Vital cellular processes such as nutrient uptake, receptor signaling, and cell migration are controlled by a balance between cell surface receptor internalization and recycling. Clathrin-mediated endocytosis is the major mechanism of receptor internalization in which cargo-enriched endocytic vesicles form at, and are released from, the plasma membrane before maturing into early endosomes. The receptors can then be sorted into fast and slow recycling pathways that replenish receptor levels at the cell surface. A major fast recycling pathway is controlled by the small GTPase Rab4a, which plays a central role in cell migration and cancer cell invasion through regulation of integrin αvβ3 recycling. Recent studies have discovered a family of clathrin-coated vesicle proteins, known as adaptin-ear-binding coat-associated proteins (NECAPs), that consists of two family members, NECAP1 and NECAP2. NECAP1 functions in endocytosis and cooperates with the clathrin adaptor AP-2 to control endocytic vesicle size, number and cargo. Importantly, NECAP2 did not rescue the knock-down phenotype of NECAP1, revealing that NECAPs are not functionally redundant. The studies described in this dissertation show that NECAP2 controls the fast recycling of epidermal growth factor receptor and transferrin receptor. Furthermore, NECAP2 specifically functions in Rab4a-mediated fast recycling together with the clathrin adaptor AP-1. In contrast, NECAP2 has no effect on AP-1-mediated transport from the Golgi or on other Rab4a-dependent sorting events that utilize additional clathrin adaptors and effector proteins. Thus, NECAP2 regulates a sub-route within the Rab4a recycling pathway and, in fact, is the first protein known to date to show this level of specificity. NECAP2 knock-down revealed that this sub-route controls cell migration and cancer cell invasion. Specifically, NECAP2 knock-down impaired the recycling of integrin αvβ3 to the cell surface, leading to decreased Rac1 activation and integrin αvβ3-dependent persistent cell migration. NECAP2 depletion also alleviated the inhibitory effect on integrin α5β1 recycling, switching cells to integrin α5β1-dependent cell migration. Notably, loss of NECAP2 function in breast cancer cells inhibited invasive migration in a 3D invasion model system. Therefore, the NECAP2 pathway may provide a therapeutic target, in particular for the 25% of breast cancers with amplification of Rab4a.

Peroxisomes compartmentalize a variety of important metabolic reactions including fatty acid β-oxidation and the related process of IBA β-oxidation. Peroxisomal proteins are encoded by nuclear genes and must be post-translationally imported. A dynamic import process is vital for proper matrix protein localization and is dependent on the family of peroxin (PEX) proteins. The delivery and peroxisomal import of cargo from a loaded receptor, PEX5 or PEX7, is carried out by the early-acting peroxins, including PEX13 and PEX14, and receptor recycling is carried out by the late-acting peroxins, including PEX4 and PEX6. In this thesis, I describe the use of double mutant analysis to differentiate early-acting and late-acting pex mutants by phenotypic and molecular analysis. I found that double mutants made with two early-acting or two late-acting pex mutants showed enhanced phenotypes in β-oxidation and import defects. In contrast, defects of double mutants made with a weak early-acting mutant and a late-acting mutant were suppressed. Additionally, I found that receptor localization is central to proper peroxisomal function. My results suggest that when the receptor is not removed from the peroxisome, stabilized peroxisomal pores may be formed, perhaps impairing peroxisomal function due to leaching of peroxisomal contents. Together my data suggest that balance between import and receptor recycling is fundamental for peroxisomal function. In humans, peroxisomal biogenesis disorders are most often caused by defects in late-acting peroxins. Peroxisomal defects occur in plants and humans as a result of the same lesions in PEX proteins. The understanding of how these late-acting defects can be ameliorated in plants, may inspire new approaches to human therapeutics.

Yes / Inhibitory GABAB receptors (GABABRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABABRs are heterodimers of GABAB1 and GABAB2 subunits and here we show that the trafficking and surface expression of GABABRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABABR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABABRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABAB1 but decreases GABAB2 surface expression. The increase in surface GABAB1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABAB2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABABR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival. / BBSRC, MRC and the European Research Council

