The Mu Opioid Receptor (MOR) is a G protein-coupled receptor (GPCR) important for pain regulation. Opioid agonists have long been the most effective treatment for most types of pain; however, this class of drugs is highly problematic due to the combination of several dangerous side effects like addiction, tolerance, and respiratory depression. Recently, a dramatic rise in opioid prescriptions has led to a nationwide opioid epidemic. Efforts to develop novel opioids with improved therapeutic profiles have led to work suggesting that MOR signaling through G proteins leads to analgesia while signaling through arrestin leads to respiratory depression and tolerance. However, more recent work has raised questions about which aspects of arrestin signaling and function contribute to these side effects. Additionally, the overall complexity of arrestin function especially with regard to trafficking at the cell membrane has recently come in to clearer view. Here, we use single-molecule tracking to describe membrane diffusion behavior of single MORs before and after agonist treatment in heterologous cells. By tracking individual MORs, we have revealed cell-context specific rules for MOR immobilization and endocytosis and shown that these processes depend on receptor density as well as the local availability of arrestin molecules.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

La protéine suppresseur de tumeurs PTEN (Phosphatase and tensin deleted on chromosome 10) est une phosphatase lipidique. En déphosphorylant le phosphatidylinositol (3,4,5) trisphosphate (PIP3) en PI(4,5) P2, PTEN contre-régule la voie PI3K/Akt et inhibe la prolifération. D’autres fonctions de PTEN peuvent être indépendantes de son activité phosphatase lipidique, notamment l’inhibition de la migration. Bien que PTEN soit, après p53, le suppresseur de tumeurs le plus muté dans un large panel de cancers (gliomes, prostate, sein, endomètre…), les mécanismes par lesquels ses fonctions sont régulées ne sont pas entièrement élucidés. Par une approche de double-hybride, notre équipe a identifié que les β-arrestines (β-arrs), des protéines d’échafaudage, interagissent avec PTEN. Nos travaux mettent en évidence que l’interaction entre PTEN et les β-arrs permet de moduler ses deux activités dépendantes ou non de son activité phosphatase lipidique. D’une part, les β-arrs augmentent l’activité phosphatase lipidique de PTEN in vitro. La GTPase RhoA et sa kinase d’aval ROCK activent PTEN, et ceci se fait par l’intermédiaire des β-arrs. La stimulation du récepteur à l’acide lysophosphatidique (LPA), qui active la voie RhoA/ROCK, augmente la formation du complexe PTEN/β-arrs et permet le recrutement du complexe à la membrane. Par l’effet positif sur l’activité phosphatase lipidique de PTEN, les β-arrs participent à l’inhibition d’Akt et de la prolifération dans les fibroblastes embryonnaires de souris (MEF). A l’inverse dans les gliomes U373, les β-arrs lèvent l’inhibition de la migration exercée par le domaine C2 de PTEN, indépendamment de son activité phosphatase lipidique. En aval de l’activation de RhoA induite par blessure du tapis cellulaire, les β-arrs interagissent davantage avec PTEN et rétablissent la migration des gliomes. De ce fait, les β-arrs régulent différentiellement les fonctions de PTEN importantes pour le contrôle de la prolifération cellulaire et la migration. Enfin, l’activité et la localisation de PTEN sont modulées par des interactions intramoléculaires entre ses domaines catalytiques, C2 et sa queue C-terminale régulatrice. Ces interactions régulent le passage d’une conformation fermée vers une conformation ouverte et active de PTEN. Grâce au développement d’un biosenseur de PTEN basé sur le transfert d’énergie par résonnance (RET), nous pouvons suivre pour la première fois les changements conformationnels de PTEN dans les cellules vivantes. En utilisant ce biosenseur nous montrons que la mutation des résidus impliqués dans les interactions intramoléculaires entraine des changements de conformation détectés par des variations de RET. De plus, l’activation de voies de signalisation connues pour activer PTEN, entrainent des changements conformationnels qui corrèlent avec l’augmentation de l’activité phosphatase lipidique de PTEN. Nos données montrent que le biosenseur peut être utilisé comme outil pour détecter les changements d’activité de PTEN dans les cellules vivantes. L’axe suppresseur de tumeurs/oncogène PTEN/PI3K/Akt joue un rôle essentiel dans la progression tumorale et constitue une cible thérapeutique pour le cancer. L’ensemble de nos travaux permet d’ajouter un degré de compréhension dans la régulation de PTEN, tant par les β-arrs que par l’interaction intramoléculaire et les changements conformationnels. / The Tumour Suppressor protein PTEN (Phosphatase and tensin deleted on chromosome 10) is a lipid phosphatase. By converting phosphatidylinositol (3,4,5) trisphosphate (PIP3) to PI(4,5)P2, PTEN inhibits the PI3K/Akt signalling pathway and cell proliferation. Other functions attributed to PTEN, including the inhibition of cell migration, can occur independently of its lipid phosphatase activity. Although PTEN function is dysregulated in a broad range of cancers (gliomas, prostate, breast, endometrium…), the mechanisms by which it is regulated are far from being completely elucidated. Using a two-hybrid approach, our team identified that the molecular scaffolds, β-arrestins (β-arrs), interact with PTEN.Our studies demonstrate that β-arrs modulate distinct functional outputs of PTEN that in turn are dependent or independent on its lipid phosphatase activity. β-arrs increase the lipid phosphatase activity of PTEN in vitro. The small GTPase RhoA and its downstream effector ROCK activate PTEN and this effect requires β-arrs. The stimulation of the lysophosphatidic acid receptor 1 (LPA1-R) receptor, that activates the RhoA/ROCK pathway, was found to increase the association of β-arrs with PTEN and induced plasma membrane translocation of the complex. Through their stimulatory effect on the lipid phosphatase activity of PTEN, β-arrs inhibit the PI3K/Akt pathway and proliferation of mouse embryonic fibroblasts. In contrast, in U373 glioma cells, βarrs release the brake on cell migration, which is mediated by the C2 domain of PTEN independently of its lipid phosphatase activity. Following wounding of a cell monolayer, and RhoA activation, β-arrs show increased association with PTEN, and rescue glioma cell migration. β-arrs therefore differentially regulate functions of PTEN important in the control of cell proliferation and migration.The activity and localization of PTEN are under tight control of intramolecular interactions between its regulatory C-terminal tail, and catalytic and C2 domains. These intramolecular interactions regulate a switch between a closed form of PTEN, and an open and active form that is targeted to the membrane. We have developed a resonance energy transfer (RET)-based biosensor that permits the monitoring of PTEN conformational change in live cells. Using the biosensor we demonstrate that mutation of residues implicated in the intramolecular switch produce conformational rearrangement of PTEN, detected by changes in RET. Furthermore, activation of signalling pathways known to activate PTEN, elicit conformational changes that parallel increased PTEN lipid phosphatase activity in living cells. Combined, these data demonstrate that the biosensor can be used as a tool to detect changes in PTEN tumour suppressor activity in live cells.The tumour suppressor/oncogene PTEN/PI3K/Akt axis plays a key role in tumour progression and represents a major therapeutic target in the treatment of cancer. Our studies help to further our understanding of how tumour suppressor PTEN is controlled by inter- and intramolecular interactions and provide a biosensor that can report changes in PTEN activity.

