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Rozprawy doktorskie na temat „?-arrestin”

Autor: Grafiati

Data publikacji: 4 czerwca 2021

Data aktualizacji: 9 czerwca 2025

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Sharmeen, Cynthia. "Involvement of Beta-arrestin 1 and Beta-arrestin 2 in store operated calcium entry." Mémoire, Université de Sherbrooke, 2016. http://hdl.handle.net/11143/9499.

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Résumé : La variation de la [Ca2+] intracellulaire participe à nombreux de processus biologiques. Les cellules eucaryotes expriment à la membrane plasmique une variété de canaux par lesquelles le calcium peut entrer. Dans les cellules non excitables, deux mécanismes principaux permettent l'entrée calcique; l'entrée capacitative de Ca2+ via Orai1 (SOCE) et l'entrée calcique activé par un récepteur (ROCE). Plusieurs protéines clés sont impliquées dans la régulation de ces voies d'entrée calcique, ainsi que dans l'homéostasie calcique. TRPC6 est un canal calcique impliquée dans l'entrée calcique dans les cellules à la suite d’une stimulation d’un récepteur hormonal. TRPC6 transloque à la membrane cellulaire et il y demeure jusqu'à ce que le stimulus soit retiré. Les mécanismes qui régulent le trafic et l'activation de TRPC6 sont cependant encore peu connus. Des découvertes récentes ont démontré qu'il y a un rôle potentiel de Rho kinase dans l'activité de TRPC6. Rho kinase est activée par la petite protéine G RhoA qui peut être activée par les protéines G hétérotrimériques Gα12 et Gα13. En plus de Gα12 et Gα13, les protéines de désensibilisation des GPCR β -arrestin 1 et / ou β-arrestin 2 peuvent aussi activer RhoA. Le but de notre étude est d'examiner la participation des protéines Gα12/13 et β-arrestin 1/ β-arrestin 2 dans l'activation de TRPC6 et de la protéine Orai1. Nous avons utilisé des ARN interférant (siRNA) spécifiques pour induire une réduction de l'expression de Gα12/13 ou β-arrestin 1/β-arrestin 2. La conséquence sur l’entrée de Ca2+ dans les cellules a été ensuite déterminée par imagerie calcique en temps réel suite à une stimulation par la vasopressine (AVP), thapsigargin ou carbachol. Nous avons donc identifié que dans des cellules A7r5, une lignée cellulaire de musculaires lisses vasculaires où le canal TRPC6 exprimé de manière endogène, la diminution de l’expression des protéines Gα12 ou Gα13 ne semble pas modifier l’entrée Ca2+ induit par l’AVP par rapport aux cellules témoins. D'autre part, la diminution de l’expression β-arrestin 1 ou β-arrestin 2 dans des cellules HEK 293 ainsi que des cellules HEK 293 exprimant de façon stable TRPC6 (cellules T6.11) ont augmenté l’entrée de Ca2+ induite par thapsigargin, un activateur pharmacologique de SOCE. Des études de co-immunoprécipitation démontrent une interaction entre la β-arrestin 1 et STIM1, alors qu'aucune interaction n'a été observée entre les β-arrestin 1 et Orai1. Nous avons de plus montré à l'aide d'analyse en microscopie confocale que la diminution de l’expression β-arrestin 1 ou β-arrestin 2 n’influence pas la quantité d’Orai1 à la périphérie cellulaire. Cependant, des résultats préliminaires indiquent que la diminution de l’expression β-arrestin 1 ou β-arrestin 2 augmente la quantité de STIM1-YFP dans l'espace intracellulaire et diminue sa quantité à la périphérie cellulaire. En conclusion, nous avons montré que les β-arrestin 1 ou β-arrestin 2 sont impliquées dans l'entrée capacitative de Ca2+ (SOCE) et contrôlent la quantité de STIM1 dans le réticulum endoplasmique. Abstract : In an organism, intracellular [Ca2+] takes part in many biological processes. Eukaryotic cells express a variety of channels in the plasma membrane through which calcium can enter. In non-excitable cells, two main mechanisms allow calcium entry; the store-operated calcium entry via Orai1 (SOCE) and receptor-operated calcium entry (ROCE). Several key proteins are involved in the regulation of these calcium entry pathways as well as in calcium homeostasis. TRPC6 is a calcium channel implied in calcium entrance into the cells following hormonal stimulation and translocates to the plasma membrane. TRPC6 channel appear to the plasma membrane until the stimulus is present. Although, the mechanisms that regulate the trafficking and activation of TRPC6 are still little known. Recent findings have demonstrated that there is a potential role of Rho kinase in activity of TRPC6. Rho kinase is activated by the small G protein RhoA that itself can be activated by the heterotrimeric G proteins Gα12 and Gα13. In addition to Gα12 and Gα13 proteins, cytosolic GPCR desensitizing proteins β-arrestin 1 and/or β-arrestin 2 could also activate RhoA. The purpose of our study is to investigate the involvement of the proteins Gα12/13 and β-arrestin 1/β-arrestin 2 in the activation of TRPC6 and Orai1 protein. We used siRNA specific to Gα12/13 or β-arrestin 1/β-arrestin 2 to knockdown their endogenous expression. Then, calcium imaging in real time was performed in order to see the quantity of calcium entered into the cell following stimulation by vasopressin (AVP), thapsigargin, or carbachol. We hence identified that in A7r5 cell, vascular smooth muscle cell where TRPC6 channel expressed endogenously; reduced expression of Gα12 or Gα13 proteins does not seem to modify the AVP-induced Ca2+ entry compared to control cells. On the other hand, calcium imaging experiment in knocked down β-arrestin 1 or β-arrestin 2 in HEK 293 cells as well as HEK 293 cells stably transfected with TRPC6 (T6.11 cells) resulted in an increased thapsigargin-induced calcium entry. The co-immunoprecipitation studies demonstrate an interaction between β-arrestin 1 and STIM1, a calcium sensor in SOCE influx, while no interaction was observed between β-arrestin 1 and Orai1.We moreover showed by confocal microscopy that reduced expression of β-arrestin 1/ β-arrestin 2 does not influence the quantity of Orai1 at the cell periphery. Preliminary results showed that reduced expression of β-arrestin 1 or β-arrestin 2 increases the quantity of STIM1-YFP in the intracellular space and less it’s in peri-membrane space. In conclusion, we showed that β-arrestin 1 or β-arrestin 2 are involved in the store-operated calcium entry (SOCE) and control the quantity of STIM1 in the endoplasmic reticulum.

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Saxena, Kunal. "Arrestin interactions with the μ-opioid receptor". Thesis, University of Bristol, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.559233.

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Upon agonist binding the u-opioid receptor (MOPr) is phosphorylated and can recruit arrestin-2 (beta-arrestin-l) and arrestin-3 (p-arrestin-2). Apart from promoting the desensitization and intemalization of the MOPr, the binding of arrestins can also lead to the triggering of alternative sigvalling pathways. It has been shown that kinases including GRK2, PKC and CaMKII can regulate MOPr. Here we used GST -fusion constructs of the intracellular regions of MOPr to investigate whether arrestin-2 and -3 can bind to these sequences in vitro, and secondly to determine whether phosphorylation of these sequences by various kinases can alter their ability to bind arrestins. Arrestin-3 bound to the unphosphorylated intracellular loops (ICL2, ICL3) and the COOH-tail of the MOPr, whereas arrestin-2 displayed preferential binding to the COOH-tail. Phosphorylation of the MOPr COOH-tail by PKC and CaMKII increased arrestin-2 and -3 binding but to a lesser extent than GRK2. The full agonist DAMGO was able to increase the interaction of both GFP-arrestin-2 and -3 to the full length MOPr. Previous published work, suggests that mice lacking arrestin-3, showed reduced opiate-induced constipation and respiratory suppression and the development of tolerance to opiates such as morphine. During my masters degree at Moscow State University (Russia), I designed and synthesized small molecule inhibitors (Kt, K2 and K3; see appendix-I-Ill for the chemical structures) of the arrestin-MOPr interaction using computer-aided drug design. In the GST pull down assay, only K3 affected the arrestin-MOPr interaction. It decreased the association of arrestin-2 b~t increased the association of arrestin-3 to the COOH-tail of the MOPr. K3 did not affect the interaction of arrestin-3 with the GRK2-phosphorylated MOPr COOH-tail fusion protein. Similar results were obtained in the full length receptor, where the association of GFP- arrestin-3 with the HA-tagged-MOPr remained unaffected in the presence of K3 and DAMGO stimulation. On the other hand, in the absence of DAMGO, K3 did increase the interaction of GFP-arrestin-3 with MOPr. In addition, K3 did not bind to the MOPr orthosteric binding sites and did not affect G-protein activation in the presence and absence of DAM GO. Taken together these data suggest that even though the small molecules were unable to inhibit the arrestin-3/MOPr interaction, such a direct modulation of the arrestin-MOPr interaction is possible and specific small molecules can be designed to modulate this interaction of other GPCRs.

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Carter, Alison A. "Molecular pharmacology of agonist-stimulated arrestin-receptor interactions." Thesis, University of Nottingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.440126.

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Eidhoff, Ulf Benno. "Heterologe Expression, Kristallisation und Untersuchungen zur Struktur von Bos taurus [beta]-Arrestin-1 [Beta-Arrestin-1] und Rattus norvegicus PAR-4." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=961407883.

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Correll, Jennifer A. "Nicotine Sensitization in β-Arrestin 2 Knockout Adolescent Mice". Digital Commons @ East Tennessee State University, 2007. https://dc.etsu.edu/etd/2050.

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ß arrestin-2 is a protein involved in signaling of D2 receptors and plays a mediating role in sensitization to psychostimulants and the opiate morphine. In this study, 3-4 week old BA-2 KO and wild type C57/B6 mice received nicotine tartarate (s.c, 0.5 mg/kg free base) for 7 or 14 consecutive days followed by a drug-free period. An acute nicotine challenge followed the drugfree period. Results indicated that the absence of ß-arrestin-2 reduced sensitization to nicotine in Experiment 1. BA-2 KOs eventually demonstrated sensitization in Experiment 2. However, absence of ß-arrestin-2 blocked expression of sensitization on the challenge. After the challenge, brain tissue was removed and the nucleus accumbens was dissected and analyzed for brainderived neurotrophic factor (BDNF). Results showed that BDNF positively correlated with behavioral results. These results appear to indicate the importance of the ß-arrestin-2 protein in locomotor sensitization and that dopamine signaling is related to BDNF.

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Lally, Ciara. "Structural and functional characterization of the arrestin-rhodopsin complex." Doctoral thesis, Humboldt-Universität zu Berlin, 2017. http://dx.doi.org/10.18452/18570.

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Die Aufgabe des Proteins Arrestin ist die Beendigung der Signalweitergabe über den GPCR Signalweg. In Stäbchenzellen bindet Arrestin an Licht-aktiviertes phosphoriliertes Rhodopsin um die Signalweitergabe zu unterdrücken. Die Bindung von Arrestin an Rhodopsin erfolgt in zwei Schritten. Zunächst wechselwirkt Arrestin mit dem phosphorilierten C-Terminus von Rhodopsin und bildet einen prä-Komplex, dies induziert Konformationsänderungen im Arrestin wodurch die Bildung eines High-affinity Komplex unter Kopplung an den helikalen Kern des aktivierten Rezeptors erfolgen kann. Biochemische Untersuchungen und Kristallstrukturen haben einen Einblick in die Konformation des Komplexes aus Arrestin und Rhodopsin ermöglicht. In dieser Arbeit werden site-directed Fluorezenz Experimente angewandt um die strukturellen Änderungen zu untersuchen, die bei der Bindung von Arrestin an Rhodopsin ablaufen und der nterschiedlichen Bindungsmodi innerhalb der Wechselwirkung zwischen Arrestin und Rhodopsin. Insbesondere wird hier eine, bisher nicht beschriebene, Assoziation von Arrestin an die Membran untersucht. Des Weiteren wurden Erkenntnisse über die Struktur des prä-Komplexes gewonnen. Die Konformation vom Arrestin im prä-Komplex scheint die Konformation im Basalzustand nachzubilden unter Beteiligung zweier Kontaktstellen: Interaktion mit dem phosphorilierten C-Terminus des Rezeptors und Assoziation mit der Membran. Beim Übergang in den High-affinity Komplex durchläuft Arrestin eine Konformationsänderung in eine aktivere Konformation: der C-Terminus wird verdrängt, es erfolgt eine Neuausrichtung der zentralen flexiblen Schleifen und die Orientierung des Membranankers ändert sich. Die Aufgabe des prä-Komplexes ist somit Arrestin und den Rezeptor zusammen zu bringen sowie die korrekte Orientierung sicherzustellen um einen schnellen Übergang in den High-affinity Komplex zu ermöglichen. The protein arrestin is responsible for termination of GPCR signalling. In the rod cell, arrestin binds light-activated phosphorylated rhodopsin in order to block further signal transduction. The binding of arrestin to rhodopsin is a two-step process. Arrestin first interacts with the phosphorylated receptor C-terminus in a pre-complex, which induces conformational changes in arrestin that allow coupling to the helical core of the active receptor in a high-affinity complex. Biochemical studies and crystal structures have provided insights into the conformation of the arrestin-rhodopsin complex. This dissertation describes site-directed fluorescence experiments, which were carried out to further investigate the conformational changes occurring upon arrestin binding to rhodopsin and the nature of different binding modes of the arrestin-rhodopsin interaction. In particular this involved characterization of a previously unidentified association of arrestin with the membrane, as well as further elucidation of the structure of the pre-complex. The conformation of arrestin in the pre-complex is indicated to resemble that of the basal state of arrestin, and involves two sites of contact: interaction with the phosphorylated receptor C-terminus, and association with the membrane. Upon transition to the high-affinity complex, arrestin undergoes a conformational change to a more active conformation: the auto-inhibitory C-tail is displaced, there is movement within the central flexible loops, and the orientation of the membrane anchor changes. The pre-complex therefore most likely functions to bring arrestin and the receptor into close contact, and in the correct orientation, to allow for fast transition to the high-affinity complex.

