Academic literature on the topic 'Beta arrestin signaling'

G protein–coupled receptors mediate their complex functions through activation of signaling cascades from receptors localized at the cell surface and endosomal compartments. These signaling pathways are modulated by heterotrimeric G proteins and the scaffold proteins beta-arrestin 1 and 2. However, in contrast to the events occurring at the cell surface, our knowledge of the mechanisms controlling signaling from receptors localized at intracellular compartments is still very limited. Here we sought to investigate the intracellular signaling from cannabinoid 2 receptor (CB2R). First, we show that receptor internalization is required for agonist-induced phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Then we demonstrate that ERK1/2 activation is mediated by beta-arrestin 1 from receptors localized exclusively at Rab4/5 compartments. Finally, we identify the retromer complex as a gatekeeper, terminating beta-arrestin 1–mediated ERK phosphorylation. These findings extend our understanding of the events controlling signaling from endocytosed receptors and identify the retromer as a modulator of beta-arrestin–mediated signaling from CB2R.

The vascular endothelium is the innermost layer of blood vessels and is a key regulator of vascular tone. Endothelial function is controlled by receptor signaling through G protein-coupled receptors, receptor tyrosine kinases and receptor serine-threonine kinases. The β-arrestins, multifunctional adapter proteins, have the potential to regulate all of these receptor families, although it is unclear as to whether they serve to integrate signaling across all of these different axes. Notably, the β-arrestins have been shown to regulate signaling by a number of receptors important in endothelial function, such as chemokine receptors and receptors for vasoactive substances such as angiotensin II, endothelin-1 and prostaglandins. β-arrestin-mediated signaling pathways have been shown to play central roles in pathways that control vasodilation, cell proliferation, migration, and immune function. At this time, the physiological impact of this signaling has not been studied in detail, but a deeper understanding of it could lead to the development of novel therapies for the treatment of vascular disease.

Abstract GPR17 is a G protein-coupled receptor (GPCR) implicated in the regulation of glucose metabolism and energy homeostasis. Our genetic knockout studies in rodents suggest that GPR17 is a potential therapeutic target for the treatment of metabolic diseases. However, the contributions of GPR17 to human metabolism and metabolic deficits are not well understood. Here, we analyzed the human GPR17 coding sequences of individuals from control and metabolic disease cohorts that were comprised of patients with clinical phenotypes including severe insulin resistance, hypercholesterolemia, and obesity. Across cohorts, 18 nonsynonymous GPR17 variants were identified, including eight variants that were exclusive to the disease cohort. We characterized the protein expression levels, cellular localization, and downstream functional signaling profiles of nine human GPR17 variants (F43L, V96M, V103M, D105N, A131T, G136S, R248Q, R301H, and G354V). We found that the protein expression levels and subcellular localization for each of the nine GPR17 variants were similar to that of the wild type GPR17. As the endogenous GPR17 ligand is still elusive, we used a synthetic GPR17 agonist to quantitatively measure the functional signaling profiles of GPR17 variants. We found some of the variants had distinctly altered signaling profiles. GPR17-G136S lost agonist-mediated cAMP, Ca2+, and beta-arrestin signaling. GPR17-V96M retained cAMP inhibition similar to GPR17-WT but had impaired Ca2+ and beta-arrestin signaling. GPR17-D105N displayed impaired cAMP and Ca2+ signaling but enhanced agonist-stimulated beta-arrestin recruitment. Also, GPR17-G354V retained cAMP and Ca2+ signaling function but had attenuated beta-arrestin recruitment. The identification and functional profiling of naturally occurring human GPR17 variants from individuals with metabolic diseases revealed receptor variants with distinct signaling profiles, including differential signaling perturbations that resulted in receptor signaling bias. These results are expected to contribute to our understanding of the molecular signaling mechanisms underlying GPR17 in metabolic regulation.

Physiological concentrations of angiotensin II (ANG II) upregulate the activity of Na+/H+ exchanger isoform 3 (NHE3) in the renal proximal tubule through activation of the ANG II type I (AT1) receptor/G protein-coupled signaling. This effect is key for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the beta-arrestin-biased AT1 receptor signaling pathway 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. 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. We found that 10−7 M TRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro. 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. These findings indicate that biased signaling of the beta-arrestin pathway through the AT1 receptor inhibits NHE3 activity in the proximal tubule at least in part due to changes in NHE3 subcellular localization.

