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Résumé : L’expression de surface des récepteurs couplés aux protéines G (GPCRs) est un processus hautement régulé et très important dans le maintien de l’homéostasie cellulaire. En effet, un déséquilibre dans leur niveau d’expression est souvent relié à différentes pathologies comme le cancer, le diabète, l’obésité, les maladies cardiovasculaires et les maladies neurodégénératives. C’est pourquoi la compréhension des mécanismes moléculaires influençant ce phénomène est si importante et nous permettra d’élaborer et/ou d’améliorer les médicaments ciblant la régulation de ce processus. Il est bien connu qu’un des acteurs importants dans le trafic vésiculaire des GPCRs est représenté par la famille des Rab GTPases. Effectivement plusieurs de ces dernières, soit les Rabs 1, 2, 4, 5, 6, 7, 8 et 11 pour ne nommer que les plus connues, modulent l’expression de surface des GPCRs. De plus, certaines études soulèvent la possibilité qu’un GPCR soit lui-même capable de réguler son propre trafic intracellulaire, et ce grâce à son interaction avec les Rab GTPases. Toutefois, le mécanisme emprunté par le GPCR pour atteindre cette fin reste à élucider. Dans le présent travail, je démontre que le GPCR, β2AR, module non seulement la maturation de la petite protéine G Rab11a grâce à son interaction avec la Rab GéranylGéranylTransférase (RGGT), mais influence également son activation en modulant son ubiquitination via son association avec la E3-ubiquitine ligase, HACE1. De plus, je révèle que la sous-unité alpha de la RGGT (RGGTA) accroît significativement la maturation et le transport antérograde du récepteur β2AR, ce qui souligne ainsi un nouveau rôle cellulaire pour cette protéine. L’ensemble des résultats générés appuie l’hypothèse qu’un GPCR puisse contrôler son propre routage intracellulaire, et éclaircit les mécanismes utilisés pour réguler l’activé de la Rab GTPase avec laquelle il interagit. // Abstract : Cell surface expression of G Protein-Coupled Receptors (GPCRs) is a highly regulated and very important phenomenon for keeping cellular homeostasis. In fact, dysregulation of their cell expression is related to many diseases like cancer, neurological disorders, obesity, diabetes and cardiovascular diseases. These facts illustrate how important understanding the molecular mechanisms involved in cell surface transport of those receptors is, which will help us in designing or improving drugs which actually target this pathway. Rab GTPases are proteins known for being essential regulators of GPCR vesicular trafficking. Indeed, an increasing number of studies report the implication of Rab1, 2, 4, 5, 6, 7, 8 and 11 (to cite the most frequently studied) cell surface transport of GPCRs. Moreover, some studies also put forward the possibility that a GPCR might be able to regulate its own cellular trafficking by interacting and controlling activation of Rab GTPases. However, the mechanism involved in this process remains to be clarified. In the present study, I demonstrate that the prototypic GPCR, β2AR, not only modulates prenylation/maturation of the small G protein Rab11a by interacting with Rab GeranylGeranylTransferase (RGGT), but also influences Rab11a activation by modulating its ubiquitination via its association with the E3-ubiquitin ligase, HACE1. Furthermore, I reveal that the α subunit of the RGGT (RGGTA) also promotes the maturation and anterograde transport of the receptor, which highlight a new cellular role for this protein. Altogether, those results support the hypothesis that GPCRs control their own trafficking, and shed light on some of the mechanisms that might be employed by those receptors in activation of Rab GTPases.