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Obeid, Joëlle. "Caractérisation de la fonction des β-arrestines dans les cellules β pancréatiques : recherche de nouvelles stratégies thérapeutiques pour le diabète de type 2". Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT066.

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Les pertes de la fonction et de la masse des cellules beta pancréatiques jouent un rôle central dans le diabète de type 2 (DT2). Les beta-arrestines 1 et 2 (ARRB1 et ARRB2), sont impliquées dans la sécrétion et/ou la survie des cellules beta pancréatiques.Dans une première étude, afin de caractériser précisément la fonction d’ARRB1 dans les cellules beta pancréatiques, nous avons eu pour objectif de générer des souris invalidées spécifiquement dans ces cellules en utilisant le système Cre/lox sous le contrôle du promoteur Ins1. Des études avaient été publiées à partir des deux lignées Ins1Cre-/+ et Arrb1f/f. Nous avons généré et travaillé sur les souris Arrb1f/f :Ins1Cre-/+. Le phénotype des souris Arrb1f/f :Ins1Cre-/+ était faible et surtout non reproductible comparé aux souris Arrb1f/f :Ins1Cre-/- utilisées comme témoins. Le faible niveau d’expression d'Arrb1 dans les cellules beta et le manque d'anticorps spécifique pour l'immunocytochimie ont rendu difficile la vérification de l'absence d'expression de ARRB1 dans ces cellules. Après séquençage du gène modifié Arrb1 des souris “floxées“, nous avons pu montrer que l'insertion du premier site loxP avait induit un décalage du cadre de lecture introduisant un codon stop et, par conséquent, la non-expression du gène Arrb1. Étant donné que les souris Arrb1 “floxées“ utilisées comme témoins étaient déjà knockout (KO), le projet utilisant ces souris a dû être arrêté.Notre équipe a rapporté l'implication d'ARRB2 dans la régulation de la masse des cellules bêta pancréatique, mais son rôle dans la signalisation du récepteur du Glucagon-Like Peptide-1 (GLP-1R), une cible thérapeutique majeure du DT2, n'avait pas encore été exploré.Nous avons montré, dans une deuxième étude, une meilleure tolérance orale au glucose ainsi qu’une augmentation de la sécrétion d’insuline chez les souris Arrb2 KO par rapport aux souris témoins sur les îlots en présence des concentrations physiologiques circulantes de GLP-1. Ceci est corrélé à une production d’AMPc et un recrutement de la PKA plus élevés dans les cellules beta Arrb2 KO. A l’inverse, l’activation des kinases ERK1/2 est diminuée indiquant un recrutement majeur des ERK1/2 par ARRB2 au GLP-1R. En parallèle, j’ai montré que les taux de ARRB1 et ARRB2 des îlots pancréatiques sont altérés par des conditions diabétogènes et diabétiques. Mes résultats démontrent clairement un rôle critique de ARRB2 dans la signalisation du GLP-1R. Un défaut d’expression de la protéine pourrait participer au déficit des mécanismes de compensation de la masse fonctionnelle des cellules beta conduisant au DT2 The loss of function and mass of pancreatic beta-cells play a central role in type 2 diabetes (T2D). Beta-arrestin 1 and 2 (ARRB1 and ARRB2) are involved in insulin secretion and/or beta-cell survival. In a first study, in order to characterize the role of ARRB1 in beta-cells, we aimed to invalidate the Arrb1 gene specifically in these cells using the Cre/lox system under the control of the Ins1 promoter. Studies had been published with both Ins1Cre-/+ and Arrb1f/f lines. We generated Arrb1f/f:Ins1Cre-/+ mice. The phenotype of Arrb1f/f :Ins1Cre-/+ mice was weak with a lack of reproducibility compared to Arrb1f/f :Ins1Cre-/- mice used as controls. The low expression level of Arrb1 in beta-cells and the lack of specific antibody for immunocytochemistry made it difficult to verify the absence of expression of ARRB1 in these cells. After sequencing the modified Arrb1 gene of the “floxed” mice, we observed that the insertion of the first loxP site induced a shift in the reading frame introducing a stop codon and, consequently, the non-expression of the Arrb1 gene. Since the “floxed“ Arrb1 mice used as controls were already knockout (KO), the project using these mice was stopped.Our team has reported the involvement of ARRB2 in the regulation of beta-cell mass, but its role in Glucagon-Like Peptide-1 (GLP-1) receptor signaling, a major therapeutic target for T2D, remained to be explored. In a second study, we showed a better glucose tolerance and an increase in insulin secretion from isolated islets in Arrb2KO compared to control mice in the presence of physiological circulating concentrations of GLP-1. This was correlated with higher cAMP production and PKA activation in Arrb2KO beta-cells. By contrast, the activation of ERK1/2 kinases was decreased indicating a major recruitment of ERK1/2 by ARRB2 to GLP-1R. In parallel, we showed that the expression levels of ARRB1 and ARRB2 in pancreatic islets were altered in diabetogenic and diabetic conditions. My results clearly demonstrate a critical role of ARRB2 in GLP-1R singaling which could impact the function, maintenance and plasticity of beta-cell mass in response to GLP-1. A lack of expression of ARRB2 could participate in the deficit of compensatory mechanisms of the functional beta-cell mass leading to T2D

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Puca, Loredana. "Role of the arrestin family in notch pathway in mammals." Paris 6, 2013. http://www.theses.fr/2013PA066350.

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La voie de signalisation Notch est une voie conservée au cours de l’évolution impliquée dans le développement embryonnaire et dans l'homéostasie tissuetaire chez les adultes. Un certain nombre de modifications post-traductionnelles ont été impliquées dans la régulation de l'activité du récepteur Notch et certaines d'entre elles ont une incidence sur la dégradation du récepteur Notch non activé. Le point de départ de mon projet de thèse est la découverte chez la Drosophile montrant que la protéine adaptateur Kurtz, l'unique arrestine non-visuelle de la Drosophile, est un régulateur essentiel de la voie de signalisation Notch. Chez les mammifères, la famille des arrestines (à l'exclusion des arrestines visuelles) est composée de deux sous-familles: β-arrestines et α-arrestines. Les principaux résultats que j'ai obtenus montrent que les deux arrestines α-β sont recrutées par le récepteur Notch non-activé et permettent l’ubiquitination de Notch médiée par Itch chez les mammifères. Une hétérodimérisation entre une β-arrestine et l’ α-arrestine ARRDC1 est nécessaire pour promouvoir l'ubiquitination de Notch et la dégradation du récepteur Notch non activé dans le lysosome. Pour conclure, nous montrons pour la première fois que α et β-arrestines agissent comme des régulateurs négatifs de la signalisation Notch, mettant en évidence l'existence d'une coopération entre ces protéines adaptatrices pour réguler le trafic des récepteurs Notch signaling is an evolutionary conserved pathway implicated in embryonic development and in adult tissue homeostasis. A number of post-translational modifications have been implicated in regulating the activity of Notch receptor and some of them affect the degradation of non-activated Notch receptor. The starting points of my PhD project are Drosophila findings showing that the adaptor protein Kurtz, the unique non-visual arrestin in Drosophila, is an essential regulator of Notch signaling. In mammals the arrestin family (excluding the visual arrestins) is composed of two sub-families: -arrestins and-arrestins. The main results that I have obtained show that both -arrestins and -arrestins are recruited to non-activated Notch receptor and allow Itch-mediated Notch ubiquitination in mammals. Biochemical evidence shows that an heterodimerization between -arrestins and the -arrestin ARRDC1 is required to promote Notch ubiquitination and its lysosomal degradation. To conclude, we show for the first time that the --arrestin heterodimer is functionally involved in the degradation of Notch receptor, highlighting the existence of a cooperation between these adaptor proteins to regulate receptor trafficking

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Celver, Jeremy Phillip. "Molecular mechanisms of opioid receptor regulation by GRK and arrestin /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/6299.

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Armando, Sylvain. "Structure quaternaire des récepteurs de chimiokines CXCR4 et CCR2 et interaction avec leur effecteurs." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20208/document.

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Les récepteurs couplés aux protéines G (RCPG) sont la famille de récepteurs membranaires la plus représentée chez les vertébrés, et la plus grande cible thérapeutique chez l'Homme. L'évolution du paradigme initial qui énonçait une stœchiométrie récepteur : protéine G : effecteur de 1 :1 :1 sera présentée sur le modèle des récepteurs aux chimiokines CXCR4 et CCR2. Grâce à la technique de transfert d'énergie par bioluminescence (BRET), les travaux réalisés durant cette thèse montrent (1) que c'est par un couplage alternatif de CXCR4 à Gα13 au lieu de la voie classique Gαi que les cellules de cancer du sein migrent pour former des métastases, (2) que la désensibilisation de CXCR4 implique le recrutement d'une combinaison définie de protéines (GRK et arrestines) permettant l'arrêt sélectif des multiples voies engagées en réponse à l'agoniste, et (3) que le protomère CXCR4 a un rôle déterminant dans l'engagement de la protéine Gαi et le recrutement de la β-arrestine par l'hétéro-oligomère CXCR4/CCR2 lorsque CCR2 est activé. Dans cette dernière et principale étude, les résultats montrent également que le dimère CCR2 peut s' assembler au dimère CXCR4 pour former un tétramère, et que l'activation de CCR2 influence la conformation du dimère CXCR4. Les phénomènes de coopérativité et d'activation asymétrique déjà rapportés pour cet hétérodimère pourraient donc impliquer l'interaction de quatre protomères. En conclusion les travaux effectués durant cette thèse démontrent une régulation supplémentaire de l'activité des récepteurs chimiokines au niveau de leur structure quaternaire, de leur signalisation, et de l'arrêt de cette signalisation G protein coupled receptors (GPCR) are the most represented cell surface receptors among vertebrates, and the major therapeutic target in humans. The initial paradigm stating a 1 :1 :1 stoichiometry for receptor :G protein :effector has evolved to a more complex model, as illustrated here with the example of the chemokine receptors CXCR4 and CCR2. Bioluminescence resonance energy transfer (BRET) was used to demonstrate that (1) CXCR4 is able to couple Gα13 instead of Gαi to promote breast cancer metastasis, (2) the multiple pathways engaged by stimulation of CXCR4 are selectively desensitized by the specific recruitment of a defined combination of proteins (GRKs and arrestins) and (3) the CXCR4 protomer plays a crucial role during Gαi engagement and β-arrestin recruitment by the CXCR4/CCR2 heterodimer upon CCR2 activation. In this last and main study, the results shown also demonstrate that CCR2 dimers could assemble with CX CR4 dimers into hetero-tetramers, and that CCR2 activation leads to a conformational change in the CXCR4 dimer. Former results showing cooperativity and asymmetric activation of a simple CXCR4/CCR2 heterodimer could then be applied to a tetramer. To conclude, the work done during this thesis demonstrates a more sophisticated regulation of chemokine receptors than previously suspected at 3 different levels: quaternary structure of the protomers, G protein signalling, and signalling termination

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Vogt, Vivien. "Untersuchungen zur Phosphorylierung von Rhodopsin und deren Einfluss auf die Arrestin-Rhodopsin-Bindung." Doctoral thesis, Humboldt-Universität zu Berlin, 2021. http://dx.doi.org/10.18452/22841.