La douleur chronique est une maladie fréquente et grave qui, pour de nombreuses personnes, ne peut pas être entièrement traitée avec les choix thérapeutiques actuels. Des agonistes des récepteurs opioïdes delta (DORs) ont été proposés comme interventions thérapeutiques pour ces maladies. Des recherches précliniques ont montré que l'activation des DOR produit des effets anti-hyperalgiques et antidépresseurs avec moins d'effets secondaires associés aux analgésiques opioïdes disponibles sur le plan clinique. Cependant, de nombreux agonistes DOR induisent une tolérance analgésique, entravant ainsi leur développement en tant que médicaments. Les travaux de cette thèse visent à mieux comprendre les causes cellulaires et moléculaires de la tolérance ainsi que ce qui rend certains agonistes plus résistants à la tolérance que d'autres. Dans le premier projet, nous nous sommes concentrés sur la superactivation de l'adénylyl cyclase induite par un ligand, un modèle de réponse adaptative médiée par les isoformes de l'adénylyl cyclase (AC). La superactivation de l'adénylyl cyclase (SA) a été associée à l’hyperalgésie, la tolérance analgésique et à des symptômes de sevrage. Ainsi, nous étions curieux de voir si les profils de signalisation cellulaire créés pour la découverte de médicaments pouvaient nous fournir des informations sur la capacité d'un ligand à induire la SA. Pour répondre à cette question, nous avons généré des profils de signalisation complets pour six agonistes différents du DORs (Met-enképhaline, deltorphine II, DPDPE, SNC-80, ARM390 et TIPP) tout en surveillant 12 différents résultats de signalisation avec des biocapteurs à base de BRET. L'analyse des profils de signalisation a montré une sélectivité fonctionnelle remarquable parmi les ligands étudiés. Ensuite, nous avons pu classer les agonistes DOR en fonction de la similarité de leurs profils en utilisant l'approche que nous avons adaptée de notre laboratoire. Nous avons par la suite démontré que, à l'exception de TIPP, dont la réponse SA était Ca2+-indépendante, les catégories de médicaments résultant du regroupement sont corrélées avec la capacité du ligand à provoquer une SA. Une investigation plus approfondie des mécanismes a révélé que Gαi/o était essentiel tant pour la SA déclenchée par TIPP que par Met-Enkepkaline, mais les mécanismes en aval étaient assez distincts pour ces ligands. Ensemble, nos résultats indiquent que les mécanismes sous-jacents à la tolérance cellulaire induite par les agonistes DOR sont spécifiques au ligand. Dans le deuxième projet, nous nous sommes principalement intéressés aux mécanismes de tolérance aux agonistes DOR qui peuvent être en partie expliqués par la désensibilisation et la régulation négative des récepteurs. Il a été établi que, les ligands qui induisent le recyclage du récepteur après l'internalisation ont été trouvés pour fournir une analgésie de longue durée. Par conséquent, les expériences menées dans cette étude ont été menées pour révéler davantage les déterminants moléculaires sous-jacents au recyclage du récepteur et sur la manière dont l'interaction agoniste-récepteur pourrait produire des modèles distincts de régulation des récepteurs. Nous avons évalué l'activation de l'agoniste et la désensibilisation du signal DOR-Gαi1. Nos données ont rapporté que le DPDPE était pratiquement sans effet sur la désensibilisation de l'activation de Gαi1, tandis que la désensibilisation par la deltorphine II était plus importante que celle induite par le DPDPE mais moins que celle induite par l'ARM390 et le SNC-80. Ensuite, nous avons établi que les DORs stimulés par le DPDPE se recyclaient de manière plus efficace que ceux activés par la deltorphine II. De plus, nous fournissons des preuves phénoménologiques que des interventions similaires ont des effets distincts sur le recyclage évoqué par chaque ligand. En particulier, la truncation du DOR ou la surexpression de βarr2 avaient des effets différentiels sur le recyclage par le DPDPE et la deltorphine II. Il est admis que les mécanismes sous-jacents à ces différences restent à être pleinement décrits, mais la phénoménologie de nos observations soutient l'idée que le DPDPE et la deltorphine II mettent en œuvre des processus de recyclage distincts. / Chronic pain is a common and severe disease that, for many people, cannot be fully treated with current therapeutic choices. Agonists of the delta opioid receptor (DOR) have been proposed as therapeutic interventions for this illness. Preclinical research has shown that DORs produce antihyperalgesic and antidepressant-like effects with fewer side effects than the ones associated with clinically available opioid analgesics. However, numerous DOR agonists induce analgesic tolerance, hampering their development as medications. Thus, further investigations are needed to understand the mechanisms underlying the tolerance associated with chronic opioid use. This thesis aimed to further understand the cellular and molecular mechanisms that causes tolerance as well as what makes some agonists more resistant to tolerance than others. In the first project, we focused on ligand-induced cyclase superactivation (SA), a pattern of adaptive response mediated by adenylyl cyclase (AC) isoforms. Cyclase SA has been associated with hyperalgesia, analgesic tolerance, and withdrawal symptoms. Therefore, we were curious to assess weather cell-based signalling profiles created for drug discovery could provide us with information on the ability of a ligand to induce cyclase SA. To address this question, we generated comprehensive signalling profiles for six different DOR agonists (Met-enkephalin, deltorphin II, DPDPE, SNC-80 and ARM390) while monitoring 12 different signalling outcomes with BRET-based biosensors. Analysis of the signalling profiles showed remarkable functional selectivity among the investigated ligands. Next, we were able to classify DOR agonists based on the similarity of their profiles using the approach we adapted from our lab. We subsequently demonstrated that except for TIPP, whose SA response was Ca2+-independent, the drug categories resulting from clustering are correlated with ligand capacity to cause SA. Further investigation of the mechanisms revealed that Gαi/o was essential for both TIPP and Met-Enkepkalin-driven cyclase SA. However, downstream mechanisms were quite distinct for these two ligands. Altogether, our findings indicate that mechanisms underlying cellular tolerance induced by DOR agonists are ligand-specific. In the second project, we were primarily concerned with the mechanisms of tolerance to DOR agonists that may be, in part, explained the receptor desensitization and downregulation. Obviously, ligands that induce receptor recycling after internalization have been found to provide long-lasting analgesia. Therefore, the objectives of the experiments in this project were to assess the molecular determinants affecting receptor recycling and how agonist-receptor interaction can result in different patterns of receptor regulation. We assessed agonist inducing activation and desensitization of DOR-Gαi1 signal. Our data showed that DPDPE was efficient in activating the receptor without noticeable desensitization effect. On the other hand, deltorphin II exerted a significant desensitization effect. However, this effect was low when compared to ARM390 and SNC-80. Then, we established that DORs stimulated by DPDPE recycle more efficiently than those activated by deltorphin II. We also provided phenomenological evidence on receptor recycling elicited by each ligand. In particular, DOR truncation or the overexpression of βarr2 had differential effects on receptor recycling by DPDPE and deltorphin II. While our data shed light on the mechanism underlying these differences, further investigation is needed for the mechanism to be fully elucidated. Admittedly, our observations support the notion that DPDPE and deltorphin II engage distinct recycling processes.