Abstract Sepsis, a major clinical problem with high morbidity and mortality, is caused by overwhelming systemic host-inflammatory response. β-arrestin 2, a key regulator and scaffolds of various signaling, modulates cell survival and cell death in various systems. However, the effect of β-arrestin 2 on sepsis-induced cardiac dysfunction is not yet known. Here, we show that β-arrestin 2 overexpression significantly enhances animal survival following cecal ligation and puncture (CLP)-induced sepsis. Importantly, overexpression of β-arrestin 2 in mice prevents CLP-induced cardiac dysfunction. In addition, β-arrestin 2 overexpression dramatically attenuates CLP-induced myocardial gp130 and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CLP. Therefore, β-arrestin 2 prevents CLP-induced cardiac dysfunction through gp130 and p38. These results suggest that modulation of β-arrestin 2 might provide a novel therapeutic approach to prevent cardiac dysfunction in patients with sepsis.

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.

The Notch receptor is part of a highly conserved signaling pathway shared in Drosophila, C. elegans and mammals. Extensive studies of Notch signaling have revealed its participation in the development of diverse organ systems including brain, blood cells, blood vessels, gut, and skin. Many genetic modifiers of the Notch signaling pathway have been identified, including some which act at the membrane and others in the nucleus. One such member is Deltex, an E3 ubiquitin ligase, which was originally identified as a modifier of Notch in a Drosophila genetic screen. In early lymphoid development, Deltex has been demonstrated functionally to antagonize Notch signaling but the precise molecular mechanism for this functional antagonism between Notch and Deltex is not understood. However, in Drosophila, recent data supports the formation of a trimeric complex between Deltex, Kurtz and Notch that promotes Notch ubiquitin-mediated proteosomal degradation. Beta-arrestin1 is one of the closest mammalian homologues of Kurtz and functions as an adaptor protein in a variety of cellular processes such as endocytosis, ubiquitination and nuclear shuttling. We hypothesize that a similar interaction occurs in mammalian cells between Notch, beta-arrestin1 and Deltex to negatively modulate the Notch signaling pathway. Our data reveal a physical interaction between beta-arrestin1 and the Notch receptor. We could not, however, detect an interaction between Deltex and beta-arrestin1 by co-immunoprecipitation. We also demonstrate that Notch and beta-arrestin1 physically associate with both a membrane-bound form of activated Notch, as well as the intracellular form of Notch after membrane cleavage. Using RNA interference, as well as overexpression of beta-arrestin1, we demonstrate that beta-arrestin1 negatively regulates a Notch/CSL dependant reporter assay. We also show that the presence of Deltex enhances the negative modulation of the Notch signaling pathway mediated by beta-arrestin1. Therefore, we reveal a new Notch interacting protein and a novel role for beta-arrestin1 in the Notch signaling pathway.

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.