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Die phosphorylierungsabhängige Bindung von Arrestin an G-Protein-gekoppelte Rezeptoren (GPCRs) ist ein weit verbreiteter Mechanismus, um aktive Rezeptoren zu inhibieren. Für die Bindung von Arrestin an lichtaktiviertes, phosphoryliertes Rhodopsin (pRho), einem GPCR, der sich in der Diskmembran der Stäbchenzellen der Netzhaut befindet, ist in Lipidmembranen die Phosphorylierung von 2 der insgesamt 7 Phosphorylierungsstellen im Rezeptor-C-Terminus nötig, wohingegen 3 Phosphate das Arrestin quantitativ aktivieren. Welchen Einfluss höhere Phosphorylierungsniveaus auf die Rezeptor-Arrestin-Interaktion haben, ist bisher ungeklärt. In dieser Arbeit wurde daher eine Methode zur quantitativen Bestimmung der Rhodopsin-Phosphorylierung etabliert, die unterschiedlich pRho-Spezies präparativ getrennt und mit den isolierten pRho-Spezies verschiedene Parameter der Rezeptor-Arrestin-Interaktion, wie z.B. die Bindungsstöchiometrie der resultierenden pRho-Arrestin-Komplexe, untersucht. Für die Charakterisierung der Arrestinbindung wurden Titrationen mit 3 verschiedenen fluoreszenzmarkierten Arrestinmutanten und pRho in gemischten Phospholidipd/Detergens-Mizellen durchgeführt. Die in dieser Arbeit gewonnenen Daten zeigen, dass der Phosphorylierungsgrad von Rhodopsin neben der Affinität von Arrestin auch die Bindungsstöchiometrie beeinflusst. So haben die Komplexe bei geringem Phosphorylierungsgrad eine 2 : 1 (pRho : Arrestin) Stöchiometrie, während bei einem sehr hohen Phosphorylierungsgrad bevorzugt 1 : 1 pRho-Arrestin-Komplexe gebildet werden. Zudem weisen ein unterschiedliches Elutionsverhalten bei der säulenchromatographischen Trennung und Abweichungen in der pRho-Arrestin-Stöchiometrie verschiedener pRho-Präparationen mit ähnlicher Gesamtanzahl an Phosphaten auf einen zusätzlichen Einfluss unterschiedlicher Phosphorylierungsmuster hin. Insgesamt deuten die Daten auf eine komplexe Beziehung zwischen dem Phosphorylierungsgrad der Rezeptoren und dem Arrestin-Bindungsmodus hin. The phosphorylation-dependent binding of arrestin to G protein-coupled receptors (GPCRs) is a widely used mechanism to inhibit active receptors. In lipid membranes, the binding of arrestin to light-activated, phosphorylated rhodopsin (pRho), a GPCR located in the disc membranes of retinal rod cells, requires the phosphorylation of 2 of the 7 phosphorylation sites in the receptor C-terminus, whereas 3 phosphates are required to completely activate arrestin. The influence of higher phosphorylation levels on the receptor/arrestin interaction is still unclear. In this thesis, a method for the quantitative determination of rhodopsin phosphorylation was established, rhodopsin species with varying degrees of phosphorylation were separated preparatively and different parameters of the receptor/arrestin interaction, such as the binding stoichiometry of the resulting pRho/arrestin complexes, were investigated for each isolated pRho species. For the characterization of arrestin binding, titrations with 3 different fluorescently labeled arrestin mutants and pRho in mixed phospholipid/detergent micelles were performed. The data obtained in this work show that the phosphorylation level of rhodopsin influences the binding stoichiometry in addition to the affinity of arrestin binding to rhodopsin. Thus, complexes with a low level of phosphorylation have a 2 : 1 (pRho : arrestin) stoichiometry, whereas 1 : 1 pRho/arrestin complexes are preferably formed at a very high degree of phosphorylation. In addition, a different elution behaviour during chromatographic separation and variances in the pRho/arrestin stoichiometry of different pRho preparations with similar overall number of phosphates indicate an additional influence of distinct phosphorylation patterns. Overall, the data indicate a complex relationship between receptor phosphorylation level and arrestin binding mode.

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Mendez, Martinez-David Indira. "Effets anxiolytiques/antidépresseurs et neurogéniques des ligands du récepteur 5-HT4 chez la Souris : rôle de la protéine β-arrestin 1". Thesis, Paris 11, 2013. http://www.theses.fr/2013PA114843/document.

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Les inhibiteurs sélectifs de recapture de la sérotonine (ISRS), agonistes indirects des récepteurs de la sérotonine (5-HT), ont un début d'effet antidépresseur retardé de plusieurs semaines. Des travaux antérieurs suggèrent que le récepteur 5-HT4 de la sérotonine serait une cible directe pour traiter la dépression et un nouvel espoir pour traiter plus rapidement ces pathologies anxio-dépressives. Toutefois, l'hypothèse « 5-HT4 » doit encore être validée dans des modèles animaux d'anxiété/dépression. Les questions posées étaient : la stimulation des récepteurs 5-HT4 centraux est-elle nécessaire aux effets comportementaux des ISRS ? la neurogenèse hippocampique adulte contribue-t-elle à ces effets ? En utilisant le modèle de stress chronique à la corticostérone (CORT) chez la souris, nous avons évalué les effets sur ces paramètres d’un traitement chronique avec un agoniste du récepteur 5-HT4 (RS67333, 1,5 mg/kg/jour pendant 4 semaines) comparé à un traitement à la fluoxétine (18 mg/kg/jour). Nous avons ensuite utilisé ce modèle murin combiné à l’ablation de la neurogenèse hippocampique par rayons-X afin d’examiner si la neurogenèse est nécessaire aux effets comportementaux d’un traitement subchronique (7 jours) ou chronique (28 jours) avec le RS67333. Nous avons également évalué le blocage des effets de la fluoxétine par un antagoniste du récepteur 5-HT4 (GR125487, 1 mg/kg/jour). Le traitement chronique avec RS67333, comme celui de la fluoxétine, induit une activité anxiolytique/antidépressive et stimule la neurogenèse hippocampique adulte. Cependant, contrairement à la fluoxétine , les effets anxiolytiques du RS67333 sont déjà présents après 7 jours de traitement, sans nécessité l’activation de la neurogenèse. Le traitement chronique avec le GR125487 empêche les deux effets anxiolytique/antidépresseur et neurogènique de la fluoxétine, indiquant que l'activation du récepteur 5-HT4 est nécessaire à ces effets de l’ISRS. Nous avons ensuite cherché à savoir si le court délai d’action antidépresseur du RS67333 peut être prédit par l'expression d'un biomarqueur périphérique. Des données de la littérature indiquent que la cascade de signalisation de β-arrestine 1 (impliquée dans la désensibilisation et l’internalisation du récepteur 5-HT4) serait un biomarqueur potentiel pré-clinique/clinique des états dépressifs et des effets d’un traitement antidépresseur. À cette fin, nous avons développé une nouvelle méthode d’évaluation des taux de protéines circulantes grâce à une analyse par immunoblot des leucocytes (PBMC) isolés à partir du sang total de souris. Les taux de β-arrestine 1 sont diminués dans les leucocytes des souris pré-traitées à la CORT. Il faut 7 jours de traitement avec le RS67333, mais 28 jours avec la fluoxétine chez ces animaux pour restaurer un taux de β-arrestine 1 comparable à celui des animaux contrôles. Ces résultats suggèrent que le taux sanguin de β-arrestine 1 est un biomarqueur de la rapidité de la réponse antidépressive. Enfin, l'activation du récepteur 5-HT4 dans le cerveau peut représenter une approche thérapeutique innovante d’apparition pour traiter plus rapidement des symptômes dépressifs associés à l’anxiété Selective serotonin reuptake inhibitors (SSRIs) display a delayed onset of action of several weeks. Past work demonstrated evidence that the 5-HT4 receptor may be a direct target for treating depression and a new hope for fast acting antidepressant treatment. However, the 5-HT4 hypothesis still needs to be validated in models of anxiety/depression.We decided to investigate whether 5-HT4 receptor stimulation was necessary for the effects of SSRIs in a mouse model of anxiety/depression and whether hippocampal neurogenesis contributed to these effects. Using the mouse corticosterone model of anxiety/depression, we assessed whether chronic treatment with a 5-HT4 receptor agonist (RS67333, 1.5 mg/kg/day) had effects on anxiety and depression-related behaviors as well as on hippocampal neurogenesis in comparison to chronic fluoxetine treatment (18 mg/kg/day). Then, using our model combined with ablation of hippocampal neurogenesis, we investigated whether neurogenesis was necessary for the behavioral effects of subchronic (7-days) or chronic (28-days) RS67333 treatment. We also assessed whether a 5-HT4 receptor antagonist, (GR125487, 1 mg/kg/day) could prevent the behavioral and neurogenic effects of fluoxetine. Chronic treatment with RS67333, similar to fluoxetine, induced anxiolytic/antidepressant-like activity and stimulated adult hippocampal neurogenesis. However, unlike fluoxetine, the anxiolytic effects of RS67333 were already present after 7 days and did not require hippocampal neurogenesis. Chronic treatment with GR125487 prevented both anxiolytic/antidepressant-like and neurogenic effects of fluoxetine, indicating that 5-HT4 receptor activation is necessary for these effects of SSRIs. We then explored whether the fast onset of action of the 5-HT4 receptor agonist RS67333 could be predicted by expression of a peripheral biomarker. The β-arrestin-signaling cascade which is involved in 5-HT4 receptor desensitization and internalization, has recently gained attention as a potential pre-clinical/clinical bridging biomarker for depressive states and treatment effects. To this end, we developed a new method to assess levels of circulating proteins through immunoblot analyses of mouse PBMCs isolated from whole blood of anesthetized animals. While we did not detect any change in β-arrestin 1 in mouse leukocytes after 7 days of fluoxetine in corticosterone-treated animals, a short term treatment with RS67333, restored the level of this protein to control levels. In fluoxetine-treated animals, a restoration was only observed in the corticosterone model after a longer exposure. These results suggest that blood levels of β-arrestin 1 may be a useful biomarker to predict antidepressant/anxiolytic activities. Finally, the activation of 5-HT4 receptors in the brain may represent an innovative and rapid onset therapeutic approach to treat depression with comorbid anxiety

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Lee, Kyu Joon. "Molecular Mechanisms of Beta-Arrestin-1 Dependent Regulation of LIMK and Cofilin." Thesis, University of California, Riverside, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10109679.

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Beta-arrestins are adaptor proteins that can scaffold a number of signaling proteins to promote localized activity within the cell. Downstream of some GPCRs, β-arrestins can promote activation of the actin filament severing protein, cofilin, through two mechanisms: one involving inhibition of LIM Kinase (LIMK) which negatively regulates cofilin activity through phosphorylation on serine 3. The mechanism by which β-arrestin-1 regulates LIMK activity has not been elucidated; however, it has been shown to be important for cell migration downstream of protease-activate-receptor-2 (PAR-2), dendritic spine formation and opioid receptor function. Here my work demonstrate that β-arrestin-1 directly binds both cofilin and LIMK, and inhibits LIMK activity directly and investigate the mechanism by which inhibition of kinase activity occurs. Using serial truncations and site-directed mutagenesis, I identify crucial residues for cofilin and LIMK interaction within amino acids 1-99 of β-arrestin-1 and show that charged residues at 50 and 51 are crucial for binding to LIMK and R51 is required for LIMK inhibition, PAR2 stimulated cofilin dephosphorylation and cell migration. Additionally, our work reveals that amino acids 1-99 aminos of β-arrestin-1 bind both cofilin and LIMK with a higher apparent affinity than the full length and blocks PAR2-stimulated cofilin dephosphorylaton in HEK293 cells, suggesting it functions as a selective dominant negative β-arrestin-1, inhibiting specifically the cofilin pathway. Thus, residues in the N-terminus of β-arrestin-1 are involved in LIMK inhibition and cofilin activation and this, in turn, is important for cell migration downstream of PAR-2.

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Webb, K. F. "Elucidating an essential role for β-arrestin 1 in regulating Golgi morphology". Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1418064/.