The discovery that bile acids act as endogenous ligands of the membrane receptor TGR5 and the nuclear receptor FXR increased their significance as regulators of cholesterol, glucose and energy metabolism. Activation of TGR5, expressed on enteroendocrine L cells, by bile acids caused secretion of GLP-1, which stimulates insulin secretion from pancreatic β cells. Expression of TGR5 on pancreatic islet cells and the direct effect of bile acids on the endocrine functions of pancreas, however, are not fully understood. The aim of this study was to identify expression of TGR5 in pancreatic islet cells and determine the effect of bile acids on insulin secretion. Expression of TGR5 was identified by quantitative PCR and western blot in islets from human and mouse, and in α (αTC1-6) and β (MIN6) cells. Release of insulin, glucagon and GLP-1 were measured by ELISA. The signaling pathways coupled to TGR5 activation were identified by direct measurements such as stimulation of G proteins, adenylyl cyclase activity, PI hydrolysis and intracellular Ca2+ in response to bile acids; and confirmed by the use of selective inhibitors that block specific steps in the signaling pathway. Our studies identified expression of TGR5 receptors in β cells and demonstrated that activation of these receptors by both pharmacological ligands (oleanolic acid (OA) and INT-777) and physiological ligand (lithocholic acid, LCA) induced insulin secretion. TGR5 receptors are also expressed in α cells and, activation of TGR5 by OA, INT-777 and LCA at 5 mM glucose induced release of glucagon, which is processed from proglucagon by the selective expression of prohormone convertase 2 (PC2). However, under hyperglycemia, activation of TGR5 in α cells augmented the glucose-induced increase in GLP-1 secretion, which in turn, stimulated insulin secretion. Secretion of GLP-1 from α cells reflected TGR5-mediated increase in PC1 promoter activity and PC1 expression, which selectively converts proglucagon to GLP-1. The signaling pathway activated by TGR5 to mediate insulin and GLP-1 secretion involved Gs/cAMP/Epac/PLC-ε/Ca2+. These results provide insights into the mechanisms involved in the regulation of pancreatic α and β cell function by bile acids and may lead to new therapeutic avenues for the treatment of diabetes.

Charles university in Prague, Pharmaceutical faculty in Hradci Králové, Department of biological and medical sciences Rheinische Friedrich-Wilhelms-University Bonn, Institute of Pharmacology and Toxicology Candidate: Michaela Čajková Supervisor: PharmDr. Miroslav Kovařík, Ph.D. Consultant: Dr. Linda Sarah Hoffmann Title of diploma thesis: Role of Gq-coupled receptors in brown adipocytes In my diploma thesis, we focused on four Gq-coupled receptors (F2R, LPHN1, α1DAR, TSHR) in brown adipocytes (BAs), which were identified in the screen as the highest expressed in immature and mature BAs. Our goal was to validate suggestion, that Thyroid stimulating hormone receptor (TSHR) plays a key role in differentiation of BAs and that F2R, LPHN1, α1D-AR might be important for BAs. In our study, we investigated gene expression of these four receptors in BAs, using analytical methodsquantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot. Results from analysis revealed, that expression of TSHR was increased in mature BAs, it means, that TSHR induce differentiation of BAs. The BAs transduced with short hairpin RNA (sh-RNA) against TSHR were less differentiated, this we proved also with Oil Red-O staining. Expression of adipocyte Protein 2 (aP2), peroxisome proliferator-activated...

