different receptors, it has been described ligands capable of selectively activating one of these signaling pathways, G protein or -arrestin, called biased agonists. The AT1 receptor is a particularly interesting GPCR for the study of biased agonism, either due to its wide tissue expression as well as also due the existence of known and established biased ligands, such as SII and TRV120027. The aim of our study was to comparatively analyze the AT1 receptor signaling pathways profiles after activation by SII or TRV120027, using kinases arrays, and expression modulation of genes related to GPCRs signaling. AngII is the natural and full agonist of this receptor (activates both G protein and -arrestin signaling pathways) was used for comparison. Our data show that the signaling profile mediated by AT1 receptor can be distinct not only when comparing the profiles from AngII and the biased agonists, but also when comparing the profiles from the two biased ligands SII and TRv120027; revealing that the complex ligand-receptor can influence the downstream signaling pathways in a fine-tune way, further to the activation of -arrestin or G-protein. This data show that there are perspectives for the future development of ligands with even higher degree of selectivity.

Recently, several studies have demonstrated that G protein coupled receptors exist as homo/heterodimers or oligomers. Adenosine A2A receptors and dopamine D2 receptors are present as both homo- and heterodimer. In the GABAergic striatopallidal neurons A2AR are co- localized with D2 receptors (D2R), and establish functional A2AR-D2R heteromers, which modulates dopaminergic activity. Due to be involved in physiological processes, these receptors bear critical roles. Dopamine receptors play critical role in dopaminergic pathways in regulation of memory, food intake and psychomotor activity, etc. On the other hand, adenosine A2A receptors are involved in the regulations of neurotransmission, immune response and cardiovascular systems. Dopamine D2R andadenosine A2AR have been shown to interact in striatum and modulate dopaminergic activity The purpose of this study is to assess the functionality of EGFP (enhanced green fluorescent protein) and mCherry (a red fluorescent protein) tagged adenosine A2A and dopamine D2 receptors and to detect homo/ hetero-dimerization of these receptors in live cells via Fluorescence Resonance Energy Transfer (FRET). Understanding the mechanisms of the interaction between adenosine and dopamine signaling will help us to figure out some molecular mechanism of neurophysiological disorders. Furthermore, the fluorescence based live cell model could be used to observe the effects of potential anti-psychotic drugs on the interaction of these two receptors.

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.

Os receptores acoplados à proteína G (GPCRs), também chamados de receptores 7TM, são conhecidos por regular virtualmente todos os processos fisiológicos em mamíferos e cerca de 40% de todas as drogas comerciais agem através destes receptores. A sinalização mediada por eles é classicamente atribuída à proteína G, que é ativada pela troca de GDP por GTP, promovendo a separação das subunidades G e G, e leva à produção de mensageiros secundários como cAMP, Ca2+ e DAG. Após a resposta os GPCRs são fosforilados pelas quinases de GPCRs (GRKs), sinalizando para recrutamento das -arrestinas citoplasmáticas, que por sua vez desencadeiam a formação de endossomos internalizando e dessensibilizando o receptor. Entretanto, estudos mostram que este endossomo, contendo o complexo ligante-receptor--arrestina, pode interagir com proteínas sinalizadoras no citoplasma desencadeando vias de sinalização independentes de proteína G. Recentemente foram descritos para diferentes receptores, ligantes capazes de ativar seletivamente uma das duas vias, proteína G ou -arrestina, chamados agonistas seletivos. O receptor AT1 é um GPCR particularmente interessante no estudo do agonismo seletivo, tanto por sua vasta expressão em tecidos quanto pelo conhecimento de agonistas seletivos já estabelecidos, tais como os ligantes SII e TRV120027. O objetivo deste trabalho foi analisar comparativamente os perfis de sinalização decorrente da ativação de AT1 por SII ou TRV120027 através do uso de arranjos de quinases e da modulação de genes relacionados a sinalização de GPCRs. Ang II que é ligante natural e total (ativa via dependente de proteína G e de -arrestina) neste receptor foi usada como controle para fins de comparação. Nossos dados mostraram que o perfil da sinalização mediada pelo receptor AT1 varia não só entre AngII e os agonistas seletivos, mas também entre os dois ligantes seletivos SII e TRV120027, mostrando que