G protein-coupled receptors (GPCRs) play a major role in physiological functions by transducing extracellular information into intracellular responses, thereby, allowing auto-, para , and endocrine communication. In combination with their ubiquitous expression, GPCRs are of special interest for developing therapeutic approaches. Due to their high targetability, GPCRs are also interesting for obesity research. Even though the prevalence for obesity and its comorbidities like type 2 diabetes mellitus is rapidly rising worldwide, the physiological function of many GPCRs is not well resolved in adipose tissue (AT). Here, the high percentage of orphan GPCRs (65 %), meaning GPCRs characterized by lacking information about an endogenous ligand, signal transduction and/or physiological functions, is an enormous restricting factor to understand AT (patho)physiology. In this study the overall goal was the identification and characterization of GPCRs hitherto unrecognized in AT and adipocyte functionality. Therefore, the first part of this study focused on incorporating a web application for the analysis of publicly available RNA-seq data of human and mouse AT and adipocytes, called FATTLAS. With this application, the GPCRome was analysed to identify GPCRs connected to AT function. Furthermore, those GPCRs were characterized in 3T3-L1, a murine preadipocyte cell line with adipogenic potential. Following, the main results are described: 1. Analysing the GPCR expression patterns in AT of lean individuals and individuals with obesity lead to the identification of a multitude of highly and differentially expressed genes, many of them being already characterized in AT or adipocyte function. Nevertheless, four GPCRs were identified being either highly expressed in both conditions (Gpr146) or differentially expressed in individuals with obesity (Fzd5, Mrgprf, and Ptger2) having an unknown function in AT. For all four receptors an agonist has been previously described. 2. Besides expression in AT, all four receptors are also present in primary and 3T3-L1 (pre)adipocytes. Thus, GPR146, FZD5, MRGPRF, and PTGER2 are suitable for characterizing their role in adipocyte function. 3. Investigating receptor overexpression in the heterologous expression system HEK293T, FZD5, MRGPRF and PTGER2 were found to be expressed at the cell surface, whereas GPR146 was mainly located in endosomes. 4. While analysing receptor signalling in HEK293T cells high basal receptor activity was detected for PTGER2 (Gɑs protein) and GPR146 (Gɑi protein). As the published agonists could not induce receptor activation in 3T3-L1 (pre)adipocytes, siRNA-mediated knockdown was the method of choice for the characterization of endogenously expressed receptors in 3T3-L1 preadipocytes. 5. Receptor knockdown analysis for these four receptors revealed a reduced adipogenesis acting via the PPARγ axis. Here, MRGPRF and PTGER2 act via a cAMP-dependent mechanism. Furthermore, the receptors are involved in preadipocyte viability, which also contribute to the regulation of adipogenesis. As the receptor knockdown in preadipocytes leads to a reduced viability and adipogenesis, adipocyte function was analysed after knockdown in adipocytes. 6. Before the investigation of adipocyte function, the effect of receptor knockdown in mature adipocytes on adipocyte viability was analysed with only Mrgprf knockdown showing an effect. 7. In adipocyte function, MRGPRF was involved in adiponectin secretion most likely by changing intracellular cAMP accumulation. Moreover, GPR146 regulates lipolysis via basal Gɑi-protein signalling. PTGER2 and FZD5 did not show an involvement in the analysed adipocyte functionalities. Taken together, in this study an interactive public database (FATTLAS) was implemented, incorporating publicly available RNA-seq data for AT and adipocytes for improved access and analysis of these complex datasets. Using this database, four hitherto unrecognized GPCRs were identified and their involvement in adipogenesis and AT function was proved. In the second part of this thesis, the subgroup adhesion GPCRs (aGPCRs) of the GPCRome, were analysed. Genome-wide association studies have linked this orphan GPCR class to AT function and metabolic dysfunction (e.g. obesity). However, they have not been thoroughly investigated yet. By performing a comprehensive study, the following main findings about aGPCR expression in AT and adipocytes and about their role in adipogenesis and adipocyte function were revealed: 1. In mice 30 aGPCRs are genomically encoded. 25 receptors were identified to be expressed in subcutaneous AT (scAT), 28 in visceral AT (vAT). Only Emr4, Gpr133 and Gpr116 showed a differential expression between scAT and vAT. 2. Under high-fat diet seven receptors (Lphn1-3, Eltd1, Emr1, Gpr124, Gpr116, and Gpr56) were significantly higher expressed in scAT, whereas in vAT three receptors were upregulated (Emr4, Gpr124, and Celsr3) and four downregulated (Gpr113, Gpr116, Gpr64, and Gpr97). 3. As AT consists of diverse cell types, cell-specific expression of aGPCRs in adipocytes and the stroma vascular fraction (SVF) was determined. Here, four receptors were significantly higher expressed in adipocytes (Lphn2, Gpr125, Gpr111, and Gpr64), six receptors were lower expressed (Emr1, Emr4, Gpr133, Gpr113, and Gpr97, Gpr126). However, most aGPCRs are expressed in adipocytes making them interesting for investigating their role in adipocyte function. 4. In human scAT CELSR1, CELSR2, EMR2, and GPR126 were upregulated comparing lean and obese conditions, GPR64 and GPR97 were downregulated. The expression of aGPCRs in human scAT was comparable to mouse scAT indicating a transferability of aGPCR function in mouse and human samples. 5. Analysing aGPCR expression during adipogenesis in 3T3-L1, three distinct expression patterns were found: no changes (Lphn1-3, Cd97, Gpr124, Gpr125, Gpr116, Gpr56, Gpr64, Gpr97, and Gpr126), steady upregulation (Gpr124 and Gpr126), and downregulation (Cd97, Gpr116, Gpr56, and Gpr64). 