G-protein coupled receptor 50 (GPR50) is a genetic risk factor for psychiatric illness. It is a member of the melatonin receptor family, which includes the well characterised melatonin receptors 1 and 2 (MT1 and MT2). However, the ligand for GPR50 remains elusive and little is known about GPR50 signalling pathways. Despite this, GPR50 is known to enhance neurite outgrowth and inhibit the actions of the neurite outgrowth inhibitor NOGO-A. Existing evidence also indicates a role in lipid metabolism; GPR50 knockout mice displayed abnormalities in energy homeostasis and weight control, whilst sequence variants are associated with altered lipid levels in humans. Further, a yeast-2-hybrid screen identified SREBF2 and ABCA2, regulators of lipid homeostasis, as GPR50 interactors. This thesis explores the role of GPR50 in neuronal development and lipid metabolism. The work presented in this thesis shows that GPR50 promotes neuronal differentiation. Overexpression significantly increased the number of neurites per cell in SH-SY5Y cells. Further, dendritic branching was enhanced by GPR50 transfection in hippocampal and cortical neurons (DIV 14). In hippocampal neurons, GPR50 transfection also lead to a shift towards spine maturity although it had no effect on spine morphology, suggesting GPR50 enhances spine development but may not alter synaptic strength. The effect of GPR50 on neuronal morphology may be driven by actin remodelling. Immunocytochemistry showed an enrichment of GPR50 in highly dynamic regions of the membrane, i.e. the lamellipodia and dendritic spines. Overexpression in SH-SY5Y cells also resulted in an increase in WAVE-2 and phosphorylated RAC1/CDC42, key modulators of actin dynamics. Additionally, GPR50 transfection altered the protein level and localisation of α- catenin, another regulator of actin organisation, in HEK293 and SH-SY5Y cells respectively. An involvement of GPR50 in lipid metabolism has also been demonstrated in this thesis. Verification of the Y2H study suggested GPR50 does not physically interact with SREBF2 or ABCA2. However, ABCA2 appears to induce the intracellular localisation of GPR50 in several cell lines. In SH-SY5Y cells, this was mimicked by the inhibition of cholesterol trafficking, suggesting the translocation of GPR50 to the plasma membrane is dependent on cholesterol transport. Further, the depletion of lipoproteins resulted in the downregulation of GPR50, indicating a responsiveness to lipid levels. Finally, GPR50 increased lipid metabolism, as seen by a decrease in intracellular lipid droplets upon GPR50 overexpression. The data presented here extends previous work indicating a role of GPR50 in neurodevelopment. It also highlights a potential mechanism by which GPR50 regulates neuronal morphology, i.e. via actin remodelling. Reports that GPR50 is involved in energy homeostasis is also supported in this thesis, further, results presented here suggest GPR50 is specifically involved in lipid metabolism. These processes are often disrupted in mental illness, thus this work may provide a functional link between GPR50 and psychiatric disorders.

La protéine GPR50, qui fait partie de la famille des récepteurs de la mélatonine, est classée, avec une centaine d’autres protéines à sept domaines transmembranaires (7TM), dans la catégorie des récepteurs couplés aux protéines G hétérotrimériques (RCPG) orphelins, c’est-à-dire pour lesquels aucun ligand n’a pu être identifié. De plus en plus d’études montrent que les 7TM peuvent avoir des fonctions indépendantes d’un ligand. C’est le cas de GPR50 qui inhibe les fonctions du récepteur de la mélatonine MT1 en interagissant directement avec lui. Nous avons cherché à identifier d’autres partenaires associés à GPR50 en appliquant la technique de purification par affinité en tandem et avons mis en évidence son interaction avec un récepteur du facteur de croissance Transforming Growth Factor ß (TGFβ), le récepteur de type I (TβRI).Nous décrivons ici la formation d’un complexe entre GPR50 et le récepteur TβRI au niveau de la membrane plasmique, avec pour conséquence l’induction d’une activité constitutive du récepteur et des voies de signalisation en aval en l’absence de TGFβ, mais également en l’absence du récepteur TßRII qui est habituellement indispensable pour l’activation de TβRI par phosphorylation. Cette activité constitutive se traduit par la phosphorylation des protéines Smad2 et Smad3, leur intégration dans