a interação receptor-ligante pode influenciar a sinalização em um grau mais refinado, além da ativação dependente de -arrestina ou proteína G. Estes dados mostram que existem perspectivas para o desenvolvimento futuro de ligantes com ainda maior grau de seletividade. / G protein coupled receptors (GPCRs), also known as 7TM receptors, are known to regulate virtually all physiological processes in mammals and approximately 40% of all current clinical drugs act by modulating such receptors. The signaling mediated by them is classically by coupling to G protein, which is activated by exchanging bound GDP for GTP, dissociation of G and G subunits, then leading to production of second messengers such as cAMP, Ca2+, and DAG. After the signal transduction, GPCR are phosphorylated by GPCR kinases (GRKs), followed by recruitment of cytoplasmic -arrestins, which initiate the endosome formation with consequent internalization and desensitization of the receptor. However, is has been demonstrated that the endosome assembling the ligand-receptor--arrestin complex can interact with cytoplasmic signaling proteins, therefore activating signaling pathways independently of G protein coupling. Recently, for different receptors, it has been described ligands capable of selectively activating one of these signaling pathways, G protein or -arrestin, called biased agonists. The AT1 receptor is a particularly interesting GPCR for the study of biased agonism, either due to its wide tissue expression as well as also due the existence of known and established biased ligands, such as SII and TRV120027. The aim of our study was to comparatively analyze the AT1 receptor signaling pathways profiles after activation by SII or TRV120027, using kinases arrays, and expression modulation of genes related to GPCRs signaling. AngII is the natural and full agonist of this receptor (activates both G protein and -arrestin signaling pathways) was used for comparison. Our data show that the signaling profile mediated by AT1 receptor can be distinct not only when comparing the profiles from AngII and the biased agonists, but also when comparing the profiles from the two biased ligands SII and TRv120027; revealing that the complex ligand-receptor can influence the downstream signaling pathways in a fine-tune way, further to the activation of -arrestin or G-protein. This data show that there are perspectives for the future development of ligands with even higher degree of selectivity.

Mon travail de thèse s’est focalisé sur l’étude des récepteurs CXCR4 et ACKR3, deux récepteurs aux chimiokines. Ceux-ci jouent des rôles majeurs dans différentes fonctions physiologiques notamment le chimiotactisme des cellules immunitaires. Le dérèglement de leur activité est souvent associé à différents pathologies, notamment des cancers. Outre le fait que ces récepteurs lient tous les deux la même chimiokine, CXCL12, il a été montré qu’ils étaient capables d’exercer l’un sur l’autre des régulations croisées. Les mécanismes sous-tendant celles-ci sont très mal connus. Ils pourraient être liés à une compétition entre les récepteurs pour lier le même ligand CXCL12 ou à des régulations au niveau des voies de signalisation activées lors de la liaison d’une chimiokine. Ces régulations croisées pourraient également résulter de la formation de complexes de récepteurs appelés oligomères, complexes qui disposeraient de propriétés pharmacologiques particulières. De tels complexes ont été décrits dès les années 1990 dans des systèmes d’expression hétérologues mais leur existence et leurs rôles dans des systèmes natifs reste très largement débattus. Une des raisons est la difficulté à élaborer des outils moléculaires permettant l’étude de ces oligomères dans les tissus natifs.Le premier objectif de ma thèse a été l’élaboration d’outils permettant d’étudier l’existence de ces oligomères dans les systèmes natifs. En collaboration avec des laboratoires d’Amsterdam, j’ai développé des nanobodies fluorescents, petits anticorps produits par le lama, afin de marquer spécifiquement les récepteurs endogènes exprimés à la surface des cellules. Pour rendre fluorescent ces nanobodies, nous avons utilisé une technique originale permettant de greffer un fluorophore sur l’extrémité C-terminale de la molécule. Ces nanobodies conservent des propriétés pharmacologiques remarquables puisqu’ils conservent de hautes affinité et spécificité pour leur cible. J’ai ainsi pu utiliser ces molécules et démontrer l’existence des oligomères CXCR4 dans des lignées cellulaires exprimant de façon endogène le récepteur CXCR4. Des analyses similaires sont en cours sur le récepteur ACKR3.Le second objectif de ma thèse a été de définir les rôles potentiels de ces oligomères. J’ai