6. Knockdown of Lphn2, Gpr124, Gpr125, Gpr116, Gpr64, and Gpr126 in 3T3-L1 lead to a reduced lipid accumulation and droplet size indicating an impaired differentiation into mature adipocytes. 7. Exemplarily, GPR64 was selected to study the role of aGPCRs in adipocyte function due to its significantly reduced expression in obesity and its role in adipogenesis. Knockdown and stimulation by a tethered agonist-derived peptide uncovered Gαs protein-mediated signalling. Furthermore, peptide stimulation resulted in a reduced adiponectin secretion and glucose uptake in 3T3-L1 adipocytes. Lipolysis was induced in 3T3-L1 and primary adipocytes after peptide stimulation indicating a transferability of cell culture experiments to ex vivo analysis. In summary, this comprehensive study describes aGPCR expression in human and mouse AT, adipocytes and SVF in depth. Furthermore, their involvement in adipogenesis under physiological and knockdown conditions was studied in the model cell line 3T3-L1. The functional role of aGPCRs was exemplarily analysed for GPR64. As the model cell line 3T3-L1 is difficult to transfect and knockdown only reduces gene expression, the suitability of CRISPR/Cas technology for generating a receptor knockout (KO) in 3T3-L1 cell line was tested. For validation, the aGPCRs, Gpr64 and Gpr126, were chosen. The main results of the third part of this study are the following: 1. Gpr64- and Gpr126 KO cells were generated using a commercially avaible 3T3-L1 cell line constitutively overexpressing Cas9 (3T3-L1 Cas9). 2. An impaired adipogenesis and adipocyte function was found already in 3T3-L1 Cas9 compared to 3T3-L1 wildtype. Creating a self-made Cas9-overexpressing 3T3-L1 was not feasible. Thus, Cas9-overexpressing 3T3-L1 cells are not suitable for analysing adipocyte function after KO. 3. Evaluating other approaches for CRISPR/Cas9-mediated KO strategies, transient overexpression of Cas9 using plasmid-based methods were not feasible, too. However, introducing Cas9 protein was successful and did not interfere with adipogenesis making this approach the method of choice for CRISPR/Cas9-mediated KO in 3T3-L1. In brief, KO studies in 3T3-L1 cell line using CRISPR/Cas technology should not be carried out using plasmid-based approaches. However, transfecting the Cas9 protein or its ribonucleoprotein complex is feasible to create 3T3-L1 KO cell lines without interfering with adipogenesis and adipocyte function. In conclusion, this thesis provides a comprehensive study about GPCR expression in AT of lean individuals and individuals with obesity. The identification and characterization of GPCRs hitherto unrecognized in AT or even orphan GPCRs supports the understanding of AT (patho)physiology. In particular, the expression of all GPCRs in AT of lean individuals and individuals with obesity was uncovered for human scAT as well as mouse scAT and vAT. Furthermore, three rhodopsin-like GPCRs (GPR146, MRGPRF, and PTGER2), one Fzd-like receptor (FZD5) and the adhesion GPCR class were basally characterized regarding adipogenesis and (pre)adipocyte functionality using the model cell line 3T3-L1. These data are fundamental for understanding the importance of the whole repertoire of GPCR in AT (patho)physiology and can be used as a starting point for their characterization in AT and adipocyte function in depth, possibly even leading to novel therapeutic approaches in the future.

Introduction Adhesion GPCRs (aGPCRs) constitute the second largest family of the GPCR superfamily, and yet their properties are also the least understood. Growing research on the biological functions of aGPCRs suggest their implications in various (patho)physiological processes, such as cell migration, organ development and cancers. Moreover, due to the unique architecture of a large extracellular region (ECR) containing a plethora of adhesion motifs, aGPCRs are vital as a mechanosensor which transduces extracellular mechanical stimuli into intracellular signal transduction. One distinct feature of aGPCRs among the GPCR superfamily is the possession of a conserved extracellular fold termed GPCR autoproteolysis-inducing (GAIN) domain in perhaps all members within the class. The cleavage at the last loop of the GAIN domain leads to the formation of two non-covalently associated N- and C-terminal fragments (NTF/CTF). A peptide stretch in the start of the CTF acts as a tethered agonist (TA) which is responsible for at least part of the signaling volumes of an activated receptor. Despite the strict conservation of the GAIN domain and its importance in the activation mechanism of aGPCRs, some other fundamental properties of the receptors, with reference to GAIN domain cleavage, have not been rigorously analysed in a biological context. Thus, this study aims to: 1. Explore the structural and molecular determinants that affects GAIN domain cleavage; 2. Investigate the consequences of GAIN domain cleavage towards (i) surface trafficking, and (ii) phosphorylation of receptors. Results Abolishment of GAIN domain cleavage in Polycystin-1, the only other protein family possessing the GAIN domain, was found to eliminate its surface expression, which is a cause of polycystic kidney/liver disease. However, whether such relationship is also true for aGPCRs has not been systematically analysed. Therefore, the study started with profiling the kinetics of surface delivery of several members of aGPCRs. Mutations on the -2 or +1 residues of the GPCR proteolytic site (GPS) (thereby abolishing GAIN domain cleavage) affected the steady-state surface and total expressions of the receptors differently, and had variable effect towards different receptor members. However, the observations from steady-state kinetics are also a resultant output from numerous processes involved in proteostasis. To further dissect whether GPS mutations affect the surface trafficking of the receptors, a pulse-chase assay called the ‘Retention Upon Selective Hook’ (RUSH) assay was employed, wherein the synthesised receptor molecules conjugated to a streptavidin-binding peptide are withheld