un complexe avec Smad4, la translocation du complexe dans le noyau et finalement l’activation de la transcription de leurs gènes-cibles. Nous avons décrypté les mécanismes moléculaires de cette activation constitutive en montrant que GPR50 entre en compétition, pour l’interaction avec TβRI, avec le régulateur négatif FKBP12, une protéine inhibitrice de l’activité basale du récepteur en l’absence de ligand. Nous avons identifié dans la queue intracytoplasmique de GPR50 un motif répétitif similaire à la séquence de FKBP12 impliquée dans son interaction avec TβRI , motif qui constitue la base moléculaire de cette compétition.Nous avons étudié les conséquences fonctionnelles de cette activation en surexprimant GPR50 de manière stable dans la lignée cellulaire MDA-MB-231, dérivée d’un cancer de sein. Nous avons observé dans ces cellules des effets pro-migratoires et anti-prolifératifs similaires à ceux causés par l’administration de TGFβ.En conclusion, ce travail décrit un nouveau mode d’activation du récepteur TβRI en l’absence de ligand, mais identifie également une nouvelle fonction indépendante d’un ligand pour le RCPG orphelin GPR50. En perspective de ce travail, nous allons essayer d’identifier des conditions biologiques où cette interaction pourrait prendre place afin de confirmer ces résultats dans un contexte plus physiologique. / During the last years, it became more and more accepted, that orphan G Protein coupled receptors (GPCRs) with a transmembrane spanning heptahelical core (7TM) can have ligand-independent functions. One of those 100 orphan GPCRs is GPR50, a 7TM protein with a long cytosolic domain. Recently, studies revealed ligand-independent functions for GPR50, where it has the capacity to modulate the activity of other proteins upon complex formation. By applying a tandem affinity purification approach we sought to identify further putative interacting partners of GPR50. One of the identified binding partners is the transforming growth factor β (TGFβ) receptor type I (TβRI).The TGFβ-dependent signal transduction pathway of serine/threonine kinases is a pathway with direct signal flow from ligand over the receptor to its substrates, the Smads which translocate into nucleus where they bind DNA and regulate gene expression. An important question concerns the generation of specificity and fine-tuning of TGFβ-dependent signaling. Throughout the years, an important number of proteins which regulate the activity of the TGFβ signal transduction pathway in a positive or negative manner have been identified. Most of them act in a cell-context-dependent manner, allowing the regulation of TGFβ signaling adapted to the particular circumstances.We report here the complex formation of GPR50 and TβRI on the plasma membrane. The consequence of this interaction is the GPR50-mediated induction of a constitutive activation of the TβRI and its downstream signaling in a TGFβ ligand-independent manner. This has been monitored by Smad2/3 phosphorylation, Smad2/3-Smad4 complex formation and their subsequent translocation into the nucleus, where they activate Smad-dependent gene expression. In order to decipher the molecular mechanism that allows this activation, we showed that GPR50 competes with the negative regulator, that prevents leaky TGFβ signaling, the gatekeeping molecule FKBP12, for binding to the TβRI. We identified a motif in FKBP12 involved in the interaction with TβRI with similarities to a motif in GPR50, providing a molecular basis for the replacement of FKBP12 by GPR50 in the TβRI complex. We showed that GPR50 is capable of activating the TβRI even in the absence of the TβRII, which normally is required for activating the TβRI by phosphorylation. This reveals a previously unknown mode of activation of the TβRI in absence of the TGFβ ligand and TβRII. In order to identify the functional consequences of this crosstalk, we studied migration and growth of MDA-MB-231 breast cancer cells stably overexpressing GPR50. In these cells, TGFβ-like pro-migratory and anti-proliferative effects have been observed.Future research will help to identify tissues and biological circumstances, where this crosstalk could take place for putting this novel mode of regulation of TGFβ signaling pathway into a context-dependent-manner. Additionally our work established another ligand-independent task for the orphan 7TM protein GPR50, consolidating its function as binding partner and activity modulator.