pu montrer que les hétéro-oligomères CXCR4 /ACKR3, complexes associant les deux types de récepteurs, possèdent des propriétés de liaison particulière. Ceux-ci semblent ne lier la chimiokine CXCL12 que sur le récepteur ACKR3 au sein du complexe. Cette asymétrie de liaison est très étonnante car le récepteur CXCR4 est capable de lier CXCL12 avec une cinétique bien supérieure à celle de ACKR3 pour ce même ligand. J’ai étudié les conséquences d’une telle asymétrie de liaison sur les propriétés de signalisation de ces récepteurs. Une analyse des différentes voies de couplage activées lors de la liaison de CXCL12 a été réalisée sur les récepteurs exprimés de façon isolée ou co-exprimés au sein d’une même cellule. Les résultats ne montrent pas de modifications majeures de leur propriété de couplage.Nous avons par ailleurs analysé la capacité de ces récepteurs à internaliser en absence ou en présence de ligand CXCL12. J’ai pu observer que les hétéro-oligomères CXCR4 / ACKR3 restaient vraisemblablement bloquer à la surface des cellules.Ces travaux ouvrent des perspectives intéressantes dans la mesure où elles constituent la première démonstration de l’existence d’oligomères CXCR4 dans des systèmes natifs.Par ailleurs l’observation d’une régulation différente de l’internalisation des hétéro-oligomères constitue une première piste conférant à ces complexes un rôle particulier dans les régulations croisées de l’activité que ces récepteurs exercent l’un sur l’autre. / During my PhD, I focused on two chemokine receptors, CXCR4 and ACKR3. They have several important physiological functions, such as chemotaxis of immune cells. However, on the other hand, when their function is disturbed, they are involved in different immunological pathologies and cancer. Both receptors recognise the same chemokine, CXCL12 and many studies have reported a crosstalk between CXCR4 and ACKR3. However, the mechanisms behind this crosstalk are still poorly understood. This crosstalk can occur because both receptors are competing for CXCL12, at the level of signalling pathways or due to the formation of complexes between CXCR4 and ACKR3 receptors, called oligomers. The oligomers might have specific pharmacological properties different from the receptor monomers. Oligomeric complexes have been described since the nineties. Most of the studies on these oligomers were performed on heterologous expression systems, but still a lot of debate exists about their existence and their role in native tissues. One of the reasons behind this controversy is that studying oligomers in a native context is complicated, especially because we often lack the molecular tools for these studies.The first objective of my PhD was to generate efficient tools to study the existence of CXCR4 and ACKR3 oligomers in native systems. In collaboration with laboratories from Amsterdam and Ghent, we have developed fluorescent nanobodies, small antibodies produced by llamas. These specific tools allow the detection of receptors endogenously expressed at the cell surface. In order to fluorescently label these nanobodies, we have used an original strategy that can specifically attach the fluorophore to the C-terminus of the nanobody. Interestingly, the fluorescent nanobodies retain high affinity and specificity for their target. With these nanobodies, I have demonstrated the existence of CXCR4 oligomers in cell lines that endogenously express CXCR4. We are currently investigating the existence of ACKR3 oligomers.The second objective of my PhD consists of defining the functional roles of these oligomers. I have shown that CXCR4/ACKR3 hetero-oligomers have specific binding characteristics. It seems that CXCL12 is binding only to ACKR3 within this hetero-oligomer and that ACKR3 impairs the CXCL12 binding to CXCR4 within the hetero-oligomer. This is interesting, since we also have demonstrated that CXCL12 is binding much faster on CXCR4 than on ACKR3.In addition, I have studied the consequences of this negative cooperativity within the CXCR4/ACKR3 hetero-oligomer on different signalling pathways. We have compared conditions where the receptor was expressed alone or when receptors were co-expressed. No major modifications have been found on their signalling properties. However, when investigating the internalisation of CXCR4 and ACKR3, it seems that CXCR4/ACKR3 hetero-oligomers remain blocked on the cellular surface.This opens interesting perspectives, since it is the first time CXCR4 oligomers have been detected at an endogenous level. Moreover, the observation of a different internalisation pattern of the hetero-oligomer is a first step to further investigate the specific roles of these oligomers in the crosstalk between the receptors.