in the ER by the co-expressed, ER-resident streptavidin, and are only released upon the addition of biotin that outcompete the receptor-streptavidin binding, creating a synchronised transport. By adapting the RUSH assay on some aGPCR members, the attenuation of surface trafficking by GPS mutations has become more apparent. The tested receptors were found to have a deficit in the quantity of surface population, rather than a change in rate of trafficking, upon the introduction of GPS mutations. This implies that the cells may utilise GAIN domain cleavage as a quality checkpoint for ER exit of aGPCRs. As the GAIN domains of at least some aGPCRs were found to be cleaved before ER exit, and as the rate of surface delivery was generally not affected by GAIN domain cleavage, the influence of GAIN domain cleavage may arise earlier during the receptor maturation in the ER. However, while the mechanisms of GAIN domain cleavage have been elucidated previously, they rely heavily on purified domains. The fundamental questions of when exactly the GAIN domain is cleaved and what additional determinants apart from the GPS sequence contribute to GAIN domain cleavage during receptor biogenesis have still not been answered. In combination with molecular dynamics (MD) simulation studies on the GAIN domain of rat isoform of ADGRL1, F803 was found to be crucial in the proteolysis by forming an edge-π interaction with H836 (-2 position of the cleavage site), such that H836 is in close proximity to the hydroxyl group of T838 for the initiation of the nucleophilic attack. Reconstruction of the edge-π interaction into ADGRB3, a naturally uncleavable receptor, partially reinstates its GAIN domain cleavage; but similar reintroduction on ADGRB2 has no effect on restoring the proteolysis. Nonetheless, this observation highlights the vitality of a proper folding of the GAIN domain, specifically the microenvironment of the cleavage site, in assisting in cleavage. The study continued with a systematic series of experiments that ultimately discover the roles of the CTF towards GAIN domain cleavage of aGPCRs. Firstly, to mimic the biogenesis of the receptor, the seven transmembrane (7TM) region of ADGRE2 (E2) was stepwisely truncated and then analysed for GAIN domain cleavage. It was observed that the extent of GAIN domain cleavage increases when the ECR of E2 precedes with more number of TMs. The proteolysis occurs, although less efficiently, as early as the first TM is synthesised. Interestingly, GAIN domain cleavage is unaffected when the TM region of the E2-1TM mutant was replaced by other single-pass TM, and whether it is trafficked to the surface or held in the ER, while the proteolysis of TM-less ECR mutants is largely impeded. Based on this observation, the ECR and the TM region was spaced either by a fluorophore moiety or a variable number of helical turns. Remarkably, the extent of GAIN domain cleavage of all tested receptors declined upon the increase in displacement with the lumenal side of the ER membrane, defining the importance of membrane proximity in the completion of proteolysis during the maturation of GAIN domain. In that, a new model of GAIN domain cleavage during biogenesis has been proposed, with appreciation of the GAIN domain as part of a higher-order stuctural organisation rather than an independent domain. A physiological extent of GAIN domain cleavage does not only require the folding of the GAIN domain, but also the membrane tethering property of the CTF, allowing a partial cleavage as little as one TM is generated, and a dynamic stability provided by the full CTF. In some aGPCRs, the contributions from CTF are more significant than the autonomous GAIN domain folding. The findings implicate more complex requirements for GAIN domain cleavage in a biological context, and hence supporting a possibility that GAIN domain cleavage is the rate-determining step for ER exit of the receptor, leading to the observations obtained in the kinetic study. Phosphorylation of L3 by PKC activated by distant signaling cascade(s) The last part of the study focused on characterising the mechanism of phosphorylation of ADGRL3 (L3) at Thr1140 (pT1140), which is a poorly explored field of aGPCRs. It was made possible by exploiting a phosphospecific antibody developed in collaboration. Coincidently, pT1140 was not dependent on the examined GPCR properties of the receptor, such as G protein coupling, dependence of the TA, and GRK-mediated phosphorylation. Instead, by series of pharmacological inhibitions, it was discovered that pT1140 originates from the action of novel PKCs (nPKCs). Co-expression of L3 and dominant-negative mutants or the catalytic domains of individual members of nPKCs reveals that PKC acts as a master regulator of the phosphorylation event, by directly phosphorylating the receptor and priming other members of the nPKCs for pT1140. Finally, possible origins of the PKC activation were explored. It was found that the stimulation of PKC occurs via actin disassembly, which can act downstreams of VEGF-A/VEGFR2 signaling, although the physiological relevance is still yet to be deciphered. Nonetheless, the observations opened up new directions of research in the aspect of crosstalks between different signaling cascades and the possible modulations of the signaling fidelity of aGPCRs. Additionally, the complexity of aGPCR signaling has been clearly demonstrated. Conclusion This study has further defined the importance of GAIN domain cleavage for surface trafficking of aGPCRs, a process crucial for extracellular interactions. Moreover, a novel mechanistic model of GAIN domain cleavage in relevance to biogenesis and maturation of the receptors has been postulated. Characterisation of a site-specific phosphorylation mechanism of L3 has illustrated the potential of complex interactions of aGPCRs with other signaling pathways in cells. The results collectively shed light on the structure-function relationship of aGPCRs, and pave ways for numerous potential areas for explorations in the future.