Le GPR54 et le RFSH régulent la reproduction en agissant sur l’axe hypothalamus-hypophyse-gonades (HPG). En agissant dans l’hypothalamus, GPR54 est un régulateur proximal de l’axe tandis que le RFSH contrôle la gamétogenèse dans les deux sexes. Ils représentent deux cibles thérapeutiques majeures pour le traitement des problèmes liés à la fertilité. GPR54 et RFSH appartiennent à la superfamille des récepteurs couplés aux protéines G (GPCR). GPR54 active préférentiellement la voie Gαq/PLC/Ca2+ alors que le RFSH induit principalement la voie Gαs/PKA/cAMP. Les deux récepteurs recrutent et activent les β-arrestines. Un nombre croissant de profils pharmacologiques ont été décrits pour agir sur des GPCR. En effet, des ligands biaisés, capables d’activer préférentiellement un sous-ensemble du répertoire de signalisation par rapport aux ligands endogènes, sont découverts à un taux élevé. Les ligands orthostériques et allostériques peuvent induire des biais en stabilisant des conformations particulières des récepteurs. Sur le plan thérapeutique, les ligands biaisés ont démontré leur potentiel à induire des effets thérapeutiques tout en évitant les effets indésirables. De plus, les ligands allostériques permettent le modulation positive ou négative d’un récepteur tout en conservant l’information temporelle associée au ligand endogène. Jusqu’il y a peu, des outils pharmacologiques aussi divers et intéressant n’existaient pas pour GPR54 ni pour RFSH. L’objectif de cette thèse a donc été d’identifier des ligands allostériques de GPR54 et du RFSH puis, de caractériser leur signalisation biaisée. Dans la première partie, nous avons caractérisé les propriétés pharmacologique d’un panel de composés de faible poids moléculaire, décrits récemment pour être des modulateurs allostériques du FSHR et appartenant à deux classes chimiques différentes. Nous avons profilé leurs actions sur différentes voies de signalisation dans des cellules HEK293 vivantes exprimant différents biosenseurs. Nous avons démontré que chacun de ces composés induisait des biais par comparaison à la FSH. En comparant différents modèles cellulaires, nous avons confirmé que les biais de système représentent un facteur de confusion crucial pour la détermination des biais. Nous avons également identifié des cas limites pour lesquels le modèle opérationnel n’a pas permis de calculer les facteurs de biais. En parallèle, nous avons caractérisé deux nouveaux composés appartenant à des classes chimiques qui n’avaient jamais été décrites pour activer le RFSH. Nous avons démontré qu’ils étaient allostériques et que leurs profils de biais étaient distincts de ceux des autres composés caractérisés. Dans la seconde partie, nous avons sélectionné and caractérisé des nanobodies ciblant GPR54 et RFSH. Nous avons identifié un nanobody exerçant un effet de modulateur allostérique positif (PAM) sur GPR54. Nous avons aussi identifié un nanobody ciblant le RFSH. Ce dernier présentait des propriétés biaisées originales puisqu’il était modulateur allostérique négatif (NAM) pour la voie AMPc mais PAM pour le recrutement de β-arrestine 2. Dans la dernière partie, nous avons essayé de développer des conjugués nanobody-drogue (NDC) en liant nos nanobodies à des agonistes – soit kisspeptin, soit un des composés agonistes que nous avons caractérisés dans la première partie – à l’aide d’un linker flexible. Bien que nous n’ayons pas eu le temps d’obtenir une preuve de concept pour les NDC, cette stratégie nous semble au minimum prometteuse en tant qu’outil de recherche. Dans sa globalité, cette thèse propose de nouveaux outils pharmacologiques qui vont permettre de comprendre les contributions relatives des différentes voies de couplage de GPR54 et du RFSH, in vivo, sur la fonction de reproduction. Ce travail ouvre également de nouvelles pistes thérapeutiques pour contrôler la reproduction chez les animaux d’élevage et en médecine de la reproduction
GPR54 and FSHR regulate reproduction by acting on the hypothalamus-pituitary-gonads (HPG) axis. Acting in the hypothalamus, GPR54 is an upstream regulator of the axis whereas FSHR controls gametogenesis in both sexes. They represents two major pharmacological targets for the treatment of fertility-related problems. Both GPR54 and FSHR belong to the G protein-coupled receptor (GPCR) superfamilly. GPR54 preferentially activates the Gαq/PLC/Ca2+ pathway whereas FSHR mainly activates the Gαs/PKA/cAMP pathway. Both receptors recruit and activate β-arrestins. Increasing number pharmacological profiles have been reported to act on GPCR. Indeed biased ligands capable of preferentially eliciting a subset of the full signalling repertoire, compared to the endogenous ligand are discovered at a high rate. Orthosteric and allosteric ligands can both induce biased signalling by stabilizing specific receptor conformations. Therapeutically, biased ligand have demonstrated the potential to avoid side effects while still activating the signalling pathways leading to therapeutic effects. Moreover, allosteric ligands allow positive or negative modulation of a receptor while keeping the temporal information provided by the endogenous ligand. Until recently, such diverse and valuable pharmacological tools were not available for FSHR and GPR54. The aim of this thesis was to identify allosteric ligands at the FSHR and GPR54 and to characterize their biased signalling. In the first section, we pharmacological characterized a panel of low molecular weight ligands, recently reported to allosterically activate the FSHR and belonging to two chemical classes. We profiled their actions on different signalling pathways in living HEK293 cells expressing different biosensors. We demonstrated each of these compounds induced biased signalling at the FSHR compared to FSH. Using different cell models, we confirmed that system bias is a crucial confounding factor in bias determination. We also identified limit cases in which the operational model did not allow to calculated bias factors. In parallel, we characterized two novel compounds belonging to chemical classes that were not yet reported to activate FSHR. We demonstrated that they were allosteric and that their biased profiles were distinct from the compounds characterized in the first study. In second section of the thesis, we selected and pharmacological characterized nanobodies targeting GPR54 and FSHR. We identified a nanobody that behaved as a positive allosteric modulator (PAM) at the GPR54. We also identified a nanobody against FSHR. This nanobody displayed striking biased properties as it was negative allosteric modulator (NAM) for cAMP production but PAM for β-arrestin 2 recruitment. In the last section of the thesis, we attempted to develop nanobody-drug conjugates (NDC) by linking our nanobodies to agonists - either kisspeptin or one of the low molecular weight agonist of the FSHR - through a flexible linker. Though we did not have time to achieve a proof of concept for NDC, we believe that such hybrid compound could represent at minimum a promising research tools. As a whole, this thesis provides novel pharmacological tools that should allow deciphering the relative contributions of the different transduction mechanisms operating at the FSHR and GPR54, in vivo, in the reproductive function. This work also opens possible avenues for future therapeutic strategies in the control of reproduction in farm animals and in reproductive medicine