GPR50, an X-linked orphan G protein-coupled receptor (GPCR), is a risk factor for bipolar disorder (BD) in female subjects. It has been shown that GPR50 plays a part in neurite outgrowth, glucocorticoid receptor signalling and leptin signalling by interacting with major factors involved in these events. Yeast two-hybrid screens have identified multiple putative GPR50 interactors involved in neurodevelopment, stress response and apoptosis, lipid and glucose metabolism, as well as regulation of NMDA receptors and GABA transmission. Among these interactors, RTN3, RTN4, SREBP2 and SNX6 are known regulators of β-secretase (BACE1), a key enzyme in Aβ generation, myelination of the central/peripheral nerve, and neurite outgrowth/synapse formation. Preliminary data indicated that GPR50 expression significantly increased endogenous BACE1 activity in HEK293 cells, so I hypothesised that there is a functional interaction between the two. In this thesis, I investigated the relationship between GPR50 and BACE1 by identifying the effects of GPR50 on BACE1 expression and function, which may provide an explanation of GPR50’s potential association with psychiatric disorders and Alzheimer’s disease. Firstly, studies on expression levels revealed that when GPR50 was over-expressed, BACE1 protein expression was up-regulated in SH-SY5Y cells, but down-regulated in HEK293 cells, suggesting a differentiated regulative system between cell lines. Then I confirmed the physical association between endogenous GPR50 and BACE1 in HEK293 cells by co-localisation and co-immunoprecipitation studies. Their putative interaction sites were located at the plasma membrane and the filopodia/lamellipodia-like structures in HEK293 cells, and at the neurites in mouse primary neuronal cells. Subcellular fractionation of adult mouse brain revealed that endogenous Gpr50 and Bace1 were co-fractionated in the presynaptic vesicles. Secondly, I showed that, in contrast to HEK293 cells, GPR50 overexpression had no effects on β-secretase activity in mouse primary cortical neurons. However, the BD-associated variant GPR50del significantly decreased β-secretase activity compared to the more common variant GPR50, and showed a trend of diminishing β-secretase activity compared to the control condition. Subcellular fractionation experiments showed that in HEK293 cells, there was an increased ratio of mature BACE1 against immature BACE1 localised in the plasma membrane fractions, indicating a role in regulation of BACE1 trafficking to one of its putative activity sites; whereas in mouse primary cortical neurons, GPR50del increased co-fractionation of immature Bace1 with endoplasmic reticulum (ER) marker calreticulin, thus potentially retarding the maturation of Bace1. Importantly, the regulative trend of GPR50/GPR50del on β-secretase activity is cell line-specific and is highly correlated to their effects on β-secretase intracellular distribution. Thirdly, I found that the mRNA levels of human GPR50 and BACE1 were negatively correlated in the dorsolateral prefrontal cortex of female subjects sampled after birth. Mouse Gpr50 and Bace1 mRNA levels were negatively correlated across the telencephalon regions, and had a trend of negative correlation across the hypothalamic regions. Co-localisation of the two proteins was detected in multiple mouse brain regions, with the strongest co-localised signals occurring in CA2 pyramidal neurons, arcuate hypothalamic nucleus and dorsomedial nucleus of the hypothalamus. Finally, preliminary experiments in Alzheimer’s disease model TgSwDI mice, suggested that the expression level of Gpr50 in layer V of the entorhinal cortex was positively correlated with Aβ deposition. Decreased Gpr50 expression was identified in the hippocampus of 9 months transgenic animals compared with age-matched controls. This indicates that Gpr50 expression might be altered in this mouse model co-ordinately with Aβ deposition. The findings in this thesis provide further evidence of GPR50’s correlation to psychiatric illnesses and its interaction with enzyme BACE1 highlights a potential link to neurodegenerative disease.