La communication cellulaire est un phénomène important pour le maintien de l’homéostasie des cellules. Au court des dernières années, cette sphère de recherche sur la signalisation cellulaire a connue des avancées importantes au niveau de l’identification des acteurs principaux impliqués dans la reconnaissance extracellulaire des signaux, ainsi que la compréhension des voies de signalisation engagées par les cellules pour répondre aux facteurs extracellulaires. Malgré ces nouvelles informations, les diverses interrelations moléculaires entre les acteurs ainsi que les voies de signalisation cellulaire, demeurent mal comprises. Le transfert d’énergie de résonance de bioluminescence (BRET) permet la mesure d’interactions protéiques et peut être utilisé dans deux configurations, le BRET480-YFP (connu aussi comme le BRET1) et le BRET400-GFP (connu aussi en tant que BRET2). Suite à l’oxydation de son substrat, la luciférase de renilla peut transférer son énergie à une protéine fluorescente, uniquement si elles sont à proximité l’une de l’autre (≤100Å). La combinaison dans un seul essai des BRET480-YFP et BRET400-GFP, a permis de suivre trois paires d’interactions, sur une même population cellulaire. Par contre, l’utilisation de deux substrats pour la réaction de bioluminescence rend impossible la mesure simultanée des différents signaux de BRET, pour ce trois nouvelles configurations de BRET ont été mises au point en utilisant des nouvelles protéines fluorescentes. Ainsi deux des nouvelles couleurs de BRET ayant des émissions résolues, le BRET400-BFP et le BRET400mAmetrine ont pu être combinées pour mesurer l’engagement par un RCPG d’une protéine G, ainsi que l’accumulation du second messager. La combinaison de ces BRET a également permis de révéler la formation d’un complexe entre le récepteur α2A adrénergique (α2AAR), Gαi1, le dimère Gβγ ainsi que la kinase des récepteurs couplés aux protéines G (GRK2), suite à l’activation du récepteur. De plus, seule l’entrée de GRK2 semble être en mesure de causer la désensibilisation du α2AAR, en s’intercalant entre Gαi1 et Gβγ. Par contre, la stabilisation de l’interaction entre α2AAR et la β-arrestine2 semble nécessiter l’activité kinase de GRK2. Une autre étude a révélé l’importance de différentes Gα pour la mobilisation du calcium, suite à l’activation du récepteur aux opioïdes de type delta (DOR). Suite à la surexpression de Gα de la famille Gαq, il a été possible de mesurer une influence de ces Gα sur la mobilisation du calcium. Toutefois, cette réponse calcique mesurée en présence des Gαq demeure sensible aux prétraitements à la toxine de Bordetella pertussis, qui inhibe sélectivement l’activité des Gαi. De plus, la co-expression de Gαi et Gαq permet de potentialiser la mobilisation de calcium, démontrant une interrelation entre ces deux familles de protéine Gα, pour la signalisation du DOR. Afin de démontrer l’interrelation directe, des expériences de BRET ont été réalisées entre différentes Gα. En plus de montrer la formation de complexes sélectifs entre les Gα, les expériences de BRET réalisées en parallèle d’analyses de séquences de Gα, ont également mis à jour un site de sélectivité d’interaction entre les Gα, l’hélice α4. Suite à la transposition de cette hélice α4 de Gα12 sur Gαi1, qui normalement n’interagissent pas, il a été possible de forcer l’interaction entre Gα12 et Gαi1, confirmant ainsi que cette hélice α contient l’information permettant une sélectivité d’interaction. Au cours de cette thèse, il a été possible de générer de nouvelles méthodes de mesure d’interactions protéiques qui permettent de multiplexer différents signaux, ce qui a permis de mettre à jour de nouvelles interactions entre divers effecteurs de la signalisation de RCGP / Cellular communication is an important phenomenon for the maintenance of cellular homeostasis. Recently, important progress has been made in the cell signalling research field concerning the identification of the major actors and the cellular pathways engaged in response to these extracellular factors. However, in spite of this new information, the interrelationships at the molecular level between the various cellular actors and the different signalling pathways remain badly understood. Bioluminescence resonance energy transfer (BRET) monitors interactions between proteins and can be used in two configurations, the BRET480-YFP (also known as BRET1) and the BRET400-GFP (also known as BRET2). Following oxidation of its substrate, renilla luciferase transfers its energy to a fluorescent protein, only if they are in close proximity (≤100Å). By combining the BRET480-YFP and BRET400-GFP in one assay, it is possible to follow three pair-wise interactions in the same cellular population. However, using two bioluminescence reaction substrates limits the possibility of measuring the different BRET signals simultaneously. In order to measure multiple BRET signals simultaneously, three new BRET configurations, based on the BRET400-GFP, were developed using fluorescent proteins with different emission wavelengths. Two of the new BRET colors which have resolved emission wavelengths, the BRET400-BFP and BRET400mAmetrine, were combined for measuring the heterotrimeric G protein engagement by the vasopressin V2 receptor, as well as the accumulation of the second messenger. Combining these new BRET techniques reveals for the first time the formation of a complex between the α2A adrenergic receptor (α2AAR), Gαi1, the Gβγ dimer and G protein-receptor kinase (GRK2) following receptor activation. Moreover, only the entry of GRK2 into the receptor complex is required for the α2AAR desensitization, by inserting between Gαi1 and Gβγ. On the other hand, the stabilization of the interaction between α2AAR and β-arrestin2 requires the kinase activity of GRK2. Another study revealed the importance of multiple Gα subunits for calcium mobilization induced upon activation of the delta opioid receptor (DOR). Gαq subfamily member overexpression altered the DOR-induced calcium mobilization, but this Gαq calcium mobilization remained sensitive to pre-treatement pertussis toxin, through selective inhibition of the activity of Gαi members. Moreover, Gαi and Gαq co-expression potentiated calcium mobilization, suggesting an interrelationship between these two Gα families in DOR signaling. This Gαi and Gαq interrelationship could result from the formation of a complex close to the receptor. In order to test this hypothesis, BRET experiments were performed, with the aim of measuring the presence of complexes between different Gα. In addition to demonstrating complex formation between Gα subunits, the BRET experiments in parallel with sequence analysis, also revealed a selective interaction site between the Gα, the α4 helix. By swapping the a4 helix of Gαi with the α4 helix of Gα12, which doesn’t normally interact with Gα12, it was possible to force the interaction between Gα12 and Gαi to confirm that this α helix contains information concerning the selectivity of interactions between Gα subunits. During this thesis, new methods were to detect protein interactions and multiplexing these methods allowed the detection of novel interactions between signalling effectors of GPCRs.