Transmembrane signal transduction by ligand-activated G protein-coupled receptors (GPCRs) controls virtually every aspect of mammalian physiology, and this receptor class is the target of 40-50% of currently marketed pharmaceuticals. In addition to the clinical use of direct GPCR agonists and antagonists, it is now believed that GPCR effectors and regulators may also be viable drug targets with improved therapeutic efficacy and specificity. The prototypic role of Regulator of G protein Signaling (RGS) proteins is inhibition of G protein signaling through acceleration of GTP hydrolysis by GΑ, which promotes re-association of GΑ and GΒΓ subunits with the receptor at the cell membrane. In this way, RGS proteins determine the magnitude and duration of the cellular response to GPCR stimulation. Though RGS protein biochemistry has been well elucidated in vitro, the physiological functions of each RGS family member remain largely unexplored. RGS6 belongs to the R7 subfamily of RGS proteins originally identified in brain. Our acquisition of an RGS6-/- mouse allowed us to survey RGS6 expression in all tissues of the body revealing the greatest expression of RGS6 in brain. Despite robust neural RGS6 expression, little is known regarding functional roles of RGS6 in the brain and spinal cord. In addition, we identified several novel, higher molecular weight RGS6 immunoreactive bands specifically present in the nervous system. The plan of this thesis work was multifaceted. We sought to elucidate novel GPCR signaling cascades modulated by RGS6 in brain while simultaneously characterizing the expression patterns and identity of the novel RGS6 species specifically detected in the nervous system. Considering the large diversity of RGS6 isoforms present in brain, the abundance of potential RGS6 binding partners, and the possibility of discovering new mechanisms involved in RGS6 regulation, elucidation of the novel RGS6 molecular species is of paramount importance. Utilizing RGS6-/- mice we identified RGS6 as a critical modulator of two GPCRs in brain. First, by inhibiting the serotonin receptor 1A (5-HT1AR)-adenylyl cyclase (AC) axis, RGS6 functions to promote anxiety- and depression-related behaviors in mice. As a result, RGS6-/- mice exhibit a robust anxiolytic and antidepressant phenotype remarkably similar to that of animals treated chronically with therapeutic doses of selective serotonin reuptake inhibitors (SSRIs). RGS6 also inhibits GABAB receptor (GABABR)-G protein- activated inwardly rectifying potassium (GIRK) channel current in cerebellar granule cells, and loss of RGS6 results in cerebellar ataxia and gait abnormalities reversible by GABABR blockade. Furthermore, evaluation of voluntary alcohol drinking behaviors in WT versus RGS6-/- mice revealed a striking reduction in alcohol intake resulting from RGS6 loss in both acute and chronic alcohol consumption paradigms due, at least in part, to potentiation of GABABR signaling. Thus, RGS6 inhibitors have potential clinical utility in the treatment of mood disorders and alcoholism. We have shown that one novel RGS6 immunoreactive band expressed in the brain and spinal cord is a phospho-protein sensitive to Λ phosphatase-mediated dephosphorylation. Further, new information acquired from PCR amplification of RGS6 mRNA species from human brain cDNA libraries has necessitated substantial revisions to the RGS6 splicing scheme devised by the Fisher laboratory in 2003. To the 36 isoforms generated from two alternate transcription start sites (RGS6L vs. RGS6), the inclusion or exclusion of exons 14 and 17, and variable splicing to one of 7 different 3' terminal exons, we have added the possible insertion of three novel internal exons (A1, A2, A3), a retained intron, and two new 3' terminal exons. As a result, the number of RGS6 mRNAs present in brain could be as many as 248 unique species, an astonishing diversity unprecedented in the RGS protein family.

Die Wahrnehmung von Geschmacksempfindungen beruht auf dem Zusammenspiel verschiedener Sinneseindrücke wie Schmecken, Riechen und Tasten. Diese Komplexität der gustatorischen Wahrnehmung erschwert die Beantwortung der Frage wie Geschmacksinformationen vom Mund ins Gehirn weitergeleitet, prozessiert und kodiert werden. Die Analysen zur neuronalen Prozessierung von Geschmacksinformationen erfolgten zumeist mit Bitterstimuli am Mausmodell. Zwar ist bekannt, dass das Genom der Maus für 35 funktionelle Bitterrezeptoren kodiert, jedoch war nur für zwei unter ihnen ein Ligand ermittelt worden. Um eine bessere Grundlage für tierexperimentelle Arbeiten zu schaffen, wurden 16 der 35 Bitterrezeptoren der Maus heterolog in HEK293T-Zellen exprimiert und in Calcium-Imaging-Experimenten funktionell charakterisiert. Die Daten belegen, dass das Funktionsspektrum der Bitterrezeptoren der Maus im Vergleich zum Menschen enger ist und widerlegen