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β-Arrestins are multifunctional signalling adaptors, facilitating and regulating many signalling pathways in a variety of sub-cellular locales. This thesis work describes a novel role for β-arrestin 1 at the Golgi where it acts as a homeostatic regulator of Golgi morphology and function. β-Arrestin 1 knock-out MEFs have a more dispersed Golgi morphology when compared to WT MEFs, a difference in Golgi morphology that cannot be explained by differences in cell size or the cytoskeleton. Consistent with this observation, the overexpression of β-arrestin 1 in MEFs and HT29 cells causes Golgi compaction, suggesting a regulatory role for β-arrestin 1 at the Golgi. Furthermore, immunofluorescence experiments confirm that endogenous β-arrestin 1 is localised to this organelle. Mutants of β-arrestin 1 with characterised disruptions in protein binding abilities were expressed in HT29 cells and Golgi morphology was assessed by immunofluorescence. A mutant of β-arrestin 1 previously shown to lack the ability to bind AP2 failed to induce the compact Golgi phenotype associated with wild-type β-arrestin 1 expression. We confirmed by co-immunoprecipitation that β-arrestin 1 binds to AP1 suggesting that the association of β-arrestin 1 with AP1 is required for maintenance of a tightly packed Golgi structure. Conversely, the expression of a previously reported non-Src binding mutant produced an excessively compact Golgi. Inhibition of ERK, but not Src, activity recapitulated this phenotype suggesting this mutant may be defective in both Src and ERK binding, and implicating ERK in β-arrestin 1-mediated Golgi dispersal. Trafficking of the ts045-VSV-G protein is also perturbed when these β-arrestin 1 mutants are expressed, suggesting a functional role for β-arrestin 1 at the Golgi. β-Arrestin 1/AP1 complex formation is potentiated by active Src and serves to negatively regulate anterograde trafficking in addition to preventing Golgi dispersal. Conversely, β-arrestin 1 mediates ERK activation to facilitate anterograde trafficking and promote Golgi dispersal. β-Arrestin 1 thus likely plays a homeostatic role at the Golgi maintaining Golgi structure in the face of highly variable membrane flux through this organelle.

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Groer, Chad E. "Agonist-selective regulation of the mu opioid receptor by βarrestins". The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1284396146.

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Nkwayana, Nonhlanhla. "β-arrestin interacting domains on the type II gonadotropin-releasing hormone (GnRH) receptor". Master's thesis, University of Cape Town, 2006. http://hdl.handle.net/11427/3178.

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Includes abstract. Includes bibliographical references. Includes bibliographical references (leaves 74-81). Over-expression of β-arrestin 1 in COS-l cells revealed that the mammalian type GnRH receptor can internalise in a β-arrestin dependent manner whereas the internalisation of the mammalian type I GnRH receptor is β-arrestin independent. investigate which domains on the mammalian type II GnRH receptor are required for β~arrestin dependent internalisation, chimeric receptors were created. The mammalian type II GnRH receptor possesses an intracellular C-terminal tail that is known to play a role in desensitisation, internalisation and overall signalling in GPCRs. On the other hand, the mammalian type I GnRH receptor, which lacks a C-terminal tail, does not readily desensitise and undergoes slow internalisation compared to the mammalian type II GnRH receptor. Over-expression of ß-arrestin 1 in COS-l cells revealed that the mammalian type GnRH receptor can internalise in a ß-arrestin dependent manner whereas the internalisation of the mammalian type I GnRH receptor is ß-arrestin independent. investigate which domains on the mammalian type II GnRH receptor are required for ß-arrestin dependent internalisation, chimeric receptors were created. Firstly, a chimera in which the full length type II GnRH receptor C-terminal tail was added to the tail-less type I GnRH receptor (TI/T2tail) was created. This chimera internalised in a ß-arrestin and GRK dependent manner, demonstrating that the type II GnRH receptor C-terminal tail confers ß-arrestin JGRK dependent internalisation on the originally ß-arrestin/GRK insensitive GnRH receptor. Mutating the putative GRK and casein kinase phosphorylation sites (serines 338 and 339) on the C-terminal tail of TI/T2tail to alanine residues did not abolish ß-arrestin dependent internalisation but eliminated GRK dependent internalisation, suggesting that other regions on the C-terminal tail are required for ß-arrestin dependent internalisation. A second chimera, in which the whole third intracellular loop of the type II GnRH receptor was replaced with that of the type I GnRH receptor (T2/TIICL3), was created. This chimera could not utilise ß-arrestin in its internalisation, indicating that the third intracellular loop of the type II GnRH receptor is required for ß-arrestin dependent internalisation. An alignment of the amino acid sequences of the two mammalian GnRH receptor third intracellular loops identified a basic residue rich area (R234, R236 and K237) on the type II GnRH receptor that was absent on the type I GnRH receptor. Interestingly, the triple mutant (R234,236,K237 A) still internalised in a ß-arrestin dependent manner, however, truncation of the C-terminal tail of R234,236,K237A abolished the ability of the receptor to internalise in a ß-arrestin dependent manner. This result indicated that the C-terminal tail of the type II GnRH receptor was compensating for the absence of the three basic residues. To summarise, this thesis demonstrates that the C-terminal tail of the type II GnRH receptor can confer ß-arrestin dependent intemalisation on the type I GnRH receptor. Furthermore, the third intracellular loop, and more specifically, basic residues R234, R236 and K237 on the mammalian type II GnRH receptor are required for ß-arrestin dependent intemalisation.

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Jones, Kymry Thereasa. "The role of beta-arrestin in regulating the muscarinic acetylcholine type II receptor." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/24815.

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Thesis (Ph.D.)--Biology, Georgia Institute of Technology, 2008. Committee Chair: Dr. Nael A. McCarty; Committee Co-Chair: Dr. Darrell Jackson; Committee Member: Dr. Alfred H. Merrill; Committee Member: Dr. Barbara D. Boyan; Committee Member: Dr. Harish Radhakrishna; Committee Member: Dr. Marion B. Sewer

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Cancellieri, C. "BETA-ARRESTIN DEPENDENT REGULATION OF CYTOSKELETON DYNAMICS AND SIGNALLING OF CHEMOKINE RECEPTOR ACKR2." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/229565.

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Chemokines promote leukocyte migration through the activation of dedicated G-protein coupled receptors. Beyond conventional chemokine receptors, which directly induce cell migration through heterotrimeric Gαi-mediated signalling events, a set of atypical chemokine receptors (ACKRs) have been described. ACKRs do not activate Gαi-mediated signalling activity, but they are mainly involved in shaping the chemokine gradient. The best characterized member of this family is ACKR2. ACKR2, previously referred to as D6, is a scavenger receptor that binds with high affinity to 13 inflammatory CC chemokines. The scavenging activity of ACKR2 relies on its intracellular traffic properties. Under homeostatic conditions, ACKR2 is mainly localized in intracellular stores associated with both early Rab4/5-positive and recycling Rab11-positive endosomes. At increasing levels of chemokines, ACKR2 increases plasma membrane abundance through an acceleration in the rate of Rab11-depedent recycling pathway, in order to optimize its chemokine scavenging activity. Here, I demonstrated that the intracellular distribution of ACKR2 is maintained by cytoskeletal dynamics. After chemokine engagement, ACKR2 activate a G-protein-independent and β-arrestin-dependent Rac1-PAK1-LIMK1 signalling cascade to finely regulate the actin cytoskeletal and the microtubules network reorganization, to promote receptor up-regulation and scavenging function. ACKR2 is able to recruit and associates both β-arrestins in basal condition, at membrane and intracellular levels, but only β-arrestin1 is recruited after active ligand stimulation, in order to promote a β-arrestin1-dependent signalling pathway, required for supporting the myosin Vb-dependent ACKR2 up-regulation and scavenging properties.

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Darcet, Flavie. "Rôle du récepteur 5-HT4 et de la protéine beta-arrestine 1 dans la modulation des processus émotionnels et cognitifs dans un modèle d'anxiété-dépression." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS126/document.

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Les troubles cognitifs constituent des symptômes quasi constants parmi les patients souffrant de dépression caractérisée. De récentes études indiquent que ces troubles mentaux pourraient bénéficier de la modulation de la signalisation du récepteur sérotoninergique 5-HT4. Dans ce travail de thèse, nous avons dans un premier temps caractérisé les troubles cognitifs dans un modèle d’anxiété/dépression chez la Souris, le modèle CORT. Nous avons ensuite évalué l’efficacité d’un traitement chronique par un agoniste du récepteur 5-HT4, le RS 67333, en comparaison à la fluoxetine, un antidépresseur monoaminergique de référence sur les altérations émotionnelles et cognitives. D’autre part, des données de la littérature indiquent que la cascade de signalisation de la protéine β-arrestine 1 (impliquée dans la désensibilisation et l’internalisation du récepteur 5-HT4) serait un biomarqueur potentiel préclinique/clinique des états dépressifs et de la réponse au traitement antidépresseur. Nous avons donc cherché à définir le phénotype anxio/dépressif et cognitif chez des souris tissus-spécifiques conditionnelles, dont l’expression de la protéine β-arrestine 1 dans les cellules souches du gyrus dentelé a été supprimée. L’ensemble de ces travaux de thèse met en avant le rôle prépondérant de l’activation du récepteur 5-HT4 non seulement dans sa capacité à corriger les symptômes d’anxiété/dépression mais aussi les troubles cognitifs associés à la dépression dans un modèle d’anxiété/dépression chez la Souris. D’un point de vue mécanistique, ces données confirment l’implication de la protéine β-arrestine 1 dans la réponse à la fluoxetine au niveau comportemental et directement au sein du processus de neurogenèse hippocampique chez la souris adulte. Enfin, l’expression de la protéine β-arrestine 1 dans les jeunes neurones de l’hippocampe se révèle aussi être un acteur déterminant dans les processus cognitifs et la réponse à la fluoxetine et au RS 67333 Cognitive disturbances are often reported as serious incapacitating symptoms by patients suffering from major depressive disorders. Recent studies showed that these mood disorders could benefit from the modulation of 5-HT4 receptor pathway. Here, we performed a complete characterization of cognitive functions in a neuroendocrine mouse model of depression, the CORT model. We then evaluated emotional and cognitive effects of either a chronic 5-HT4 receptor agonist treatment, RS 67333 or fluoxetine, a classical monoaminergic antidepressant. Recent data indicate that β-arrestins proteins could be an important molecular determinant in depressive states and in the effects of antidepressants. We determined emotional and cognitive phenotypes in conditional tissue-specific mice in which expression of β-arrestin 1 in adult hippocampal stem cells was deleted. This work suggests that the 5-HT4 receptor plays a major role to correct not only anxiety/depression-related symptoms but also cognitive alterations in a mouse model of anxiety/depression model. Moreover, these data confirm the involvement of β-arrestin 1 protein in behavioral and neurogenic responses to fluoxetine treatment in adult mice. Finally, β-arrestin 1 protein expression in adult born neuron is critical for cognition and also to fluoxetine and RS 67333 response

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Wilham, Laura Elizabeth. "The role of [beta]-arrestin in agonist-induced down-regulation of the M₁mAChR." CONNECT TO THIS TITLE ONLINE, 2006. http://etd.lib.umt.edu/theses/available/etd-03022007-104437/.

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Codega, Paolo. "Prolonged illumination up-regulates arrestin and two GCAPs: a novel mechanism for light adaptation." Doctoral thesis, SISSA, 2008. http://hdl.handle.net/20.500.11767/4644.

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Habourdin, Clemence. "Caractérisation des membres du clan arrestine chez l'amibe Dictyostelium discoideum : Etude de la protéine AdcA et de son partenaire FrmC." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV074/document.

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Le clan « arrestine » est une superfamille de protéines adaptatrices regroupant les arrestines conventionnelles (β-arrestines et arrestines visuelles), bien décrites pour leur rôle dans la régulation des récepteurs membranaires couplés aux protéines G hétéro-trimériques et la signalisation associée et des protéines de type arrestin-like identifiées plus récemment, qui semblent partager des fonctions communes dans la régulation et le trafic de cargos membranaires. Ce travail de thèse a porté sur l'étude de la protéine arrestin-like AdcA de Dictyostelium discoideum dans l'objectif d'en définir le rôle fonctionnel. En plus de son module arrestine, caractéristique de cette famille de protéines, AdcA possède un domaine FYVE qui permet son association à la voie endocytaire, un domaine C-terminal riche en tyrosines et un domaine N-terminal riche en histidines impliqué dans son oligomérisation. Mes travaux ont permis d'établir que la protéine AdcA répond à des stress de natures diverses dont l'hyper-osmolarité, par une multi-phosphorylation massive, notamment au niveau du domaine N-terminal. La sensibilité d'AdcA à la dépolymérisation du réseau d'actine suggère un lien fonctionnel entre le cytosquelette d'actine et la cascade de signalisation menant à la phosphorylation de la protéine. En conditions de stress modéré, la phosphorylation d'AdcA est transitoire et sa déphosphorylation, en partie dépendante du facteur de transcription STATc, corrèle avec l'adaptation des cellules aux conditions de stress. Cette modification post-traductionnelle transitoire pourrait permettre de contrôler l'activité d'AdcA et d'optimiser la réponse des cellules au stress. Parallèlement, la caractérisation fonctionnelle d'un partenaire d'AdcA, la protéine FrmC, a été entreprise. FrmC est une nouvelle protéine, encore non caractérisée, présentant plusieurs domaines fonctionnels dont un domaine FERM capable de lier l'actine in vitro et un domaine à motifs LRR. Mes travaux ont notamment mis en évidence que FrmC est recrutée à la membrane plasmique et joue un rôle dans l'adhésion cellulaire. Par ailleurs, l'absence de FrmC affecte l'adaptation des cellules et la réponse d'AdcA en situation de stress hyper-osmotique The arrestin clan represents a large group of adaptor proteins which includes the canonical arrestins - β-arrestins and visual arrestins – well described for their role in the regulation of membrane receptors coupled to heterotrimeric G-proteins and associated signaling as well as arrestin-like proteins identified more recently that seem to share functions in the regulation and trafficking of membrane cargoes. This work focused on the study of the arrestin-like protein AdcA of Dictyostelium discoideum, to determine the functional role of this atypical member. In addition to the arrestin module common to all the members of the arrestin family, AdcA harbors a FYVE domain responsible for its association to the endocytic pathway, a C-terminal tyrosine-rich domain and an N-terminal extension rich in histidine residues mediating its oligomerization. I have established that AdcA responds to a variety of stresses such as hyperosmolarity by a massive multi-phosphorylation of the protein. Sensitivity of AdcA to changes in F-actin polymerization status suggests a link between the signaling cascade leading to AdcA phosphorylation and the actin cytoskeleton. In conditions of moderate stress, AdcA response is transient and its dephosphorylation depends on the transcription factor STATc and correlates with cell adaptation to the stress conditions. This post-translational modification of AdcA could modulate its activity and optimitize the cell response to stress. In parallel, the functional characterization of a partner of AdcA, the protein FrmC, has been undertaken. This so-far uncharacterized protein presents a multimodular structure with a FERM domain able to bind F-actin in vitro and several leucine-rich repeats (LRR). I have shown that FrmC is recruited to the plasma membrane and is involved in cell-substrate adhesion. In addition, disruption of FrmC affects cell adaptation and AdcA response in conditions of hyperosmotic stress

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Gillis, Alexander. "µ-opioid receptor ligand pharmacology in cell and animal models." Thesis, University of Sydney, 2020. https://hdl.handle.net/2123/23113.