Les β-arrestines jouent un rôle important dans la transduction du signal par les récepteurs couplés aux protéines G (RCPG). Nous montrons dans cette thèse que les β-arrestines exercent des régulations plus complexes et subtiles qu'on ne le pensait jusque-là sur la voie AMPc/PKA/CREB qui est activée par les RCPGs couplés à Gs. Nous montrons que les β-arrestines interagissent directement la PKAcat et contribuent à sa translocation nucléaire. De plus, nous mettons en évidence une interaction β-arrestine/CREB qui conduit à la formation d'un complexe transcriptionnellement actif sous l’action de l’agoniste. D’autre part, nous avons constaté que les β-arrestines interagissent directement avec PKAcat, p70S6K et Src via un même site et lesquelles sont donc potentiellement mutuellement exclusives. Nous avons ensuite mesuré l’impact d’une mutation et d’un polymorphisme du R-FSH sur la signalisation dépendante des β-arrestines, notamment grâce à l’utilisation de senseurs FRET et BRET. / Β-arrestins play an important role in G protein-coupled receptor (GPCR)-induced signal transduction. In this thesis, we show here that β-arrestins exert more complex and subtle regulation than previously thought on the cAMP/PKA/CREB pathway which is activated by Gs-coupled GPCRs. We demonstrate that β-arrestins directly interact with PKAcat and promote its translocation to the nucleus. Moreover, we provide evidence that β-arrestins directly interact with CREB thereby forming a transcriptionally active complex upon agonist stimulation. We also found that PKAcat, p70S6K and Src all directly interact with β-arrestins through the same interaction site and are therefore potential mutually exclusive interactions. We then measured the impact of a point mutation and of a polymorphism in the FSH-R on β-arrestin-dependent signaling, in part using FRET and BRET sensors.