Sonic hedgehog (Shh) is a critical secreted signaling molecule that regulates many aspects of organogenesis. In the absence of Shh, many organs, including the foregut, larynx, palate, cerebellum and heart do not form properly. However, the cellular details of the roles of Shh, including the relevant domains of Shh expression and reception, have not been elucidated for many of these processes.

The single embryonic foregut tube must divide into the trachea and esophagus, which does not occur in the Shh-null mutant. In Chapter 5, I use Cre-Lox technology to determine that the ventral foregut endoderm is the relevant source of Shh for this process and the mesoderm must directly receive that Shh signal. Surprisingly, this signaling event appears to occur two days before the foregut begins to divide, indicating an early essential role for Shh in foregut division.

Shh is also expressed at later stages in the maturing trachea and esophagus. In Chapter 6, I demonstrate that these domains serve to establish differentiated mesoderm. In the trachea, Shh from the endoderm signals directly to the mesoderm to form the tracheal cartilage rings. In the esophagus, the roles of Shh are more complex. Shh regulates the size of the esophagus and controls patterning of the concentric rings of esophageal mesoderm, however this process seems to be indirect, requiring autocrine Shh signaling within the esophageal endoderm.

The laryngeal apparatus is entirely absent in the Shh-null mouse. I n Chapter 3, I dissect the domains of Shh expression and reception required for laryngeal development and demonstrate that loss of endodermal Shh expression causes laryngotracheoesophageal clefts and malformed laryngeal cartilages. As much of laryngeal morphogenesis poorly understood, I also utilize dual mesodermal and neural crest fate maps to determine the embryonic origins of various laryngeal tissues. Finally, as Shh signaling often occurs in concert with Bone Morphogenic Protein (BMP) signaling, I investigate the roles of BMP signaling in laryngeal development.

Much of Shh signaling occurs at the primary cilium, to which Smoothened, a critical pathway member, must translocate upon Shh signal transduction. This process requires a Smo-Kif3a-βarretin complex in mammalian cell culture. However, the roles of βarrestins in mouse development, and their relationship to Shh signaling have not been investigated in vivo. To do so, in Chapter 4, I analyze the phenotypes of the βarr1/βarr2 double knockout embryos and demonstrate that they have palatal, cerebellar, cardiovascular and renal defects consistent with a specific impairment of mitogenic Shh signaling.

Altogether, my work dissects the cellular details of Shh signaling during multiple organ systems in the mouse embryo. I further analyze the consequences of absent or misregulated Shh signaling across multiple developmental contexts and determine that Shh plays critical and diverse roles in organogenesis.

Dissertation