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Opioid analgesics are a critical class of medications, with severe side effects such as lethal respiratory depression. The primary target of these drugs is the µ-subtype of the opioid receptor (MOR), a G protein-coupled receptor (GPCR). In addition to G protein signalling, putative signalling of the regulatory protein β-arrestin 2 has been implicated in mediating opioid side effects. Whole-body plethysmography (WBP) was used to assess respiratory changes induced by opioid agonists in mice. Contrary to previous reports, morphine respiratory depression was unaltered in β-arrestin 2 knock-out mice. Persistent opioid respiratory depression was also observed in response to fentanyl, and in a separate genetic model deficient in MOR/β-arrestin 2 interactions, employing mice expressing a phosphodeficient, arrestin non-recruiting mutant MOR. These results are not consistent with any role of MOR/β-arrestin 2 interactions in mediating or positively facilitating opioid respiratory depression. Putatively G protein-biased agonists were assessed together with opioid analgesics in a G protein-coupled potassium (GIRK) channel assay of MOR activation. Agonists proposed to be biased, oliceridine (TRV130), SR-17018, and PZM21, all showed low intrinsic efficacy relative to the prototypical morphine, fitting into a spectrum between uniquely low efficacy buprenorphine and the full agonists fentanyl and DAMGO. Mathematical analysis of this and published signalling assay data shows how low efficacy agonists might be theoretically and empirically predicted to appear to be G protein-biased, and how existing pharmacological quantification is confounded by dynamic, non-equilibrium systems. Fluorescence-based assays confirmed that rank order of arrestin-recruitment efficacy is predicted by accurate G protein efficacy estimates. These novel partial agonists were assessed in vivo against a panel of control, existing opioids. Some slight improvement relative to morphine in this pre-clinical model, comparing hotplate antinociception to WBP respiratory depression, was observed. Very low efficacy buprenorphine produced similar antinociception to other agonists but with minimal respiratory depression at very high doses, whereas high efficacy fentanyl had a narrow, pre-clinical therapeutic window. Partial agonism is a plausible alternative explanation for the pre-clinical and clinical profiles of both apparently biased lead compounds and existing opioid analgesics. Fluorescence assays of receptor activation show that the rate of activation is efficacy-dependent, whereas the rate of receptor deactivation is ligand-dependent but efficacy-independent, and correlates across several functional assays. Total-internal reflection microscopy shows that agonist-induced changes in single-receptor resolution studies of receptor mobility are efficacy-dependent. Future development of opioid analgesics should consider both intrinsic efficacy, and proposed ligand bias where it can be robustly demonstrated, in the context of receptor signalling kinetics.

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Clayton, Ashley Rebecca. "Investigation of the role of SASH1 in the regulation of TRAF6-beta-arrestin complex formation." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/48602.

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Proper activation and regulation of innate immune signaling pathways and inflammatory responses are essential to efficiently fight infection. Dysregulation of these responses, however, can result in the development of a severe hyper-inflammatory state, ultimately leading to sepsis. The endothelium has been shown to play a critical role in innate immune responses and the pathogenesis of sepsis. In the presence of Gram-negative infection, circulating LPS is recognized by TLR4 expressed on the surface of endothelial cells, mediating downstream activation of pro-inflammatory signal cascades and immune responses. SASH1 has been recently characterized as a novel scaffold protein in the regulation of endothelial TLR4 signaling. The work presented in this thesis further characterizes the role of SASH1 in the regulation of TRAF6 activation within the TLR4 pathway. LPS-induced auto-ubiquitination of TRAF6 is an essential regulatory step in the downstream activation of TLR4-mediated inflammatory signaling. Co-immunoprecipitation analyses confirmed the interaction of endogenous SASH1 with TRAF6 in an LPS-dependent manner in endothelial cells. Furthermore, SASH1 was required for LPS-induced auto-ubiquitination of TRAF6. To further investigate SASH1 function, a yeast two-hybrid approach was employed to identify novel SASH1-interacting proteins. β-arrestin 1 was identified as the top potential interactor of SASH1. Both β-arrestin 1 and β-arrestin 2 have been shown to act as negative regulators of the TLR4 pathway by impairing the oligomerization and auto-ubiquitination of TRAF6. It was speculated that SASH1 may coordinate the interaction of TRAF6 with the β-arrestins to mediate regulation of the TLR4 pathway. Reciprocal co-immunoprecipitation experiments confirmed the interaction of SASH1 with both β-arrestin 1 and β-arrestin 2. SASH1 was also found to interact with β-arrestin 1 in a complex with TRAF6. Further studies aimed to characterize the role of SASH1 in coordinating the interaction of TRAF6 with the β-arrestins. Although knockdown of SASH1 did not modulate TRAF6-β-arrestin binding, the enforced expression of SASH1 was found to specifically impair TRAF6 binding with β-arrestin 1, but not β-arrestin 2. The significance of this isoform-specific regulation remains to be determined. Overall, these studies further investigate the role of SASH1 as a critical scaffold protein in the regulation of the TLR4 pathway. Medicine, Faculty of Medicine, Department of Experimental Medicine, Division of Graduate

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Vogt, Vivien [Verfasser]. "Untersuchungen zur Phosphorylierung von Rhodopsin und deren Einfluss auf die Arrestin-Rhodopsin-Bindung / Vivien Vogt." Berlin : Humboldt-Universität zu Berlin, 2021. http://d-nb.info/1233678582/34.

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Vogt, Viola [Verfasser]. "Effekte chemisch induzierter β-Arrestin-1-Translokation an die Chemokinrezeptoren CXCR4 und CCR5 / Viola Vogt". Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://d-nb.info/1238345557/34.

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Carrat, Gaëlle. "La voie de signalisation ERK1/2 couplée au récepteur 5-HT4 et sa régulation par GRK5." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20187/document.

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Les récepteurs couplés aux protéines G (RCPG) peuvent activer des voies de signalisation indépendantes des protéines G. Cependant, la régulation de ces voies, et en particulier leur désensibilisation, est peu connue. Le récepteur de la sérotonine de type 4 (R-5-HT4) est un RCPG exprimé dans le cerveau et les organes périphériques. Il est impliqué dans des fonctions physiologiques importantes comme la mémoire, l'apprentissage, la prise de nourriture, le contrôle respiratoire et la mobilité gastro-intestinale. Le R-5-HT4 est couplé à la protéine Gs. De plus, il active la voie Src/ERK1/2, indépendamment des protéines G et des β-arrestines.Nous avons montré que GRK5, physiquement associé à la région C-terminale (C-ter) du R-5-HT4 inhibait la voie Src/ERK1/2 couplée au récepteur, mais pas la voie Gs. Ce résultat a été observé dans la lignée de cellules HEK-293 mais aussi dans des neurones de collicules en culture. Cette inhibition nécessite deux séquences d'évènements : l'association de la β-arrestine1 à une région riche en sérines et thréonines, localisée dans le domaine C-ter du récepteur et la phosphorylation par GRK5, de la β-arrestine1 (en sérine 412) liée au récepteur. La β-arrestine1 phosphorylée empêche l'activation de Src, constitutivement liée au récepteur, nécessaire à l'activation d'ERK1/2. Ceci constitue la première démonstration que la phosphorylation d'une β-arrestine par une GRK régule la signalisation indépendante des protéines G. En plus de ces résultats, nous avons démontré que l'activation d'ERK1/2 par le R-5-HT4, indépendante des β-arrestines, implique la libération d'un ligand induite par une métalloprotéase, conduisant à la transactivation d'un autre récepteur. Par une approche protéomique, nous avons également identifiés plusieurs partenaires potentiels du R-5-HT4. L'étude de ces partenaires pourrait apporter un éclairage supplémentaire sur les voies de signalisation du récepteur et leur régulation G protein-coupled receptors (GPCRs) have been found to trigger G protein-independent signalling. However, the regulation of G protein-independent pathways, especially their desensitization, is poorly characterized.The 5-Hydroxytryptamine 4 receptor (5-HT4R) is a GPCR widely expressed in the brain and at the periphery. It is implicated in important physiological functions such as memory, cognition, feeding, respiratory control and gastrointestinal motility. 5-HT4R couples to the Gs/cAMP/PKA pathway. Moreover, this receptor can activate a Src/ERK pathway independently of both G proteins and β-arrestins.Here, we show that the G protein-independent 5-HT4R-operated Src/ERK pathway, but not the Gs pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor C-terminus, in both HEK-293 cells and colliculi neurons. This inhibition requires two sequences of events: the association of β-arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C-terminal domain and the phosphorylation by GRK5 of β-arrestin1 (at Ser 412) bound to the receptor. Phosphorylated β-arrestin1 prevents in turn activation of Src constitutively bound to 5-HT4R, a necessary step in receptor-stimulated ERK signalling. This is the first demonstration that β-arrestin phosphorylation by a GRK regulates G protein-independent signalling.In addition to these results, we also demonstrated that the β-arrestin-independent activation of ERK1/2 by the 5-HT4R involves a metalloprotease-dependant ectodomain shedding and transactivation of another receptor. By a proteomic approach, we also identified several potential partners of the 5-HT4R. Study of these proteins may provide a better understanding of 5-HT4R signalling and his regulation

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Goh, Poh. "Roles of protein kinase C and arrestin in migration of cells via CXCR4/CXCL12 signalling axis." Thesis, University of East Anglia, 2018. https://ueaeprints.uea.ac.uk/67806/.

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Aim: The chemokine system not only coordinates leukocyte migration in immunity and inflammation, but it is also implicated in the pathogenesis of many human diseases, including cancer. The expression of chemokines and their receptors is altered in many malignancies and leads to aberrant chemokine receptor signalling. Emerging evidence indicates that the tumour microenvironment has critical roles in all aspects of cancer biology, including growth, angiogenesis, metastasis and progression. One of the important representatives of this system are the chemokine ligand CXCL12 and its receptor, CXCR4 as they are most commonly found on human and murine cancer cells. Our aims are to study and understand if there are any differences in activation of signalling molecules in the downstream signalling cascades in CXC- chemokine receptors in different cell types, and to identify the importance of different effector proteins in migration of cells; the two proteins of interest include Protein Kinase C (PKC) and arrestins. Methodology: Experimentation was undertaken in MCF-7 breast cancer cells and Jurkat leukemic T-lymphocytes which both naturally express the chemokine receptor CXCR4. Small molecule inhibition and protein overexpression was used in chemotaxis and calcium release assays to measure cellular responses. Immunocytochemistry was used to determine the effect of protein blocking and protein overexpression on receptor internalisation, protein localisation and the formation of cellular structures associated with migration. Results: Inhibition of PKC has no effect on Jurkat cell migration, but it blocks MCF-7 cell migration showing that there is a difference in the usage of PKC in different cell types. Arrestin 3 is important for migration in both suspension Jurkat cells and adherent breast cancer MCF-7 cells. Conclusion: Our study shows that CXCL12-induced migration may be arrestin 3 mediated. We have also shown that activation of signalling molecules needed for CXCL12-induced migration can differ between different cell lines. Overall, the research in this thesis has identified potential signalling molecules that can be targeted to interfere with migration of cells.