G protein-coupled receptors (GPCRs) form a link between the cell and their environment when signaling pathways are activated upon ligand binding. However, the ligands and functions for many GPCRs remain to be determined. G protein-coupled receptor 50 (GPR50) is one such orphan, and its exact role is yet unknown. There is however emerging functional and genetic evidence suggesting a function for GPR50 in psychiatric illness and lipid metabolism. It was hypothesised that investigating GPR50’s protein-protein interactions would lead to a greater understanding of the role of GPR50 in normal brain functioning and in mental illness. Putative protein interactors were initially isolated by a yeast two-hybid study and were further tested here. To address GPR50’s links to mental illness, the GPR50∆502-505 deletion variant associated with mood disorders was also investigated. To test this hypothesis I sought to confirm some of the key yeast two-hybrid interactions. Using co-immunoprecipitation and immunocytochemistry the interaction of GPR50 with reticulon family members Nogo-A, Nogo-C and RTN3, and with cell-cell adhesion molecule CDH8 and lipid-associated protein ABCA2 were validated. In order to identify the location of interactions, subcellular fractionation of mouse brain and rt-PCR and immunohistochemistry in developing and adult mouse brain were performed. GPR50 and several interactors were found to be enriched at the synapse by subcellular fractionation of whole adult brain, and at embryonic day 18 (E18) and 5 weeks by rt-PCR. Colocalisation of GPR50 and interactors was found in the amygdala, hypothalamus, cortex and specific brain stem nuclei by immunohistochemistry. The discovery of GPR50 expression in noradrenergic, serotonergic and dopaminergic nuclei in the adult brain stem suggests a further role for GPR50 in neurotransmitter signaling and stress. To investigate the function of GPR50 two assays were performed that measure processes which are known to be affected by Nogo and RTN3: The first assay was a neurite outgrowth assay in Neuroscreen-1 cells, a PC12 cell clone. A significant increase in neurite length was detected after transient overexpression of GPR50 and this effect was increased in the GPR50∆502-505/T532A variant. Additionally GPR50-overexpression resulted in an increase in filopodia formation suggesting a role in actin dynamics. As a second functional assay in vitro BACE1 activity assays were performed in HEK293 cells. GPR50 but not GPR50∆502-505/T532A overexpression resulted in a significant increase in BACE1 activity. Lastly a final series of pilot experiments were performed to gain insight into the secondary structure of the C-terminal domain and the effects of the polymorphisms on structure. The 35kDa GPR50 C-terminal domain was purified and Circular Dichroism studies indicated a predominantly unstructured protein with increased a- helical content in the GPR50∆502-505 variant. The results in this thesis indicate a role for GPR50 in neuronal development and synaptic functioning. The results also strengthen an association with major mental illness, with links to several disease mechanisms.

Pronounced seasonal adaptations in physiology and behaviour are exhibited by mammals living in polar and temperate habitats. These include the development of a winter coat, altered fat reserves, reproductive quiescence and food hoarding. Maintaining constant body temperature (Tb) during winter is energetically very costly, and so many small mammals periodically abandon homeothermy in favour of heterothermy. The two principal heterothermic strategies are daily torpor and seasonal hibernation, in which bouts of profound hypothermia range from a few hours to several days (respectively). It is now clear that hypothalamic thyroid hormone (TH) regulation, and specifically the availability of the active metabolite triiodothyronine (T3), is a critical regulator of seasonal reproductive cycles in many species including birds and mammals. The impact of this signal as a switch for seasonal changes in physiology has been highlighted by the demonstration that blockade of this pathway prevents seasonal adaption in hamsters. Peripheral TH signalling is also a principle regulator of metabolic rate in mammals. Despite these findings nothing is yet known about the involvement of central (hypothalamic) and peripheral TH cycles in the expression of torpor and hibernation. Within this thesis, the role of TH dynamics both in the brain and peripheral circulation is examined within three models of heterothermia: the Siberian (Phodopus sungorus) and European (Cricetus cricetus) hamsters, which employ daily torpor and hibernation, respectively, and the laboratory mouse (Mus Musculus) which exhibits torpor in response to metabolic stress such as food restriction. To delineate TH regulation and signalling in the context of both seasonal and acute physiological responses, the expression of genes involved in thyroid hormones conversion (e.g. Deiodinase type II (Dio2) and type III (Dio3) and transport (e.g. Monocarboxylate transporter 8, Mct8) within the ependymal layer of the ventral 3rd ventricle have been detailed across seasonal (long (LD) and short day (SD)) photoperiods, and during normothermic and hypothermic conditions. Furthermore, TH concentrations have been directly measured within the hypothalami of P. sungorus and C. cricetus, and TH responsive genes (e.g. Hairless (Hr) and Thyrotropin releasing hormone (TRH) to determine the potential impact of regional T3 signalling. As expected, Dio2 and Dio3 expression in P. sungorus exhibited a strong seasonal cycle indicative of elevated T3 production during SD (reduced Dio2 and elevated Dio3). Unexpectedly, total T3 measures from hypothalamic extracts revealed no significant alteration either seasonally or during torpor/hibernation in hamsters. However, Hr expression in the ependymal layer and TRH expression in the paraventricular nucleus (PVN) suggests low T3 concentrations during SD are localised to specific regions and does not encompass the whole hypothalamus per se. In addition, altered serum TH concentrations implicate seasonal and torpor associated dynamics that may play a role in seasonal adaptation and hypothermia. Finally, data from transgenic mice strongly implicate the melatonin-related receptor (GPR50) in leptin signalling and aberrant thermogenesis in mice.