La communication cellulaire est un phénomène important pour le maintien de l’homéostasie des cellules. Au court des dernières années, cette sphère de recherche sur la signalisation cellulaire a connue des avancées importantes au niveau de l’identification des acteurs principaux impliqués dans la reconnaissance extracellulaire des signaux, ainsi que la compréhension des voies de signalisation engagées par les cellules pour répondre aux facteurs extracellulaires. Malgré ces nouvelles informations, les diverses interrelations moléculaires entre les acteurs ainsi que les voies de signalisation cellulaire, demeurent mal comprises. Le transfert d’énergie de résonance de bioluminescence (BRET) permet la mesure d’interactions protéiques et peut être utilisé dans deux configurations, le BRET480-YFP (connu aussi comme le BRET1) et le BRET400-GFP (connu aussi en tant que BRET2). Suite à l’oxydation de son substrat, la luciférase de renilla peut transférer son énergie à une protéine fluorescente, uniquement si elles sont à proximité l’une de l’autre (≤100Å). La combinaison dans un seul essai des BRET480-YFP et BRET400-GFP, a permis de suivre trois paires d’interactions, sur une même population cellulaire. Par contre, l’utilisation de deux substrats pour la réaction de bioluminescence rend impossible la mesure simultanée des différents signaux de BRET, pour ce trois nouvelles configurations de BRET ont été mises au point en utilisant des nouvelles protéines fluorescentes. Ainsi deux des nouvelles couleurs de BRET ayant des émissions résolues, le BRET400-BFP et le BRET400mAmetrine ont pu être combinées pour mesurer l’engagement par un RCPG d’une protéine G, ainsi que l’accumulation du second messager. La combinaison de ces BRET a également permis de révéler la formation d’un complexe entre le récepteur α2A adrénergique (α2AAR), Gαi1, le dimère Gβγ ainsi que la kinase des récepteurs couplés aux protéines G (GRK2), suite à l’activation du récepteur. De plus, seule l’entrée de GRK2 semble être en mesure de causer la désensibilisation du α2AAR, en s’intercalant entre Gαi1 et Gβγ. Par contre, la stabilisation de l’interaction entre α2AAR et la β-arrestine2 semble nécessiter l’activité kinase de GRK2. Une autre étude a révélé l’importance de différentes Gα pour la mobilisation du calcium, suite à l’activation du récepteur aux opioïdes de type delta (DOR). Suite à la surexpression de Gα de la famille Gαq, il a été possible de mesurer une influence de ces Gα sur la mobilisation du calcium. Toutefois, cette réponse calcique mesurée en présence des Gαq demeure sensible aux prétraitements à la toxine de Bordetella pertussis, qui inhibe sélectivement l’activité des Gαi. De plus, la co-expression de Gαi et Gαq permet de potentialiser la mobilisation de calcium, démontrant une interrelation entre ces deux familles de protéine Gα, pour la signalisation du DOR. Afin de démontrer l’interrelation directe, des expériences de BRET ont été réalisées entre différentes Gα. En plus de montrer la formation de complexes sélectifs entre les Gα, les expériences de BRET réalisées en parallèle d’analyses de séquences de Gα, ont également mis à jour un site de sélectivité d’interaction entre les Gα, l’hélice α4. Suite à la transposition de cette hélice α4 de Gα12 sur Gαi1, qui normalement n’interagissent pas, il a été possible de forcer l’interaction entre Gα12 et Gαi1, confirmant ainsi que cette hélice α contient l’information permettant une sélectivité d’interaction. Au cours de cette thèse, il a été possible de générer de nouvelles méthodes de mesure d’interactions protéiques qui permettent de multiplexer différents signaux, ce qui a permis de mettre à jour de nouvelles interactions entre divers effecteurs de la signalisation de RCGP / Cellular communication is an important phenomenon for the maintenance of cellular homeostasis. Recently, important progress has been made in the cell signalling research field concerning the identification of the major actors and the cellular pathways engaged in response to these extracellular factors. However, in spite of this new information, the interrelationships at the molecular level between the various cellular actors and the different signalling pathways remain badly understood. Bioluminescence resonance energy transfer (BRET) monitors interactions between proteins and can be used in two configurations, the BRET480-YFP (also known as BRET1) and the BRET400-GFP (also known as BRET2). Following oxidation of its substrate, renilla luciferase transfers its energy to a fluorescent protein, only if they are in close proximity (≤100Å). By combining the BRET480-YFP and BRET400-GFP in one assay, it is possible to follow three pair-wise interactions in the same cellular population. However, using two bioluminescence reaction substrates limits the possibility of measuring the different BRET signals simultaneously. In order to measure multiple BRET signals simultaneously, three new BRET configurations, based on the BRET400-GFP, were developed using fluorescent proteins with different emission wavelengths. Two of the new BRET colors which have resolved emission wavelengths, the BRET400-BFP and BRET400mAmetrine, were combined for measuring the heterotrimeric G protein engagement by the vasopressin V2 receptor, as well as the accumulation of the second messenger. Combining these new BRET techniques reveals for the first time the formation of a complex between the α2A adrenergic receptor (α2AAR), Gαi1, the Gβγ dimer and G protein-receptor kinase (GRK2) following receptor activation. Moreover, only the entry of GRK2 into the receptor complex is required for the α2AAR desensitization, by inserting between Gαi1 and Gβγ. On the other hand, the stabilization of the interaction between α2AAR and β-arrestin2 requires the kinase activity of GRK2. Another study revealed the importance of multiple Gα subunits for calcium mobilization induced upon activation of the delta opioid receptor (DOR). Gαq subfamily member overexpression altered the DOR-induced calcium mobilization, but this Gαq calcium mobilization remained sensitive to pre-treatement pertussis toxin, through selective inhibition of the activity of Gαi members. Moreover, Gαi and Gαq co-expression potentiated calcium mobilization, suggesting an interrelationship between these two Gα families in DOR signaling. This Gαi and Gαq interrelationship could result from the formation of a complex close to the receptor. In order to test this hypothesis, BRET experiments were performed, with the aim of measuring the presence of complexes between different Gα. In addition to demonstrating complex formation between Gα subunits, the BRET experiments in parallel with sequence analysis, also revealed a selective interaction site between the Gα, the α4 helix. By swapping the a4 helix of Gαi with the α4 helix of Gα12, which doesn’t normally interact with Gα12, it was possible to force the interaction between Gα12 and Gαi to confirm that this α helix contains information concerning the selectivity of interactions between Gα subunits. During this thesis, new methods were to detect protein interactions and multiplexing these methods allowed the detection of novel interactions between signalling effectors of GPCRs.

