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.

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

3BP2 is a pleckstrin homology (PH) domain- and Src homology 2 (SH2) domain-containing adapter protein that has been linked through genetic evidence to a rare human disease called cherubism 146. 3BP2 was originally cloned in a screen to identify c-Abl SH3 binding proteins 23,24. In overexpression studies, 3BP2 has been implicated as a positive regulatory adapter molecule coupled to immunoreceptor on T cells 67,69,70, B cells 68, NK cells 71-73 and mast cells 74,75. It was also evident that 3BP2 forms complexes with a number of signaling molecules, such as Zap-70, LAT, phospholipase C-γ1 (PLC-γ1), Grb2, Cbl, and Fyn in Jurkat cells 67 and Vav1, Vav2, PLC-γ, and Syk in Daudi B cells 68. Despite the growing body of biochemical data to support the importance of 3BP2 in cells of the hematopoietic lineage, a clear picture of the biological function of 3BP2 has yet to emerge. To elucidate the in vivo function of 3BP2, our laboratory has generated 3BP2 gene-deficient mice through homologous recombination 452. The 3BP2-deficient (3BP2-/-) mice were born at the expected Mendelian frequency and were fertile and viable. 3BP2-/- mice accumulate splenic marginal-zone (MZ) B cells, possess a reduced frequency of peritoneal B-1 B cells, and have a diminished thymus-independent type 2 (TI-2) antigen response. 3BP2-/- B cells demonstrate diminished proliferation and cell survival following cross-linking of the B-cell receptor (BCR). Following BCR ligation, 3BP2 might be recruited to BCR complex through its inducible interaction with BCR costimulatory molecule CD19. In the absence of 3BP2, the activation of BCR downstream effectors such as MAPK Erk1/2, JNK, and c-Abl is normal; however, 3BP2 deficiency leads to defects in Syk phosphorylation and calcium flux. In addition to defects in peripheral B cell activities, 3BP2 deficiency contributes to defects in neutrophil activities. In response to the chemotactic peptide, fMLF, 3BP2-/- neutrophils fail to establish directional migration in vitro. There is a defect in the accumulation of filamentous actin at the leading edge of migrating 3BP2-/- neutrophils which might be responsible for the random movement of these cells under shallow gradient of fMLF. In vivo, there is a delay in the recruitment of circulating neutrophils to the site of chemically induced inflammation in 3BP2-/- mice. Compared to wildtype neutrophils, 3BP2-/- neutrophils fail to properly produce superoxide anion (O2-) following fMLF stimulation. Defects in both directional migration and superoxide production of 3BP2-/- neutrophils might contribute to the reduction in bacteria clearance and the increased mortality in 3BP2-/- mice post Listeria Monocytogenes infection. In Chapter 1 of this thesis, I have reviewed basic structures and functions of the domain modules found in adapter proteins. In addition, I have reviewed the findings from numerous reports on the function of 3BP2 in different cell types. A discussion of the physical appearance and some of the initial characterization of 3BP2-deficient mice (3BP2-/-) we have generated in our laboratory are included in Chapter 1. The second part of Chapter 1 consists of an introduction on B cell receptor signaling pathway and B-cell development and activation. A discussion of G protein-coupled receptor-mediated neutrophil functions can also be found in Chapter 1. Chapter 2 contains all the methods and materials used in my study. Chapter 3 includes the characterization of peripheral B cell compartment of 3BP2-/- mice as well as the role of 3BP2 downstream of B-cell antigen receptor and in T-independent immune response. In chapter 4, I present data from experiments designed to examine the role of 3BP2 downstream of a G protein-coupled receptor, fMLF receptor, of neutrophils. I also show the requirement of 3BP2 in the clearance of Listeria Monocytogenes. In chapter 5, I propose two models for 3BP2 action based on the findings in B cells and neutrophils and discuss future areas for investigation.