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Correll, Jennifer A., Daniel M. Noel, A. Brianna Sheppard, Kimberly N. Thompson, Yi Li, Deling Yin, and Russell W. Brown. "Nicotine Sensitization and Analysis of Brain-Derived Neurotrophic Factor in Adolescent Beta-Arrestin-2 Knockout Mice." Digital Commons @ East Tennessee State University, 2009. https://doi.org/10.1002/syn.20625.

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Nicotine sensitization and levels of brain-derived neurotrophic factor (BDNF) were analyzed in adolescent beta-arrestin-2 knockout (betaA-2 KO) and wild type (WT) mice. The beta-arrestin-2 protein has been shown to be important in G-protein hydrolysis and receptor internalization. Four- to five-week-old adolescent betaA-2 KO and WT C57/Bl6 mice were administered either nicotine (0.5 mg/kg free base) or saline 10 min before being placed into a locomotor arena on each of 7 (Experiment 1) or 14 (Experiment 2) consecutive days. A nicotine challenge was given 7 days after sensitization was complete. In Experiment 1, betaA-2 KO mice administered nicotine or saline and WT mice administered nicotine demonstrated significant hypoactivity during early in testing, and neither WT nor betaA-2 KO mice administered nicotine demonstrated sensitization. On the nicotine challenge, WT mice administered nicotine demonstrated significantly higher activity levels compared to all groups, and this same group demonstrated significantly higher levels of accumbal BDNF compared to all groups. In Experiment 2, betaA-2 KO mice were again hypoactive compared to WT mice, whereas WT mice administered nicotine demonstrated significant hypoactivity during initial testing and significantly higher levels of activity compared to all other groups late in testing. On the nicotine challenge, WT mice that received nicotine demonstrated a significant increase in activity compared to all groups, and showed increased accumbal BDNF compared to all groups. These results show that the beta-arrestin-2 protein is important in induction and expression of nicotine sensitization as well as nicotine's effects on accumbal BDNF.

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Mößlein, Nadja [Verfasser], and Moritz [Akademischer Betreuer] Bünemann. "Untersuchungen zu Mechanismen der Co-Internalisierung von Arrestin-3 mit GPCRs / Nadja Mößlein ; Betreuer: Moritz Bünemann." Marburg : Philipps-Universität Marburg, 2021. http://d-nb.info/1240383878/34.

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Santos, Ana Paula Carneiro dos. "Participação da via de sinalização da beta-arrestina na produção de óxido nítrico induzido pelo shear stress." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/5/5166/tde-01042015-150141/.

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As células endoteliais são capazes de converter o estímulo mecânico em sinais intracelulares e produzir fatores vasoativos como o óxido nítrico (oNO). Evidências recentes sugerem que as beta-arrestinas desempenham um papel importante não somente na dessensibilização e internalização de receptores acoplados à proteína G (GPCR) como também na mecanotransdução. Nós testamos a hipótese de que células endoteliais submetidas ao shear stress (SS) produzem oNO por meio da ativação da via de sinalização dependente de beta-arrestina. Para tal, células endoteliais de veia safena (hSVEC) foram transfectadas com siRNA contra as isoformas 1 e 2 da beta-arrestina e, posteriormente, submetidas ao SS (15 dinas/cm2) durante 10 min. Nós encontramos que as SVEC silenciadas para a beta-arrestina 1/2 (70%) exibiram uma menor produção de nitrito no meio de cultura em resposta ao SS (166±17 vs. 326±44% comparado com hSVEC transfectadas com siRNA controle). Além disso, o silenciamento da beta-arrestina 1 e 2 preveniu os níveis de fosforilação da Akt no resíduo de serina 473 e a fosforilação da eNOS no resíduo de serina 1177, enquanto que a fosforilação da ERK 1/2 manteve-se inalterada. Curiosamente, análises de imunoprecipitação mostraram que a beta-arrestina interage com caveolina-1, um mecanossensor do shear stress, mas não é influenciado pelo SS. Além disso, na situação estática, a beta-arrestina encontra-se em uma localização perinuclear e, após o SS, adquiriu um padrão mais difuso no citosol. Coletivamente, esses dados sugerem que a beta-arrestina e a sinalização downstream Akt/ eNOS são necessárias para a produção de oNO induzido por shear stress em células endoteliais vasculares humana Endothelial cells are capable of converting mechanical stimuli into intracellular signals generating vasoactive factors such as nitric oxide (oNO). Recent evidence suggests that beta-arrestins play a role not only on G protein-coupled receptors (GPCR) desensibilization but also in mechanotransduction. We tested the hypothesis that beta-arrestin and its downstream signaling influence laminar shear stress (SS)-induced oNO production by endothelial cells. Towards this end, human saphenous vein endothelial cells (hSVEC) transfected with siRNA against beta-arrestins isoforms 1 and 2 were subjected to SS (15 dynes/cm2, 10 minutes). We found that the SS-induced production of nitrite in the cell culture medium from down-expressed beta-arrestin 1/ 2 (70%) SVEC decreased (166±17 vs. 326±44% compared to wild-type hSVEC; P < 0.001). The beta-arrestin 1 and 2 down-regulation in SVEC also inhibited the phosphorylation levels of Akt at the serine residue 473 and the phosphorylation levels of eNOS at the serine residue 1177, whereas ERK phosphorylation remained unchanged. Interestingly, immunoprecipitation analysis showed that beta-arrestin interacts with caveolin-1, a shear stress mechanosensor, which is not influenced by SS despite the fact that the static perinuclear localization of beta-arrestins changed to the cytosol upon SS. Collective these data suggest that beta-arrestin and Akt/eNOS downstream signaling are required for shear stress-induced nitric oxide production in human vascular endothelial cells

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Beyrière, Florent [Verfasser], Klaus Peter [Akademischer Betreuer] Hofmann, and Peter [Akademischer Betreuer] Hildebrandt. "Spectroscopic and microscopic analysis of arrestin-rhodopsin interactions / Florent Beyrière. Gutachter: Peter Hildebrandt. Betreuer: Klaus Peter Hofmann." Berlin : Technische Universität Berlin, 2015. http://d-nb.info/1067388826/34.

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Seitz, Katharina [Verfasser], and Alexandra [Akademischer Betreuer] Schambony. "Funktionelle Charakterisierung des Frizzled-Dishevelled-Arrestin Komplexes in Frizzled abhängigen Wnt-Signalwegen / Katharina Seitz. Betreuer: Alexandra Schambony." Erlangen : Universitätsbibliothek der Universität Erlangen-Nürnberg, 2012. http://d-nb.info/102636535X/34.

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Zimmerman, Brandon. "Regulation of angiotension II type I receptor signalling by beta-arrestin and the clathrin adaptor AP-2." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107645.

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G protein-coupled receptors (GPCRs) play fundamental roles in our homeostatic balance through their involvement in numerous physiological processes. In order to maintain their responsiveness to the extracellular environment, a complex process of receptor desensitization and resensitization has evolved. To allow receptors to be resensitized, they must first undergo endocytosis, a process involving numerous adaptor proteins, which facilitate the internalization of the receptor into the cell. The most common method of GPCR internalization engages the clathrin-dependent pathway, where the two most prominent adaptors are βarrestin and AP-2. While there is a relatively clear picture of how these proteins mediate receptor endocytosis, regulatory mechanisms through which these endocytic adaptors may affect or be affected by signalling events is significantly less well described.The angiotensin II type I receptor is used as the model receptor in our studies, as previous work in our lab characterized some of the protein factors necessary for the endocytic process. While the previous work demonstrated phosphorylation-dependent regulation of βarrestin and AP-2 complex at a putative tyrosine residue, these results were solely in vitro, and had not been confirmed in living cells. As such we generated a polyclonal antibody against this putative phosphorylation site and revealed that not only did this phosphorylation event occur in multiple cell types in response to angiotensin II (AngII) treatment but that the activation of multiple receptors including a non-GPCR, the epidermal growth factor receptor, induced phosphorylation on the β-subunit of AP-2, the main subunit involved in βarrestin binding. My work, which is published in a previous manuscript, also revealed that prevention of this phosphorylation event stabilizes βarrestin/AP-2 complex dramatically. While our initial studies revealed that this phosphorylation event had little impact on receptor endocytosis, new tools have since been developed in the lab. Mainly, we generated a single chain antibody that can be expressed intracellularly targeting the phosphosite, and demonstrated that binding to this phosphorylated residue results in a prolonged endocytic block. We further established this phosphorylated tyrosine residue as a putative binding motif for certain Src homology 2 (SH2) domain containing proteins as three are capable of binding phosphorylated β2adaptin. Finally, due to the βarrestin-dependent nature of this phosphorylation event, we characterized four AngII analogs with single amino acid substitutions in their octapeptide sequence. Our findings established a correlative relationship between the strength of βarrestin to receptor avidity and the level of extracellular signal-regulated kinase (ERK) activation. Presumably, the differences in avidity would alter the trafficking of the receptor affecting its fate towards recycling or degradation. Furthermore, we establish that the conformation of βarrestin can alter the pathways activated downstream of the receptor resulting in alternative cellular outcomes like cell growth or migration. These results highlight how important regulation of these endocytic adaptors is to overall GPCR fate, not only due to their role in internalization but also for their own signalling potential. Les récepteurs couplés aux protéines G (RCPGs) jouent un rôle fondamental dans notre équilibre homéostatique par leur implication dans de nombreux processus physiologiques. Afin de maintenir leur réactivité à l'environnement extracellulaire, un processus complexe de désensibilisation et resensibilisation des récepteurs a évolué. Afin de permettre aux récepteurs d'être resensibilisés, ils doivent d'abord être internalisés par endocytose, un processus impliquant de nombreuses protéines adaptatrices. La méthode la plus commune de l'internalisation des RCPGs est la voie dépendante à la clathrine, où les deux adaptateurs les plus importants sont βarrestine et AP-2. Bien que nous ayons une image relativement claire de la façon dont ces protéines conduisent à l'endocytose du récepteur, les mécanismes de régulation où ces adaptateurs de l'endocytose peuvent affecter ou être affecté par des processus de signalisation sont nettement moins bien décrits. Le récepteur de l'angiotensine II de type I est utilisé comme récepteur modèle dans nos études. Les études antérieures de notre laboratoire ont caractérisé certains des facteurs protéiques nécessaires pour le processus d'endocytose. Bien que les études précédentes ont démontré une régulation dépendante à la phosphorylation des complexes βarrestine et AP-2, ces résultats ont été exclusivement in vitro et n'ont pas été confirmés dans les cellules vivantes. Nous avons généré un anticorps polyclonal dirigé contre le site de phosphorylation putatif et avons révélé que non seulement cet évènement de phosphorylation se produit dans plusieurs types de cellules en réponse au traitement à l'angiotensine II (AngII), mais que l'activation de récepteurs multiples, comprenant un non-RCPG, le récepteur du facteur de croissance de l'épiderme, induisait la phosphorylation sur l'unité β de AP-2. Mon travail, précédemment publié dans un manuscrit, a également révélé que la prévention de cet évènement de phosphorylation stabilise les complexes βarrestine/AP-2 de façon significative. Bien que nos études initiales aient révélé que cette phosphorylation a peu d'impact sur l'endocytose des récepteurs, de nouveaux outils ont depuis été développés dans le laboratoire. Premièrement, nous avons généré un anticorps à chaîne unique qui peut être exprimé de façon intracellulaire et cible les phosphosites, et avons démontré que la liaison à ce résidu phosphorylé résulte dans un arrêt prolongé de l'endocytose. Nous avons également démontré que ce résidu tyrosine phosphorylé est un motif putatif de liaison pour certaines protéines contenant un domaine SH2 étant donné que trois de ces protéines sont capables de se lier à une β2adaptine phosphorylée. Enfin, en raison de la nature dépendate à βarrestine de cet évènement de phosphorylation, nous avons characterise quatre analogies de AngII avec une seule substitution d'acide amine dans leur sequence octapeptidique. Nos conclusions établissent une relation corrélative entre la force d'avidité de βarrestine pour son récepteur et le niveau d'activation de la kinase ERK suite aux signaux extracellulaires. Vraisemblablement, les différences d'avidité modifient le trafic du récepteur dans son destin vers le recyclage ou la dégradation. Par ailleurs, nous établissons que la conformation de βarrestine peut altérer les voies activées en aval du récepteur, entraînant d'autres effets cellulaires comme la croissance cellulaire ou la migration. Ces résultats mettent en évidence l'importance de la régulation par ces adaptateurs de l'endocytose sur le destin du RCPG, non seulement par leur rôle dans l'internalisation, mais aussi par leur propre potentiel de signalisation.