damit die Aussage, dass humane und murine orthologe Rezeptoren durch das gleiche Ligandenspektrum angesprochen werden. Die Interpretation von tierexperimentellen Daten und die Übertragbarkeit auf den Menschen werden folglich nicht nur durch die Komplexität des Geschmacks, sondern auch durch Speziesunterschiede verkompliziert. Die Komplexität des Geschmacks beruht u. a. auf der Tatsache, dass Geschmacksstoffe selten isoliert auftreten und daher eine Vielzahl an Informationen kodiert werden muss. Um solche geschmacksstoffassoziierten Stimuli in der Analyse der gustatorischen Kommunikationsbahnen auszuschließen, sollten Opsine, die durch Licht spezifischer Wellenlänge angeregt werden können, für die selektive Ersetzung von Geschmacksrezeptoren genutzt werden. Um die Funktionalität dieser angestrebten Knockout-Knockin-Modelle zu evaluieren, die eine Kopplung von Opsinen mit dem geschmacksspezifischen G-Protein Gustducin voraussetzte, wurden Oozyten vom Krallenfrosch Xenopus laevis mit dem Zwei-Elektroden-Spannungsklemm-Verfahren hinsichtlich dieser Interaktion analysiert. Der positiven Bewertung dieser Kopplung folgte die Erzeugung von drei Mauslinien, die in der kodierenden Region eines spezifischen Geschmacksrezeptors (Tas1r1, Tas1r2, Tas2r114) Photorezeptoren exprimierten. Durch RT-PCR-, In-situ-Hybridisierungs- und immunhistochemische Experimente konnte der erfolgreiche Knockout der Rezeptorgene und der Knockin der Opsine belegt werden. Der Nachweis der Funktionalität der Opsine im gustatorischen System wird Gegenstand zukünftiger Analysen sein. Bei erfolgreichem Beleg der Lichtempfindlichkeit von Geschmacksrezeptorzellen dieser Mausmodelle wäre ein System geschaffen, dass es ermöglichen würde, gustatorische neuronale Netzwerke und Hirnareale zu identifizieren, die auf einen reinen geschmacks- und qualitätsspezifischen Stimulus zurückzuführen wären. / Taste impression is based on the interaction of taste, smell and touch. To evaluate the nutritious content of food mammals possess five distinct taste qualities: sweet, bitter, umami (taste of amino acids), sour and salty. For bitter, sweet, and umami compounds taste signaling is initiated by binding of tastants to G protein-coupled receptors. The interactions of taste stimuli, usually watersoluble chemicals, with their cognate receptors lead to the activation of the G protein gustducin, which, in turn, initiates a signal resulting in the activation of gustatory afferents. However, details of gustatory signal transmission and processing as well as neural coding are only incompletely understood. This is partly due to the property of some tastants to elicit several sensations simultaneously, unspecific effects caused by the temperature, viscosity, osmolarity, and pH of the solvents, as well as by mechanical stimulation of the tongue during stimulus application. The analysis of gustatory processing of taste information are mainly based on mouse models after stimulation with bitter taste stimuli. Even though it is known that the mouse genome codes for 35 bitter taste receptor genes only few of them had been analysed so far. For better understanding and interpretation of animal experiments 16 mouse bitter receptors had been analysed by Calcium Imaging experiments with HEK293T cells. The data reveal that mouse bitter taste receptors are more narrow tuned than human bitter taste receptors, proving that the ligand spectra of murine and human orthologous receptors are not complient. In order to avoid the disturbing effects of solvents and stimulus application on the analysis of gustatory information transfer and processing, I employ an optogenetical approach to address this problem. For this purpose I generated three strains of gene-targeted mice in which the coding regions of the genes for the umami receptor subunit Tas1r1, the sweet receptor subunit Tas1r2 or the bitter taste receptor Tas2r114 have been replaced by the coding sequences of different opsins (photoreceptors of visual transduction) that are sensitive to light of various wavelengths. In these animals I should be able to activate sweet, bitter, or umami signalling by light avoiding any solvent effects. In initial experiments of this project I demonstrated that the various visual opsins indeed functionally couple to taste signal transduction pathway in oocyte expression system, generating basic knowledge and foundation for the generation of the gene-targeted animals. The knockout-knockin strategies have been successfully realized in the case of all three mouse models, revealed by RT-PCR, in situ hybridization and immunohistochemical analysis of taste papillae. All data confirm that the particular taste receptors have been replaced by the different opsins in taste cells. Further analysis concerning the functional consequences of opsin knockin and taste receptor knockout are part of prospective work.