3BP2 is a pleckstrin homology (PH) domain- and Src homology 2 (SH2) domain-containing adapter protein that has been linked through genetic evidence to a rare human disease called cherubism 146. 3BP2 was originally cloned in a screen to identify c-Abl SH3 binding proteins 23,24. In overexpression studies, 3BP2 has been implicated as a positive regulatory adapter molecule coupled to immunoreceptor on T cells 67,69,70, B cells 68, NK cells 71-73 and mast cells 74,75. It was also evident that 3BP2 forms complexes with a number of signaling molecules, such as Zap-70, LAT, phospholipase C-γ1 (PLC-γ1), Grb2, Cbl, and Fyn in Jurkat cells 67 and Vav1, Vav2, PLC-γ, and Syk in Daudi B cells 68. Despite the growing body of biochemical data to support the importance of 3BP2 in cells of the hematopoietic lineage, a clear picture of the biological function of 3BP2 has yet to emerge. To elucidate the in vivo function of 3BP2, our laboratory has generated 3BP2 gene-deficient mice through homologous recombination 452. The 3BP2-deficient (3BP2-/-) mice were born at the expected Mendelian frequency and were fertile and viable. 3BP2-/- mice accumulate splenic marginal-zone (MZ) B cells, possess a reduced frequency of peritoneal B-1 B cells, and have a diminished thymus-independent type 2 (TI-2) antigen response. 3BP2-/- B cells demonstrate diminished proliferation and cell survival following cross-linking of the B-cell receptor (BCR). Following BCR ligation, 3BP2 might be recruited to BCR complex through its inducible interaction with BCR costimulatory molecule CD19. In the absence of 3BP2, the activation of BCR downstream effectors such as MAPK Erk1/2, JNK, and c-Abl is normal; however, 3BP2 deficiency leads to defects in Syk phosphorylation and calcium flux. In addition to defects in peripheral B cell activities, 3BP2 deficiency contributes to defects in neutrophil activities. In response to the chemotactic peptide, fMLF, 3BP2-/- neutrophils fail to establish directional migration in vitro. There is a defect in the accumulation of filamentous actin at the leading edge of migrating 3BP2-/- neutrophils which might be responsible for the random movement of these cells under shallow gradient of fMLF. In vivo, there is a delay in the recruitment of circulating neutrophils to the site of chemically induced inflammation in 3BP2-/- mice. Compared to wildtype neutrophils, 3BP2-/- neutrophils fail to properly produce superoxide anion (O2-) following fMLF stimulation. Defects in both directional migration and superoxide production of 3BP2-/- neutrophils might contribute to the reduction in bacteria clearance and the increased mortality in 3BP2-/- mice post Listeria Monocytogenes infection. In Chapter 1 of this thesis, I have reviewed basic structures and functions of the domain modules found in adapter proteins. In addition, I have reviewed the findings from numerous reports on the function of 3BP2 in different cell types. A discussion of the physical appearance and some of the initial characterization of 3BP2-deficient mice (3BP2-/-) we have generated in our laboratory are included in Chapter 1. The second part of Chapter 1 consists of an introduction on B cell receptor signaling pathway and B-cell development and activation. A discussion of G protein-coupled receptor-mediated neutrophil functions can also be found in Chapter 1. Chapter 2 contains all the methods and materials used in my study. Chapter 3 includes the characterization of peripheral B cell compartment of 3BP2-/- mice as well as the role of 3BP2 downstream of B-cell antigen receptor and in T-independent immune response. In chapter 4, I present data from experiments designed to examine the role of 3BP2 downstream of a G protein-coupled receptor, fMLF receptor, of neutrophils. I also show the requirement of 3BP2 in the clearance of Listeria Monocytogenes. In chapter 5, I propose two models for 3BP2 action based on the findings in B cells and neutrophils and discuss future areas for investigation.