The C type natriuretic peptide receptor (NPRC) also known as NPR3 is a widely expressed single transmembrane-spanning protein. NPRC functions as a homodimer at the cell surface for the metabolic clearance of a broad range of natriuretic peptides from circulation. The intracellular domain of NPRC is coupled to inhibitory G proteins and is involved in mediating signal transduction. In order to further elucidate the role of NPRC in signal transduction a proteomic approach was taken to identify putative protein binding partners for NPRC in different cell-types. An interrogation of the molecular association between NPRC and its identified protein binding partner(s) was carried out in different cell types to identify the specific interacting domains. The physiological role of the association between NPRC and its protein binding partner(s) were investigated in situ. Furthermore NPRC is subject to post translation modifications including glycosylation and phosphorylation. Although evidence suggests NPRC is phosphory ated on serine residues the specific amino acid residues that are phosphorylated and the kinases responsible for their phosphorylation has yet to be determined. A recombinant GST-NPRC fusion protein polyclonal NPRC antibody kinase prediction algorithm and several phosphospecific and substrate motif antibodies were utilized to characterize the phosphorylation state of NPRC in vitro.

La FSH est une hormone clé dans la fonction de reproduction. La compréhension de ces mécanismes moléculaires est essentielle pour comprendre ses effets biologiques. Nous montrons ici pour la première fois que non seulement la FSH induit le recrutement de polysomes dans la cellule de Sertoli, mais qu’en plus, elle stimule la traduction de deux ARNm sélectivement, c-fos et vegfa. La p70S6K est impliquée dans ce mécanisme. La FSH active et induit le recrutement de la p70S6K sur la coiffe des ARNm où elle active ses cibles traductionnelles. Les β-arrestines, des protéines d’échafaudage qui régulent la signalisation du RFSH, semblent participer à l’activation de la p70S6K. La déplétion des β-arrestines augmente massivement le recrutement de la p70S6K à la coiffe et diminue son activité enzymatique. De plus, la p70S6K et les β-arrestines interagissent mais cette interaction ne semble pas modulée par la FSH. Nos résultats suggèrent que les β-arrestines séquestreraient la p70S6K inactive pour permettre son activation et son recrutement à la coiffe m7GTP en réponse à la FSH. Ce travail apporte de nouvelles connaissances sur le rôle de la FSH dans la traduction et les mécanismes de signalisation impliqués. / FSH is a key hormone of the reproductive function. A clear understanding of its molecular mechanism is essential to fully understand its biological effects. Here, we show for the first time that FSH not only enhances the assembly of polysomes but also stimulates the translation of at least two mRNA selectively, c-fos and vegfa, in Sertoli cells. p70S6K participates in this mechanism. FSH activates and enhaced p70S6K recruitment to the m7GTP cap structure of mRNA where this kinase phosphorylates its targets. β-arrestins, which are scaffolding proteins that regulate FSH signalling, seem to participate in p70S6K activation. Accordingly, β-arrestins depletion increased p70S6K recruitment to the cap and reduced its enzymatic activity. Importantly, p70S6K and β-arrestins interact but the interaction is not FSH-dependent. We assume that β-arrestins sequester inactive p70S6K to activate it locally and then p70S6K translocates to the cap in response to FSH. In conclusion, this work brings new knowledge about FSH function in translational control and the signaling mechanisms involved.