Fundamental to the complexity of the nervous system is the precise regulation in space and time of the production, maturation, and migration of neurons in the developing embryo. This is eloquently seen in the forming cranial sensory ganglia (CSG) of the peripheral nervous system. Placodes, which are transient pseudostratified neuroepithelia in the surface ectoderm of the embryo, are responsible for generating most of the neurons of the CSG. Placodal progenitors commit to the neuronal fate and delaminate from the epithelium as immature, multipolar neuroblasts. These neuroblasts reside in a staging area immediately outside the placode. Differentiation of the neuroblasts is intimately coupled to their adoption of a bipolar morphology and migration away from the staging area to the future site of the CSG. Thus the forming CSG is a highly tractable model to anatomically separate the three phases of a neuroblast’s lifetime: from neuroepithelial progenitor (in the placode), to immature neuroblast (in the staging area), to mature neuron (in the migratory stream). In this thesis, I used the forming CSG as a model to investigate the role of Regulator of G protein Signalling 3 (RGS3) in neuroblast commitment and differentiation. Promoters within introns of the RGS3 locus generate isoforms in which N-terminal sequences are sequentially truncated, but C-terminal sequences are preserved. Intriguingly, I found that expression of these isoforms in the forming CSG is temporally co-linear with their genomic orientation: longer isoforms are exclusively expressed in the progenitor placode; a medium isoform is expressed exclusively in the neuroblast staging area; and the shortest isoforms are expressed in the neuronal migratory stream. Furthermore, through loss- and gain-of-function experiments, I demonstrated that each of these isoforms plays a specific role in the differentiation state in which it is expressed: placode-expressed isoforms negatively regulate neurogenesis; the neuroblast-expressed isoform negatively regulates differentiation; and the neuron-expressed isoforms negatively regulate neuronal migration. The negative regulatory role which all isoforms play in different cell-biological contexts is intriguing in light of the fact that they all share a C-terminal RGS domain, which canonically negatively regulates G protein signalling. Through domain mutation and deletion, I showed that the RGS and N-terminal domains are important for the function of each isoform. Thus temporally co-linear expression within the RGS3 locus generates later-expressed isoforms which lack the regulatory N-terminal domains of the earlier-expressed isoforms, giving them new license to perform different biochemical functions. Lastly, I investigated the conservation and evolution of RGS3 and its isoforms. RGS3 was found to be present in all extant metazoans, and results from this thesis implicate it as the founding member of the R4 subfamily of RGS proteins. Furthermore, in the early vertebrate lineage, a critical domain was lost. This is intriguing in light of the fact that placodes in their stereotypic forms also emerged early in the vertebrate lineage. Ectopic overexpression of the full-length invertebrate RGS3 protein prevented pseudostratification of the vertebrate placode, suggesting that the domain loss in the early vertebrate lineage was important for the evolution of pseudostratified placodes and the expansion of the vertebrate nervous system. In summary, the work in this thesis has uncovered a previously unseen model of transcriptional regulation of a single locus: intragenic temporal co-linearity. Furthermore, the demonstrated functions of this regulation have profound implications on the generation and differentiation of vertebrate neurons, as well as the evolution of the vertebrate nervous system.

No description available.