Le facteur de l’ADP-ribosylation 6 (ARF6) et Rac1 sont des petites protéines liant le GTP qui régulent plusieurs voies de signalisation comprenant le trafic de vésicules, la modification des lipides membranaires et la réorganisation du cytosquelette d’actine. Cependant, les mécanismes moléculaires par lesquels ARF6 et Rac1 agissent de concert afin de contrôler ces différents processus cellulaires restent méconnus. Dans cette étude, nous montrons que, dans les cellules HEK293, ARF6 et Rac1 sont retrouvées en complexe suite à la stimulation du récepteur à l’angiotensine. Des expériences réalisées in vitro nous indiquent que ces deux GTPases interagissent ensemble directement, et que ARF6 s’associe préférentiellement avec la forme inactive de Rac1. L’inhibition de l’expression de ARF6 par interférence à l’ARN entraîne une activation marquée en cellule de Rac1 via le facteur PIX, indépendamment de la stimulation d’un récepteur, ce qui provoque la migration non contrôlée des cellules. Les arrestines, protéines de régulation de la désensibilisation des récepteurs couplés aux protéines G, servent de protéines d’échafaudage pour Rac1 et ARF6, en interagissant directement avec les GTPases et en augmentant leur association stimulée par l’angiotensine. De plus, les arrestines permettent l’activation, en s’en dissociant, de la MAP Kinase p38 qui régule l’activité de ARF6 et son interaction précoce avec les arrestines. Mis ensemble, ces résultats montrent que les arrestines contrôlent l’activité de ARF6, en influençant p38. ARF6 joue un rôle inhibiteur sur l’activation basale de Rac1 pour permettre ensuite son recrutement et son activation dépendante de l’angiotensine. Cette étude nous a permis de préciser le mode de régulation mis en jeu dans l’initiation de la migration cellulaire, suite à l’activation d’un récepteur couplé aux protéines G. Par le fait même, nous avons identifié certains des acteurs impliqués dans ce processus, offrant ainsi de nouvelles cibles pour le traitement des déséquilibres pathophysiologiques de la migration cellulaire. / The ADP-ribosylation factor 6 (ARF6) and Rac1 are small GTP-binding proteins that regulate several signaling events ranging from vesicle trafficking, to modification of membrane lipids and reorganization of the actin cytoskeleton. However, the molecular mechanisms by which ARF6 and Rac1 act in concert to control these different cellular processes remain unclear. Here, we show that in HEK 293 cells, ARF6 and Rac1 can be found in complex upon stimulation of the angiotensin receptor (ATR). In vitro experiments indicate that these two small G proteins can directly interact together, and that ARF6 preferentially interacts with the GDP-bound form of Rac1. Depletion of ARF6 by RNA interference leads to a marked PIX-dependent Rac1 activation in cells, independently of receptor stimulation, leading to uncontrolled cell migration. Arrestins, which are known for their role in G protein-coupled receptor desensitization, act as scaffold proteins toward Rac1 and ARF6, by directly interacting with the GTPases and by increasing their agonist-promoted association. Besides, arrestins allow p38 MAP Kinase activation, by releasing it, which regulates ARF6 activity and early association occurring between arrestins and ARF6. Taken together, this study shows that arrestins control ARF6 activity, by managing p38. ARF6 is an inhibitor of basal Rac1 activation to further allow the protein to be recruited and activated following angiotensin treatment. This study allowed us to precise how cell migration induction is regulated following G protein-coupled receptor activation. As a result, we identified some of the key players implicated in this process, providing new targets in the treatment of patho-physiological inbalance in cell migration.