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Lake, David Jonathan. "A human alpha-arrestin protein with a potential role in cargo protein trafficking within the endocytic system." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/13335/.

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β-Arrestins are essential adaptors for G protein-coupled receptor (GPCR) trafficking. Evolutionary ancestors of the β-arrestins – dubbed α-arrestins – are present in yeast/fungi and, similar to β-arrestins, recognise cargo proteins and mediate their intracellular trafficking. Mammalian α-arrestins include five largely uncharacterised arrestin domain-containing (ARRDC1-5) proteins that display a predicted arrestin structure; the current study focuses on human ARRDC2. Confocal microscopy of exogenous, fluorescent protein-tagged ARRDC2 in U2OS cells in combination with compartment-specific markers indicated that ARRDC2 is dynamically distributed throughout the plasma membrane and endocytic system, predominantly to late endosomes/lysosomes. Anti-ARRDC2 immunostaining in several primary cell lines broadly supported this conclusion. ARRDC2 contains two proline-rich (PPxY) motifs that in other α-arrestins have been reported to mediate interactions with WW domain-containing NEDD4 family E3 ubiquitin ligases. Coimmunoprecipitation indicated that ARRDC2 is able to interact with several NEDD4 E3s via its PPxY motifs, and confocal microscopy suggested that this interaction may influence the subcellular targeting of the ligases. Ubiquitination of ARRDC2 was detected by coimmunoprecipitation, although this modification was independent of ARRDC2 interaction with NEDD4 E3s. ARRDC2 colocalised with agonist-stimulated, internalised GPCRs (β2-adrenergic receptor (β2AR) and δ-opioid receptor (δOR)) and colocalisation analysis indicated that this involved compartmental redistribution of ARRDC2 to receptor-containing early/recycling endosomes, suggesting a specific effect. Interaction of ARRDC2 with δOR was detected using coimmunoprecipitation, and confocal analysis suggested that ARRDC2 may influence δOR and β2AR intracellular trafficking. ARRDC2 was also found to oligomerise with itself and the β-arrestins. Confocal microscopy showed that ARRDC2 overexpression can induce the redistribution of β-arrestin1 to ARRDC2-positive vesicles, and a punctate bimolecular fluorescence complementation (BiFC) signal was detected between ARRDC2 and β-arrestin2. From this, it is speculated that α-/β-arrestins may function cooperatively or competitively to mediate discrete GPCR sorting events in the endocytic pathway.

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Parisis, Nikolaos. "Identification of PAR-2-regulated ERK substrates and (Beta)-arrestin-interacting proteins in invasive breast cancer cells." Thesis, University of Essex, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.520107.

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Simard, Élie. "Les β-arrestines et les produits de glycation avancée régulent et modulent respectivement la contraction cellulaire induite par l’activation de récepteurs couplés aux protéines G". Thèse, Université de Sherbrooke, 2015. http://hdl.handle.net/11143/6745.

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Résumé : La contraction cellulaire est une activité centrale dans plusieurs processus physiologiques. Entre autre, elle joue un rôle dans la régulation de la perméabilité vasculaire et de la pression artérielle. Il est également établi qu’une contraction anormale des cellules du muscle lisse vasculaire (VSMC) est souvent associée à l’hypertension et ses complications. Il est donc suggéré que l’étude des mécanismes impliqués dans la transduction de signaux menant à la contraction cellulaire et les mécanismes impliqués dans la régulation de celle-ci pourrait permettre d’identifier de nouvelles cibles potentielles afin d’envisager de nouveaux traitements pour cette maladie. Étant donné le rôle connu du système angiotensinergique dans l’activité contractile des VSMC et les nombreux indices suggérant une dérégulation de ce système dans de développement de l’hypertension; la présente thèse a été consacrée, dans un premier temps, à identifier de nouveaux mécanismes de signalisation impliqués dans la contraction cellulaire induite par l’angiotensine II. Ainsi, il est montré, pour la première fois, que les β-arrestines; des protéines adaptatrices impliquées dans la désensibilisation et la signalisation des récepteurs couplés aux protéines G, participent à la contraction cellulaire associée à l’activation du récepteur de l’angiotensine de type 1, et ce, en exerçant des effets opposés sur la phosphorylation de la chaîne légère de la myosine. Dans un second temps, étant donné qu’en condition diabétique une dysfonction vasculaire est observée et que cette dernière serait reliée à des changements dans le phénotype fonctionnel des VSMC; la deuxième partie de cette thèse traite des effets de l’exposition aux produits de glycation avancée (AGE) sur le phénotype et l’activité contractile des VSMC. Effet, les AGE, issus de la réaction entre les protéines et le glucose et dont la formation est particulièrement favorisée en contexte diabétique, pourraient être impliqués dans la dysfonction vasculaire observée. Ainsi, il est montré, pour la première fois, que l’exposition des VSMC aux AGE inhibe l’expression de leur phénotype contractile en affectant leurs propriétés mécaniques et diverses fonctions cellulaires telles que la signalisation, la contraction et l’organisation du cytosquelette. Dans un contexte plus large, cette thèse confirme également l’importance d’intégrer les approches biophysiques à la biologie cellulaire. En effet, les résultats obtenus par microscopie à force atomique sont uniques puisqu’ils offrent une nouvelle perspective concernant l’activité cellulaire via la caractérisation du phénotype mécanique de la cellule et la mesure de la contraction au niveau d’une seule cellule. || Abstract : Cell contraction plays a key role in a variety of physiological processes, among those; it regulates vascular permeability and blood pressure. It is known that abnormal contraction of vascular smooth muscle cells (VSMC) is often associated with hypertension and its complications. Therefore, it is suggested that studying mechanisms involved in signal transduction leading to cell contraction and mechanisms regulating the cell ability to contract may allow identifying new therapeutic targets in order to suggest new treatments for hypertension. Knowing the role of the angiotensin system in VSMC contractility and the numerous evidences suggesting a dysregulation of this system in the development of hypertension; this thesis first sought to identify new signaling mechanisms involved in cell contraction induced by angiotensin II. Therefore, it is shown here, for the first time, that β- arrestins; which are scaffolding proteins involved in the desensitisation and the signalling of protein G-coupled receptors, participate in cell contractile activity induced by angiotensin receptor type 1 activation by reciprocally regulating myosin activity through its light chain phosphorylation. Secondly, knowing that a vascular dysfunction is observed in diabetes which could be attributed to changes in VSCM functional phenotype; this thesis also investigated the effect of advanced glycation end-products (AGE) exposure on the contractile phenotype and function of VSMC. Indeed, AGE, which are the product of a reaction between proteins and glucose, are increased significantly in diabetes and are suspected to be involved in the vascular dysfunction observed in this metabolic disease. Therefore, it shown, for the first time, that AGE stimulation of the VSMC A7r5 interfere with the expression of their contractile phenotype by changing their mechanical properties and various cellular functions such as signal transduction, contraction and cytoskeletal organisation. Finally, this thesis also underlines the utility of integrating biophysical tools into studying cellular biology. Indeed, the various results obtained using atomic force microscopy are unique in a way that they provide new insights into studying cellular activity trough the measurement of single cell contraction and characterisation its mechanical phenotype

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Lally, Ciara [Verfasser], Franz [Gutachter] Bartl, Athina [Gutachter] Zouni, and Peter [Gutachter] Hegemann. "Structural and functional characterization of the arrestin-rhodopsin complex / Ciara Lally ; Gutachter: Franz Bartl, Athina Zouni, Peter Hegemann." Berlin : Humboldt-Universität zu Berlin, 2017. http://d-nb.info/1189327805/34.

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Frosch, Clara [Verfasser], Ivo [Gutachter] Quack та Hadi [Gutachter] Al-Hasani. "Die Rolle von β-Arrestin-2 in der hyperglykämieinduzierten Nierenschädigung / Clara Frosch ; Gutachter: Ivo Quack, Hadi Al-Hasani". Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2021. http://d-nb.info/1231541962/34.

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Doan, Thuy Anh. "Mammalian rod's single-photon responses : what do they tell us about rapid and reliable GPCR inactivation /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/10638.

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Hirata, Cristiane Lumi. "Thioredoxin interacting protein (Txnip) forms redox sensitive high molecular weight nucleoprotein complexes." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/264647.

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Felixberger, Johannes [Verfasser], and Armin [Akademischer Betreuer] Buschauer. "Luciferase complementation for the determination of arrestin recruitment: Investigations at histamine and NPY receptors / Johannes Felixberger. Betreuer: Armin Buschauer." Regensburg : Universitätsbibliothek Regensburg, 2016. http://d-nb.info/1082128015/34.

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Zindel, Diana [Verfasser], and Moritz [Akademischer Betreuer] Brünemann. "Die zeitliche Stabilität und zelluläre Lokalisation von Arrestin-Rezeptorkomplexen wird durch die Rezeptorphosphorylierung determiniert. / Diana Zindel. Betreuer: Moritz Brünemann." Marburg : Philipps-Universität Marburg, 2016. http://d-nb.info/108237735X/34.

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Raehal, Kirsten Michele. "Opioid-Induced Side Effects in Beta-arrestin2 adn G Protein-Coupled Receptor Kinase Knockout Mice." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1236884585.

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Fessart, Delphine. "Regulation of the endocytic adaptor proteins [beta] arrestin and AP-2 during clathrin-mediated internalization of Angiotensin II type 1 receptor." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=102501.

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G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors. They transduce the signals mediated by a diverse range of signalling molecules, including ions, amines, and peptides, as well as photons, to mediate intracellular functions. These receptors play a fundamental role in many physiological responses such as cardiovascular functions. To remain responsive to their environment, cells must find a way to rapidly desensitize and resensitize their activated GPCRs. Desensitization of receptors, for instance, involves the phosphorylation of receptors by G protein-coupled receptor kinase (GRKs) followed by the recruitment of betaarrestin. This interferes with the binding of the G protein (the signalling effector). betaarrestin then targets the receptors to the clathrin endocytosis pathway, and serves as an adaptor linking receptors to other signalling pathways. Internalization of receptors serves not only to remove desensitized receptors from the plasma membrane, but also to engage receptors in the resensitization pathway. The internalization of Angiotensin II (Ang II) type 1 receptor (AT1R) is controversial and poorly described. Therefore, our laboratory studies the mechanisms behind AT1R internalization. The agonist-induced internalization of AT1R begins with the formation of a complex including betaarrestin, the clathrin adaptor AP-2, and the tyrosine protein kinase, c-Src. In turn, this c-Src recruitment regulates the clathrin-mediated internalization of AT1R by controlling the formation of endocytic complexes during endocytosis. Indeed, the recruitment of c-Src is involved in the dissociation of AP-2 during receptor internalization. Based on our evidence that AP-2 and c-Src can be found in the same complex, we suggested that AP-2 could be phosphorylated by c-Src. Indeed, we found that Ang II induced the c-Src-mediated tyrosine phosphorylation of the beta-subunit of AP-2 (beta2-adaptin). We were able to map one of the tyrosines in beta2-adaptin and assess its role in regulating the binding of its principal partner: betaarrestin. The phosphorylation state of beta2-adaptin dictates its association profile with betaarrestin: when phosphorylated it reduces its binding to betaarrestin. Finally, we proposed a model for AT1R internalization. Overall, these studies are significant because they allow a better understanding of the underlying mechanism that regulates the initial steps of clathrin-coated vesicle endocytosis of AT1R.

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David, Indira. "Effets anxiolytiques/antidépresseurs et neurogéniques des ligands du récepteur 5-HT4 chez la Souris : rôle de la protéine β-arrestin 1". Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-00925063.

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Kilpatrick, Laura Elise. "The use of bimolecular fluorescence complementation (BiFC) to investigate the functional implications of neuropeptide Y receptor dimerisation and beta-arrestin recruitment." Thesis, University of Nottingham, 2015. http://eprints.nottingham.ac.uk/28767/.