G protein-coupled receptors (GPCRs) are one of the largest protein families in mammals. GPCRs are instrumental for hormonal and neurotransmitter signalling and are important in all major physiological systems of the body. Paper I describes the repertoire of GPCRs in Branchiostoma floridae, which is one of the species most closely related species to vertebrates. Mining and phylogenetic analysis of the amphioxus genome showed the presence of at least 664 distinct GPCRs distributed among all the main families of GPCRs; Glutamate (18), Rhodopsin (570), Adhesion (37), Frizzled (6) and Secretin (16). Paper II contains studies of the Adhesion, Methuselah and Secretin GPCR families in nine genomes. The Adhesion GPCRs are the most complex gene family among GPCRs with large genomic size, multiple introns and a fascinating flora of functional domains. Phylogenetic analysis showed Adhesion group V (that contains GPR133 and GPR144) to be the closest relative to the Secretin family among the groups in the Adhesion family, which was also supported by splice site setup and conserved motifs. Paper III examines the repertoire of human transmembrane proteins. These form key nodes in mediating the cell’s interaction with the surroundings, which is one of the main reasons why the majority of drug targets are membrane proteins. We identified 6,718 human membrane proteins and classified the majority of them into 234 families of which 151 belong to the three major functional groups; Receptors (63 groups, 1,352 members), Transporters (89 groups, 817 members) or Enzymes (7 groups, 533 members). In addition, 74 Miscellaneous groups were shown to include 697 members. Paper IV clarifies the hierarchy of the main families and evolutionary origin of majority of the metazoan GPCR families. Overall, it suggests common decent of at least 97% of the GPCRs sequences found in humans, including all the main families.

Questions have been raised about the genomic complexity of the human genome, since it was reported that it only consisted of 32,000 genes. Alternative splicing is considered the explanation of the enormous difference between the number of genes and the number of proteins. Aligning expressed sequence tags (ESTs) to the genomic sequence has become a popular approach for gene prediction, revealing alternative splice variants. The aim in this thesis is to identify and analyse splice variants of the adhesion family of G protein-coupled receptors using EST data. 75% of the genes in the data set of 33 sequences were found to have a total of 51 splice variants. About half of the variants were considered functional.

<p>Questions have been raised about the genomic complexity of the human genome, since it was reported that it only consisted of 32,000 genes. Alternative splicing is considered the explanation of the enormous difference between the number of genes and the number of proteins. Aligning expressed sequence tags (ESTs) to the genomic sequence has become a popular approach for gene prediction, revealing alternative splice variants. The aim in this thesis is to identify and analyse splice variants of the adhesion family of G protein-coupled receptors using EST data. 75% of the genes in the data set of 33 sequences were found to have a total of 51 splice variants. About half of the variants were considered functional.</p>

Current drugs used to treat Congestive Heart Failure target the renin-angiotensin and adrenergic systems. Studies showed increased mortality rates in patients treated with a combination of these medications. Angiotensin-AT1 and ?2-Adrenergic receptors were shown to form receptor heteromers. Blockade of one receptor in the complex can affect the signal transmitted by the other; suggesting that ligand-based therapy is not as selective as we might think. Modulating receptor trafficking after synthesis might prove to be a valid therapeutic strategy. Unfortunately, little is known about receptor assembly and transport from Endoplasmic Reticulum to Plasma Membrane. The objectives of this study are to identify the proteins that participate in the assembly of AT1R-?2AR heteromer and the regulators of the anterograde trafficking of G-Protein Coupled Receptors. This thesis introduces the role of important targets in those poorly understood processes. The identification of such targets could lead to developing better drugs with fewer adverse effects.