Biogene Amine sind kleine organische Verbindungen, die sowohl bei Wirbeltieren als auch bei Wirbellosen als Neurotransmitter, Neuromodulatoren und/oder Neurohormone wirken können. Sie bilden eine bedeutende Gruppe von Botenstoffen und entfalten ihre Wirkungen über die Bindung an eine bestimmte Klasse von Rezeptorproteinen, die als G-Protein-gekoppelte Rezeptoren bezeichnet werden. Bei Insekten gehören zur Substanzklasse der biogenen Amine die Botenstoffe Dopamin, Tyramin, Octopamin, Serotonin und Histamin. Neben vielen anderen Wirkung ist z.B. gezeigt worden, daß einige dieser biogenen Amine bei der Honigbiene (Apis mellifera) die Geschmacksempfindlichkeit für Zuckerwasser-Reize modulieren können. Ich habe verschiedene Aspekte der aminergen Signaltransduktion an den „Modellorganismen“ Honigbiene und Amerikanische Großschabe (Periplaneta americana) untersucht. Aus der Honigbiene, einem „Modellorganismus“ für das Studium von Lern- und Gedächtnisvorgängen, wurden zwei Dopamin-Rezeptoren, ein Tyramin-Rezeptor, ein Octopamin-Rezeptor und ein Serotonin-Rezeptor charakterisiert. Die Rezeptoren wurden in kultivierten Säugerzellen exprimiert, um ihre pharmakologischen und funktionellen Eigenschaften (Kopplung an intrazelluläre Botenstoffwege) zu analysieren. Weiterhin wurde mit Hilfe verschiedener Techniken (RT-PCR, Northern-Blotting, in situ-Hybridisierung) untersucht, wo und wann während der Entwicklung die entsprechenden Rezeptor-mRNAs im Gehirn der Honigbiene exprimiert werden. Als Modellobjekt zur Untersuchung der zellulären Wirkungen biogener Amine wurden die Speicheldrüsen der Amerikanischen Großschabe genutzt. An isolierten Speicheldrüsen läßt sich sowohl mit Dopamin als auch mit Serotonin Speichelproduktion auslösen, wobei Speichelarten unterschiedlicher Zusammensetzung gebildet werden. Dopamin induziert die Bildung eines völlig proteinfreien, wäßrigen Speichels. Serotonin bewirkt die Sekretion eines proteinhaltigen Speichels. Die Serotonin-induzierte Proteinsekretion wird durch eine Erhöhung der Konzentration des intrazellulären Botenstoffs cAMP vermittelt. Es wurden die pharmakologischen Eigenschaften der Dopamin-Rezeptoren der Schaben-Speicheldrüsen untersucht sowie mit der molekularen Charakterisierung putativer aminerger Rezeptoren der Schabe begonnen. Weiterhin habe ich das ebony-Gen der Schabe charakterisiert. Dieses Gen kodiert für ein Enzym, das wahrscheinlich bei der Schabe (wie bei anderen Insekten) an der Inaktivierung biogener Amine beteiligt ist und im Gehirn und in den Speicheldrüsen der Schabe exprimiert wird. / Biogenic amines are small organic compounds that act as neurotransmitters, neuromodulators and/or neurohormones in vertebrates and in invertebrates. They form an important group of messenger substances and mediate their diverse effects by binding to membrane receptors that primarily belong to the large gene-family of G protein-coupled receptors. In insects, the group of biogenic amine messengers consists of five members: dopamine, tyramine, octopamine, serotonin, and histamine. Besides many other effects, some of these biogenic amines were shown, for example, to modulate gustatory sensitivity to sucrose stimuli in the honeybee (Apis mellifera). I have investigated various aspects of the aminergic signal transduction in the “model organisms” honeybee and American cockroach (Periplaneta americana). So far, I have characterized two dopamine receptors, a tyramine receptor, an octopamine receptor and a serotonin receptor of the honeybee, which is well-known for its learning and memory capacities. The receptors where expressed in cultivated mammalian cells in order to analyze their pharmacological and functional (i.e., second messenger coupling) properties. The spatiotemporal expression patterns of the respective receptor mRNA were investigated in the honeybee brain by using different techniques (RT PCR, Northern blotting, in situ-hybridization). The salivary glands of the American cockroach were used as a model object in order to investigate the cellular effects of biogenic amines. Both dopamine and serotonin trigger salivary secretion in isolated salivary glands. The quality of the secreted saliva is, however, different. Stimulation of the glands by serotonin results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Serotonin-induced protein secretion is mediated by an increase in the intracellular concentration of cAMP. The pharmacological properties of dopamine receptors associated with cockroach salivary glands were investigated and the molecular characterization of putative aminergic receptors of the cockroach was initiated. Furthermore, I have characterized the ebony gene of the cockroach. This gene encodes an enzyme that is probably involved in the inactivation of biogenic amines in the cockroach (as in other insects). The ebony gene is expressed in the brain and in the salivary glands of the cockroach.