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The functional significance of G protein coupled receptor (GPCR) dimerisation remains debatable partly due to the inability to assign pharmacological properties directly to defined dimers. To address this problem, this thesis uses bimolecular fluorescence complementation (BiFC), whereby protein-protein interactions are identified by the fluorescence generated from the association of complementary fluorescent protein fragment tags. The irreversibility of BiFC constrains receptor complexes of precise composition. The 4 Y receptor family members were chosen as model GPCRs as their relative β-arrestin recruitment and regulation by endocytosis remains questionable. BiFC based high content imaging assays quantified the pharmacology of β-arrestin2 recruitment to Y receptors, which correlated with their agonist induced endocytosis (Chapter 3). Targeted mutagenesis, showed key intracellular receptor domains shared in arrestin recruitment and internalisation. Fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy measured the diffusion of fluorescent Y receptor complexes and showed agonist stimulation slowed receptor motility (Chapter 4). A novel BiFC system allowed the correlation of this slowed motility with the behaviour of defined Y receptor/arrestin signalling complexes. BiFC also constrained Y1 receptors or β2-adrenoceptors as dimers of precise composition. Quantitative platereader imaging, measured BiFC dimer internalisation as an indirect readout of β-arrestin recruitment and dimer function (Chapters 5 and 6). Selective mutagenesis showed occupation of a single ligand binding site and the presence of one phosphorylated C terminal domain was sufficient, implying symmetrical binding of β-arrestin to Y1 receptor dimers. Finally Y1/Y5 receptor BiFC heterodimers showed modified pharmacology not evident for other heterodimer combinations. The most striking alterations were switching of Y5 selective antagonists from surmountable to insurmountable antagonism and the ineffectiveness of Y1 antagonists at inhibiting Y1/Y5 dimer responses, suggesting allosteric interactions between the respective protomers. Previous anti-obesity therapies targeting either Y1 or Y5 subtypes have lacked long term efficacy. However novel responses of the Y1/Y5 dimer, and the use of BiFC to screen for selective antagonism, may help identify future treatments for obesity.

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Urs, Nikhil Mahabir. "The regulation of cellular trafficking of the human lysophosphatidic acid receptor 1: identification of the molecular determinants required for receptor trafficking." Diss., Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/16177.

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The following thesis research was undertaken to gain a better understanding of the mechanisms that regulate the cellular trafficking and signaling of the endothelial differentiation gene (EDG) family of G-protein coupled receptors, LPA1, LPA2, and LPA3. This thesis will specifically focus on the regulation of the trafficking of the LPA1 Lysophosphatidic acid receptor, which is the most widely expressed and has been shown to be a major regulator of migration of cells expressing it. The initial studies undertaken in this project were aimed at understanding the endocytic pathway followed by the LPA1 receptor. Lysophosphatidic acid (LPA), an abundant serum phospholipid, stimulates heterotrimeric G protein signaling by activating three closely related receptors, termed LPA1, LPA2 and LPA3. In the first part of the project we show that in addition to promoting LPA1 signaling, membrane cholesterol is essential for the association of LPA1 with β-arrestin, which leads to signal attenuation and clathrin dependent endocytosis of LPA1. The second phase of the project was aimed at elucidating the different structural motifs required for the trafficking and signaling of the LPA1 receptor and helping us gain a more mechanistic view of the processes involved in its regulation. In the second part of the project we show that agonist-independent internalization of the LPA1 receptor is clathrin adaptor, AP-2 dependent and PKC-dependent and that it requires a distal dileucine motif, whereas agonist-dependent internalization of the LPA1 receptor is β-arrestin and clathrin-dependent and requires a cluster of serine residues in the tail region, which is upstream of the dileucine motif. These studies collectively vastly enhance our understanding of mechanisms that regulate LPA1 trafficking and signaling. These studies can also be applied to other G-protein coupled receptors making the task easier for other scientists to understand this vast family of receptors.

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Storez, Hélène. "Identification de nouveaux partenaires d'interaction des β-arrestines : 1-oligomérisation des β-arrestines : 2-Caractérisation de l'intéraction β-arrestine- Filamine A". Paris 7, 2007. http://www.theses.fr/2007PA077098.

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Les β-arrestines sont des protéines ubiquitaires impliquées dans la régulation des récepteurs couplés aux protéines G (RCPG) ainsi que dans de nombreuses voies de signalisation. Le crible de double hybride SRS (pour "SOS Recruitment System") effectué avec la β-arrestine 2 comme appât, a permis d'identifier parmi les proies, un clone correspondant à la β-arrestine 2 elle-même. Ce résultat nous a conduits à étudier l'oligomérisation des β-arrestines, déjà postulée d'après la structure du cristal de β-arrestine 1, mais non définitivement démontrée. Grâce à des approches basées sur le transfert d'énergie par résonance (FRET et BRET), nous avons pu montrer que les p-arrestines forment des oligomères constitutifs à des concentrations physiologiques. La co-expression des deux isoformes conduit à la délocalisation extranucléaire de la β-arrestine 1, via son hétéro-oligomérisation avec la p-arrestine 2, cette dernière étant exclue du noyau par un processus actif. L'oligomérisation des β-arrestines pourrait réguler leur distribution sub-cellulaire et celle de leurs partenaires d'interaction. Nous avons également identifié la filamine A (ou FLNA) protéine de liaison à l'actine, comme partenaire d'interaction des p-arrestines. A l'état basal, cette interaction renforce l'association des β-arrestines avec les MAPKinases ERKs. En réponse à l'activation des RCPGs (récepteurs M1 muscarinique et AT1 de l'angiotensine II), les p-arrestines et la FLNA coopèrent pour une activation optimale des ERKs. Dans les cellules Hep2, la stimulation des RCPGs conduit à la colocalisation du récepteur, de la p-arrestine, de la FLNA et d'ERKs activées dans les structures membranaires, appelées "ruffles" qui témoignent de la réorganisation du cytosquelette d'actine. L'invalidation de FLNA ou de p-arrestine par interférence à ARN et l'emploi d'un mutant dominant négatif de la MAP2 Kinase MEK1, inhibent la formation de ces "ruffles". Ainsi, la coopération entre les β-arrestines et la FLNA est déterminante pour l'activation optimale des ERKs et pour la réorganisation du cytosquelette qui en dépend. Ce phénomène pourrait expliquer une des fonctions des p-arrestines au cours du chimiotactisme β-arrestins 1 and 2 (BARRIand I3ARR2) are two highly homologous proteins with multiple biological functions. First known as key regulators of G protein-coupled receptor (GPCR) desensitization and endocytosis, βARRs were shown more recently to act as scaffolding proteins for ERK1/2 and JNK3 cascades, to activate Src and to bind to ubiquitin ligases. In addition to their propensity to interact with multiple partners, βARRs might also auto-assemble to form oligomers, as suggested by the observation that both IJARR1 and visual arrestin, the isoform which is specifically expressed in the retina, form dimers in crystals. In a two-hybrid screen, based on the Sos Recruitment System and using a C-terminal truncated mutant of βARR2 as the bait, one of the preys corresponded to the C-terminal portion of βARR2 itself, suggesting that βARR2 might also form dimers in solution. To investigate whether βARR2 might function as a dimer in a more physiological context, we took advantage of the bioluminescence energy transfer (BRET) approach, which was developed recently to study protein-protein interaction in living cells. The cDNAs encoding Renilla luciferase (lue) and the yellow variant of the Green Fluorescent Protein (YFP), the BRET donor and accepter moieties, respectively, were fused 5' or 3' to the cDNAs of βARR1 or βARR2. Various appropriate combinations of the resulting constructs were transfected in COS-7 cells. A constitutive BRET was measured with βARR2 constructs. As expected for a specific non-random interaction between the BRET donor and accepter, BRET signals increased as a function of the BRET accepter concentration up to a maximal value (BRETmax), and constant BRET signals were measured when cells were transfected with increasing amounts of a fixed ratio of acceptordonor. Interestingly, significantly different BRETmax values were measured when the BRET donor construct (βARR2-luc) was co-expressed with fusion proteins in which the BRET accepter was located either at the N-terminal (YFP-βARR2) or the C-terminal (βARR2-YFP) extremity of βARR2, indicating that the βARR-2 dimer is oriented. A similar constitutive BRET was also measured for βARR1 and when the BRET donor and the accepter were fused to different UARRisoforms. These data are consistent with a model in which both βARR2 and βARR1 form constitutive homo-dimers in living cells and might also be engaged in constitutive hetero-dimers. Several important questions are still under investigation, such as the proportion of dimers versus monomers, the effect of GPCR activation on βARR dimerization, the potentially different biological function of monomers, homo-dimers and hetero-dimers

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Mekiri, Maryam. "réponse, non-réponse et résistance aux traitements antidépresseurs monoaminergiques. Etude des marqueurs neurogéniques et moléculaires dans un modèle animal d'anxiété-dépression." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS051/document.

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Environ 30% des patients ne répondent pas de manière adéquate à un traitement antidépresseur. Cette absence de rémission, voire aggravation de l’état dépressif après la mise en place du traitement est qualifiée de non-réponse au traitement. La résistance au traitement est caractérisée lorsque cet échec thérapeutique est récurrent pour différentes stratégies thérapeutiques de mécanisme d’action différents. Afin d’améliorer les stratégies thérapeutiques visant à traiter les patients résistants, une meilleure compréhension des mécanismes biologiques associés à la non-réponse/résistance est nécessaire. De nombreux travaux ont associé le phénomène de neurogenèse hippocampique adulte à la réponse antidépressive, et ont montré qu’un blocage de la neurogenèse altère la réponse antidépressive chez la Souris. Cependant, aucune étude n’a montré si la non-réponse/résistance au traitement était associée à des altérations de la neurogenèse. De plus, il n’existe à ce jour aucun modèle de résistance qui ne présente une validité translationelle à ce qui est observé chez l’Homme. Enfin, alors que 2/3 des patients dépressifs sont des femmes, la majorité des études précliniques sont réalisées chez des mâles. Le but de mon travail de thèse a donc été de modéliser la résistance au traitement antidépresseur chez la souris C57BL6 mâle et femelle.Le premier objectif de ce travail a été la modélisation chez la femelle d’un phénotype anxio-dépressif, en adaptant un modèle neuroendocrinien de la dépression élaboré chez le mâle, basée sur l’administration chronique de corticostérone. Le deuxième objectif a été l’étude de la comparaison de la neurogenèse entre les souris répondeuses et non-répondeuses à un traitement chronique de fluoxétine ou résistantes à 2 stratégies successives de traitement présentant un mécanisme d’action différent (fluoxétine puis imipramine).D’autre part, les données de la littérature clinique suggèrent qu’un marqueur périphérique, la protéine β-arrestine 1, serait un marqueur de l’état dépressif et de la réponse au traitement. Nous avons donc mesuré dans notre modèle les variations de ce potentiel biomarqueur clinique.L’ensemble de ces travaux de thèse a permis de montrer la complexité d’induire un phénotype anxio/dépressif chez la souris femelle de façon stable et robuste via l’administration chronique de corticostérone. Chez le mâle, nous avons pu modéliser la résistance au traitement antidépresseur dans le modèle CORT. Nous avons pu observer que les processus neurogéniques semblent jouer un rôle essentiel dans la réponse au traitement, puisqu’une absence de réponse est associée avec une altération de la neurogenèse hippocampique adulte. Si dans notre modèle, l’expression périphérique de la β-arrestine 1 n’est pas diminuée chez les souris présentant un phénotype anxio-dépressif, elle permet cependant de discriminer les souris répondeuses des souris résistantes au traitement, ce qui valide son intérêt en tant que biomarqueur de la réponse antidépressive Around 30% of patients do not respond adequately to chronic antidepressant treatments. This lack of response, or worsening of the depressive state after the onset of the treatment can lead to treatment resistant depression (TRD). TRD is characterized by a recurrent lack of therapeutic response to various antidepressant which display different mechanism of action. A better understanding of the mechanisms that underlies TRD is necessary to discover some new effective therapeutic strategies. Numerous studies in rodents have shown that chronic antidepressant treatment improves adult hippocampal neurogenesis, and that disrupting this phenomenon partially alters antidepressant response. However whether lack of response or resistance to antidepressant treatment is associated with altered neurogenesis has yet not been observed. Additionally, there is yet no model of TRD with a translational validity. As major depressive disorders affects women twice more than men, yet the preclinical studies are performed mostly in males. Thus, the aim of this thesis was to model non-response an resistance to antidepressant response in male and female C57BL6 mice.The first aim of this thesis work was to induce a anxio-depressive phenotype in female mice, by adapting a neurodencocrine model of depression developed in males and based on chronic administration of corticosterone (CORT). The second aim was to study adult hippocampal neurogenesis in animals that respond or not to chronic fluoxetine administration, and in animals that were resistant to two successive antidepressant treatment with a different mechanism of action (fluoxetine and then imipramine).Additionally, data from the literature suggests that peripheral β-arrestin 1 expression could be a potential biomarker of depressive state and antidepressant response in humans. Thus, we explore its validity in our model of TRD in mice.Overall, our results highlight the difficulty of inducing an anxio-depressive phenotype in female mice, using different dosage or treatment duration of corticosterone, which hampers the use of corticosterone to induce emotionality in female mice.However, in male mice, we showed that we were able model resistance to treatment in the using the CORT model. Lack of response to chronic fluoxetine and treatment resistance to fluoxetine/imipramine were associated with altered neurogenesis in the dentate gyrus of the hippocampus. This confirms that hippocampal neurogenesis is critical for a full antidepressant response. While peripheral β-arrestin 1 expression was not decreased after chronic CORT exposure, its differential expression between responder vs treatment-resistant mice confirms its validity as a biomarker for antidepressant response

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