Myriad small molecule compounds targeting metabotropic glutamate receptors (mGluRs) have been investigated for the treatment of various neuropsychiatric diseases and displayed promise in preclinical studies. At the clinical level, many of these compounds have been well tolerated by human subjects but have eluded success as promising therapeutics. There are eight subtypes of mGluRs, which express as constitutive dimers. This dimerization can occur between identical (homodimerization) or different (heterodimerization) mGluR protomer subtypes, which are subject to pairing-specific signaling mechanisms. Subtype expression of mGluRs is heterogenous between brain regions and cell types, yielding probable cell-specific homo- and heterodimer combinations that respond differently to certain drugs. While G protein recruitment to active mGluR dimers has been studied extensively, little is known about arrestin recruitment to these receptors. I used bioluminescence resonance energy transfer (BRET) assays, which provide a quantitative measure of protein-protein proximity, to observe and quantify arrestin recruitment to specific mGluR subtype pairings upon ligand administration in heterologous cells. I studied how select allosteric ligands affect communication between protomers to enhance arrestin recruitment to dimers. My findings indicate that arrestin recruitment occurs only at select mGluR homodimers upon orthosteric stimulation but is frequently stimulated or enhanced by administration of activating allosteric ligands. Additionally, I found that trans-protomer communication is highly specific to mGluR protomer subtype pairings, the ligand administered,a nd inter-protomer signal direction. Lastly, my findings reveal a cooperative effect of mGluR2 and 3 heterodimerization on arrestin recruitment that is dependent on the functional ability of each protomer to bind orthosteric agonist and responds distinctively from homodimers to stimulation by certain allosteric ligands. Taken together, this work shows that mGluR signaling can be tuned using strategic pharmacology and energizes hope for future clinical success of mGluR-targeting ligands.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

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.

Les protéines de la membrane plasmique jouent un rôle fondamental dans la détection des informations véhiculées par le milieu extracellulaire et l’adaptation des cellules aux variations de l’environnement. Elles font l’objet d’une régulation fine qui permet de moduler leur présence à la membrane et de contrôler les voies de signalisation en aval. Dans ce contexte, les arrestines qui constituent une superfamille de protéines adaptatrices, se sont imposées comme des régulateurs clés depuis la découverte des β-arrestines et arrestines visuelles, spécifiques des eucaryotes supérieurs, et de leur rôle dans la régulation des récepteurs couplés aux protéines G hétéro-trimériques, jusqu’à l’identification plus récente de nouveaux membres apparentés, présents des mammifères jusqu’aux protistes, et partageant un rôle commun de régulation de cargos membranaires. Ce travail de thèse porte sur la caractérisation fonctionnelle de deux représentants du clan arrestine de l’amibe Dictyostelium discoideum, les protéines AdcB et AdcC. Ces deux protéines partagent une même organisation multimodulaire, spécifique aux Dictyostélides, qui associe au cœur arrestine, un domaine putatif C2 de type calcium-binding et deux modules SAMs, respectivement aux extrémités N- et C-terminales des protéines. Nous avons établi que ces domaines apportent des fonctions spécifiques à ces arrestines en leur conférant la capacité de lier des lipides anioniques in vitro en réponse au calcium à travers leur module C2, et de former des structures homo- et hétéro-oligomériques via leurs domaines SAMs. En dépit de ces similarités, AdcB et AdcC présentent un comportement différent in cellulo dans la mesure où seul AdcC transloque à la membrane plasmique en réponse à une élévation du calcium cytosolique, provoquée par la stimulation des cellules par les chimioattractants AMPc et acide folique ou le calcium lui-même. Ces résultats ont été complétés par une étude phénotypique des mutants invalidés pour ces arrestines et la recherche de partenaires qui ouvrent des pistes pour des études futures. / Integral proteins of the plasma membrane play a major role in the detection of environmental cues and in the adaptation of cells to variations of their environment. Regulatory mechanisms modulate their presence at the cell surface and control the signaling cascades activated in response to their stimulation. In this context, members of the arrestin revealed to be key regulators, since the discovery of β- and visual arrestins and their well-described role in the regulation of G-protein coupled receptors in complex organisms, and the more recent identification of arrestin-related proteins, present from mammals to protists and sharing functions in membrane cargo trafficking. This work aims at the functional characterization of two arrestin-related proteins of the social amoeba Dictyostelium discoideum, the AdcB and AdcC proteins. These two members of the arrestin clan share a similar multimodular organization, specific to Dictyostelids, with a putative N-terminal calcium-binding type C2 domain and two C-terminal SAM domains surrounding the arrestin module. We showed that the C2 domain confers calcium-dependent binding properties to anionic lipids in vitro and that the SAM domains allow the self-association and hetero-interaction of the two proteins in complexes of high molecular weight. Despite these similarities, AdcB and AdcC harbor a distinct behavior in vivo as only AdcC translocates to the plasma membrane in response to an intracellular calcium rise triggered by the chemoattractants acid folic and cAMP or extracellular calcium. In parallel, a phenotypic characterization of adcB and adcC single or double null mutants and a search for partners were conducted, that open new avenues for future research on these adaptor proteins.