The mechanisms that regulate the differentiation of stem cells (SCs) into cardiomyocytes are still unclear and the role of endogenous molecules on this process remains unexplored. One such molecule is the bioactive phospholipid lysophosphatidic acid (LPA) which accumulates in the myocardium following acute infarction and exerts multiple biological functions, including the regulation of cell growth and differentiation as well as cell survival (Tigyi et al., 2003; Sengupta, et al., 2004). Experiments were therefore carried out in this thesis to reveal whether LPA can induce the differentiation of stem cells into cardiomyocytes and to identify the signalling mechanisms that mediate this effect. All experiments were carried out in the mouse P19 carcinoma stem cell line. Treatments with LPA in the absence and presence of various pharmacological compounds were conducted in embryoid bodies (EBs) formed from the P19 cells in sterile Petri dishes over 4 days. The EBs were subsequently transferred into 6-well cell culture plates and cultured for specific time points. Lysates were generated and subjected to western blotting for expression of cardiac- specific myosin light chain -1v (MLC-1v). To look at the expression of LPA receptors (LPAR1-LPAR5) experiments were carried out by RT-PCR using specific primers for each LPA receptor and the role of the latter in mediated responses to LPA were examined in the presence of the LPAR 1/3 antagonist, Ki16425, or the LPAR 4 receptor blocker suramin. In addition, experiments were carried out investigating the role of Gαi and specific signalling pathways that may be involved in the differentiation of P19 cells. These were carried out using potent inhibitors/antagonists of Gαi inhibitor (Pertussis toxin), PI3K inhibitor (LY294002), Akt inhibitor (Akt inhibitor XIII), PKC inhibitor (Bisindolylmaleimide I BIM-I), ROCK inhibitor (Y-27632), p38-MAPK inhibitor (SB203580) and ERK1/2 inhibitor (PD98059). Further experiments were carried out to establish whether the presence of LPA results in the phosphorylation of the targeted kinases. These studies were however limited to Akt, p38 MAPK and ERK1/2. Incubation of cells with LPA resulted in the differentiation of P19 cells into cardiomyocytes as reflected by the induction of MLC-1v. The latter increased significantly above basal in a time-dependent manner, reaching a maximum 10 days after plating EBs in 6-well plates. The induction of MLC-1v was more pronounced in cells incubated with 5 μM LPA at 6 days but showed little concentration differences at day 12. RT-PCR analysis confirmed the expression of LPA receptors 1 to 4 but not 5. Pre-incubating cells with suramin and Ki16425 concentration-dependently inhibited MLC-1v expression with 0.05 mg/ml and 10 μM respectively, virtually abolishing the expression of MLC-1v. Additionally, inhibitors of LPAR1/3 and LPAR4 receptors and all the signalling inhibitors except SB203580 abolished the phosphorylation of ERK1/2. Similarly, p38 MAPK activation was completely abolished by LPAR1/3 and LPAR4 receptor antagonists, Interestingly, only LY294002 (5 μM) and Y27632 (10 μM) abolished the LPA induced activation of p38 MAPK while SB203580, BIM-I, Akt inhibitor XIII and PD95080 caused no significant changes to the phosphorylation of p38 MAPK. In conclusion, the studies carried out in this thesis have shown that LPA can induce P19 stem cells to differentiate into cardiomyocytes and they are linked to the well characterised LPA receptors (LPAR1/3 and 4). These receptors are coupled to downstream signalling pathways of which those involving the ROCK, PI3K, PKC and/or Akt may be critical, and may converge on ERK1/2. Inhibition of any of these pathways has the potential to suppress differentiation. In contrast, signalling leading to p38 activation may potentially suppress differentiation but this needs further clarification.

No description available.