Les récepteurs couplés aux protéines G (RCPG) constituent la plus grande famille de récepteurs transmembranaires. Ils sont impliqués dans une large variété de processus physiologiques et par conséquent ils représentent une cible thérapeutique d'intérêt pour le développement de médicaments. Plusieurs études ont démontré que les RCPGs sont capables d'interagir entre eux pour former des complexes oligomériques. Cependant, leur existence in vivo et leur rôle fonctionnel reste sujet à débats. Afin de mieux appréhender ce phénomène, nous avons utilisé un RCPG de classe C comme modèle d'étude, le récepteur de l'acide γ-aminobutyrique (GABAB), qui est impliqué dans une grande variété de désordres neurologiques et psychiatriques. Son originalité réside dans le fait qu'il est un hétérodimère obligatoire composé de deux sous-unités : GABAB1 et GABAB2 (GB1 et GB2). La liaison de l'agoniste sur GB1 conduit à l'activation de GB2. Au cours de ma thèse, nous avons montré en utilisant une nouvelle approche biophysique basée sur un marquage fluorescent enzymatique appelé Snap-tag que, contrairement aux récepteurs métabotropiques du glutamate, le récepteur GABAB forme des dimères de dimères (tétramères). Cette organisation hétéro-oligomérique est assurée par des contacts stables entre les domaines extracellulaires des sous-unités GB1. De plus, nous avons apporté des données en faveur de l'existence physiologique de cet assemblage en utilisant des membranes de cerveau de rat et de souris. Dans une seconde partie, nous avons souhaité déterminer les conséquences fonctionnelles de cette organisation. Nos résultats suggèrent une efficacité de couplage à la protéine G réduite du récepteur GABAB lorsqu'il est associé en dimères de dimères. Collectivement, nos données rapportent pour la première fois, l'existence de larges complexes allostériques de RCPGs dans le cerveau. / The G-protein coupled receptors (GPCR) constitute the main family of transmembrane receptors. They are involved in many physiological processes and, as a consequence, they represent a therapeutic target of interest for the development of new drugs. Few studies have demonstrated that GPCRs are able to interact with each other to form oligomeric complexes. However, the existence in vivo and the functional interest of these oligomers remain a subject of intense debates. To address this issue, we have used a class C GPCR as a model, the γ-aminobutyrate B receptor (GABAB), which is involved in a wide variety of neurological and psychiatric disorders. This receptor has the particularity to be an obligatory heterodimer composed of two subunits GABAB1 and GABAB2 (GB1 and GB2). Agonist binding on GB1 leads to G-protein activation by GB2. During my thesis, we developed a new biophysical approach based on an enzyme-mediated fluorescent labeling calle d Snap-Tag and showed that, unlike metabotropic glutamate receptors, GABAB forms dimers of dimers (tetramers). This oligo-heterodimers organization is mediated via stable contacts between extracellular domains of GB1 subunits. Furthermore, we brought evidence of the physiological reality of this assembly using rat and mouse brain membranes. Then, we aimed at assessing what would be the functional rational of the GABAB dimer of heterodimers. Our results suggest that the GABAB receptor has a lower G protein-coupling efficacy when associated into dimers of dimers. Altogether, our data report for the first time, the existence of large allosteric GPCR complexes in the brain.

Selon le modèle classique, le signal reçu par les récepteurs couplés aux protéines G (RCPG) se propage suite à des interactions transitoires et aléatoires entre les RCPGs, les protéines G et leurs effecteurs. Par les techniques de transfert d’énergie de résonance de bioluminescence (BRET), de complémentation bimoléculaire de protéines fluorescentes (BiFC) et de co-immunoprécipitation, nous avons observé que les récepteurs, les protéines G et les effecteurs forment un complexe stable, avant et après l’activation des récepteurs. L’interaction entre l’effecteur Kir3 et le dimère Gbetagamma se produit initialement au réticulum endoplasmique et est sensible à un agoniste liposoluble des récepteurs beta2-adrénergiques. Bien que peu de spécificité pour les nombreux isoformes des sous-unités Gbetagamma ait été observée pour l’activation du canal Kir3, les interactions précoces au RE sont plus sensibles aux différentes combinaisons de Gbetagamma présentes. En plus de son rôle dans la régulation des effecteurs, le dimère Gbetagamma peut interagir avec de nombreuses protéines possédant des localisations cellulaires autres que la membrane plasmique. Nous avons identifié une nouvelle classe de protéines interagissant avec la sous-unité Gbeta, autant en système de surexpression que dans des extraits de cerveaux de rats, soit les protéines FosB et cFos, qui forment le complexe de transcription AP-1, suite à leur dimérisation avec les protéines de la famille des Jun. La coexpression du dimère Gbetagamma réduit l’activité transcriptionnelle du complexe AP-1 induit par le phorbol 12-,myristate 13-acetate (PMA), sans toutefois interférer avec la formation du complexe Fos/Jun ou son interaction avec l’ADN. Toutefois, le dimère Gbetagamma colocalise au noyau avec le complexe AP-1 et recrute les protéines histones déacétylases (HDAC) afin d’inhiber l’activité transcriptionnelle du complexe AP-1. / Based on the classical model of G protein activation, signal transduction occurs by transient and random interactions between the receptor, the G protein and the effectors. Bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation assay (BiFC) and co-immunoprecipitation experiments revealed that receptor, heterotrimeric G proteins and effectors were found in stable complexes that persisted during signal transduction. Kir3 channel and Gbetagamma dimer interacts first in the endoplasmic reticulum (ER) and this interaction can be modulated by the membrane-permeable beta2-adrenergic agonist cimaterol. Little specificity has been reported for several isoforms of the Gbetagamma dimer in the activation of the Kir3 channel. However, we found that the “precocious” interaction in the ER is sensitive to the presence of different combination of Gbeta and Ggamma subunits. Recently, a number of new proteins, which are not classical effectors at the plasma membrane have been shown to interact with GbetagammaThese include histone deacetylases 4 and 5 (HDAC)[1, 2] and the glucocorticoid receptor. We identified a novel interaction between Gbetagamma subunit and the Fos proteins, which form the transcription factor AP-1 following their dimerization with Jun proteins. Gbetagamma and Fos interactions can be detected in HEK 293 cells overexpressing the two proteins as well as in brains from rats pre-treated with amphetamine. Gbetagamma/Fos interaction favours the nuclear translocation of Gbetagamma dimer and inhibits AP-1 transcriptional activity. Gbetagamma did not block Fos/Jun dimerization or the interaction of AP-1 with DNA but recruited HDACs to the AP-1 complex.