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

Abstract Molecular oxygen is a prerequisite for essential metabolic processes in multicellular organisms. However, the supply of oxygen can be disturbed and tissue aerobic metabolism becomes compromised in several pathophysiological conditions. In prolonged hypoxia, cells initiate cell type-specific adaptation processes, which are typically mediated by alterations in gene expression. Changes are mainly driven by a transcription factor called hypoxia-inducible factor 1 (HIF-1). Heart muscle is a highly oxidative tissue and HIF-1 activation turns on myocardial adaptation mechanisms for enhanced survival in oxygen-deprived conditions. The aim of this study was to characterize myocardial gene expression changes during chronic hypoxia and couple the adaptational changes to cardiomyocyte function. The role of hypoxia and HIF-1 activation was studied by using in vitro mouse and rat heart cell culture models, tissue perfusions and in vivo infarction models. In this study, apelin, sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) and G protein-coupled receptor 35 (GPR35) were characterized as novel functionally important myocardial HIF-1 target genes. Apelin and GPR35 were induced in hypoxia, while SERCA2a expression was reduced HIF-1 dependently. HIF-1 activation also altered cardiac myocyte contractility through modulation of SERCA2a and GPR35 expression, leading to impairment of the cellular calcium metabolism. Reduced contractility was suggested to serve as an adaptive mechanism for reduced aerobic ATP production in hypoxic conditions. This study presents novel information about the plasticity of myocardial adaptation to prolonged hypoxia. The role of a conserved transcription factor, HIF-1, was shown to be essential in the adaptation process in the myocardial cells. / Tiivistelmä Riittävä hapensaanti on välttämätöntä monisoluisten eliöiden elintoiminnoille. Hapensaanti voi kuitenkin häiriintyä erilaisissa tautitiloissa, jolloin happea käyttävät prosessit estyvät. Hapenpuutteen (hypoksia) pitkittyessä elimistön solut aloittavat sopeutumisen tilanteeseen muuttamalla toimintaansa geenien ilmentymismuutosten kautta. Adaptaatiota ohjaa pääasiassa hypoksia-indusoituva tekijä 1 (HIF-1). Sydän käyttää runsaasti happea energiantuotannossaan. Hapenpuutteen aikana HIF-1-transkriptiotekijä muuttaa sydämen geenien ilmentymistä siten, että sydänsolut selviävät paremmin happivajaissa olosuhteissa. Tämän tutkimuksen tavoitteena oli määrittää sydämen geenien ilmentymisen hapenpuutevasteita ja yhdistää muutokset sydänsolujen toiminnallisiin muutoksiin. Hapenpuutteen ja HIF-1:n merkitystä sopeutumisessa tutkittiin käyttäen malleina rotan ja hiiren sydänsoluviljelmiä, in vitro-kudosperfuusiomalleja sekä in vivo-sydäninfarktimalleja. Tässä työssä havaittiin apeliinin, sarkoplasmisen kalvoston Ca2+-ATPaasin (SERCA2a) sekä G-proteiinikytketyn reseptori 35:n olevan toiminnallisesti tärkeitä HIF-1:n säätelemiä geenejä sydämessä. Apeliinin sekä GPR35:n ilmentyminen lisääntyi hypoksian aikana, mutta SERCA2a:n ilmentyminen sen sijaan väheni HIF-1 –aktivaation seurauksena. HIF-1 –aktivaation osoitettiin myös vähentävän sydänsolujen supistustoimintaa muuttuneiden SERCA2a:n ja GPR35:n ilmentymisten kautta. Heikentynyt supistustoiminta sopeuttaa soluja vähentyneeseen aerobiseen ATP:n tuottoon hapenpuutteen aikana. Tämä tutkimus antaa lisätietoa sydämen sopeutumiskyvyn mukautumisesta pitkittyneeseen hapenpuutteeseen. Lisäksi tutkimus osoittaa HIF-1:n roolin olevan oleellinen myös sydänsolujen hypoksia-adaptaatioprosesseissa.