Membrane proteins play a key role in numerous physiological processes such as transport, energy transduction in respiratory and photosynthetic systems, and signal transduction, and are of great pharmaceutical interest, comprising more than 60% of known drug targets. However, crystallisation of membrane proteins, and G protein-coupled receptors (GPCRs) in particular, still relies heavily on the use of protein engineering strategies, which have been shown to hamper protein activity. Here, a range of biophysical methods were used to study the structure and function of two membrane proteins, a prokaryotic peptide transporter, PepT_So and a GPCR, neurotensin receptor 1 (NTS1), using different membrane reconstitution methods to study the proteins in a native-like environment. Firstly, using the pulsed electron paramagnetic resonance (EPR) method of double electron-electron resonance (DEER) the conformation of PepT_So reconstituted into lipid bilayers was assessed and compared to previous structural data obtained from crystallography and modelling. The influence of the membrane potential and the presence of substrate on the conformational heterogeneity of this proton-coupled transporter were investigated. Secondly, NTS1 purification was optimized for biophysical study. Cysteine mutants were created and a labelling protocol was developed and optimized for fluorophore and nitroxide labelling studies. NTS1 was then studied by continuous-wave EPR, to assess the influence of ligand on local protein dynamics, and to assess the structure of a receptor segment known as helix 8, that was proposed to be an α-helix, but was only observed to be helical in one of the NTS1 crystallographic studies. Ensemble and single-molecule Förster resonance energy transfer (FRET), and DEER were combined to study the dimerisation behaviour of NTS1, showing novel dynamics of the interfacial associations. Finally, the signalling mechanism of NTS1 was also investigated using microscale thermophoresis (MST) to assess the affinity of the receptor for G protein in vitro in the absence of ligand, or in the presence of agonist or antagonist. MST measurements were performed in detergent and in nanodiscs of different lipid compositions, to assess the influence of the lipid environment on receptor function. In summary, this thesis demonstrates the potential of biophysical techniques to study various aspects of membrane protein structure and function in native-like lipid systems, complementing e.g. structural data obtained from crystallographic studies with functional data for membrane proteins in more native environments, as well as shedding light on protein dynamics. The work presented here provides novel insights into PepTSo transport, and in particular into NTS1 structure, signalling, and oligomerisation, opening up several avenues for future research.

Os receptores medeiam a maioria das respostas fisiológicas em resposta a diversidade de estímulos. A ativação da sinalização mediada pelo receptor de angiotensina II tipo 1 é o principal responsável pelos efeitos do hormônio angiotensina II (Ang II) nos tecidos alvo. No rim concentrações fisiológicas de Ang II aumentam a atividade no túbulo proximal da isoforma 3 do trocador de Na+/H+ (NHE3). Este efeito é crucial para a manutenção do volume extracelular e pressão arterial. Evidências recentes mostraram que a ativação seletiva da sinalização enviesada da beta-arrestina/ receptor AT1 induz diurese e natriurese independentemente da sinalização via proteína G. Neste estudo testamos a hipótese de que a sinalização enviesada do receptor AT1/ beta-arrestina inibe a atividade do NHE3 no túbulo proximal, bem como investigar os possíveis mecanismos moleculares que medeio este efeito. Para tal, nós determinamos os efeitos do composto TRV120023, que se liga ao receptor AT1, bloqueando o acoplamento da proteína G e estimulando a sinalização da beta-arrestina, na função do NHE3 in vivo e in vitro. A atividade do NHE3 foi medida quer em túbulo proximal nativo, por meio de microperfusão estacionária, bem como em uma linha celular de túbulo proximal de gamba (OKP), por meio de recuperação de pH intracelular dependente de Na+. Os nossos resultados mostram que o TRV120023 na concentração de 10-7 M inibe marcadamente a atividade do NHE3 em túbulo proximal quer in vivo quer in vitro, sendo que este efeito é completamente abolido nas células silenciadas para a beta-arrestina 1 e 2 através de RNA de interferência. Adicionalmente, a estimulação do NHE3 pela Ang II é completamente suprimida pelo TRV120023 quer in vivo quer in vitro. A inibição do NHE3 pelo TRV120023 foi associada com a diminuição do NHE3 expresso na superfície da membrana plasmática em células OKP e com a redistribuição entre o corpo e a base das microvilosidades em túbulo proximal de rato. A diminuição do NHE3 na superfície da membrana plasmática em células OKP estava associado com um aumento na internalização do NHE via endocitose mediada por clatrina. A inibição do NHE3 mediada pela beta-arrestina não envolve a sinalização do receptor AT2, cAMP/ PKA, Akt e ERK1/2. Estes achados indicam que a sinalização enviesada do receptor AT1/beta-arretina inibe a atividade do NHE3 em túbulo proximal, pelo menos em parte, devido a alterações na localização subcelular do NHE3 / Cell surface receptors mediate most of our physiological responses to an array of stimulus. The triggering of the angiotensin II type I (AT1) receptor signaling is the major control point in the regulation of the ultimate effects of the peptide hormone angiotensin II (Ang II) on its target tissue. In the kidney physiological concentrations of Ang II upregulate the activity of proximal tubule Na+/H+ exchanger isoform 3 (NHE3). This effect is crucial for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the betaarrestin-biased AT1 receptor signalingpathway induces diuresis and natriuresis independent of G-protein mediated signaling. This study tested the hypothesis that activation of this AT1 receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule as well as investigate the underlying molecular mechanisms mediating this effect. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G protein coupling, and stimulates beta-arrestin signaling, on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na+-dependent intracellular pH recovery. Our results showed that 10-7 MTRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro, and the effect was completely abolished in OKP cells silenced for beta-arrestin 1 and 2 by small interference RNA. Additionally, stimulation of NHE3 by Ang II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. The decreased surface NHE3 in OKP cells was associated with an increase in NHE3 internalization via clathrin mediated endocytic. Beta-arrestin mediated NHE3 inhibition did not involve AT2 receptor, cAMP/ PKA, Akt and ERK1/2 signaling. These findings indicate that biased signaling of the AT1 receptor/beta-arrestin pathway inhibits NHE3 activity in the proximal tubule at least in part due to changes in NHE3 subcellular localization