In this dissertation we studied the pharmacological and functional consequences of adenosine A2A receptor interaction with other proteins, as other neurotransmitter receptores localized in the human brain and an important enzyme regulating the extracellular concentration of adenosine, the ecto-ADA (adenosine desaminase). The first aim of this thesis was to study the molecular and functional interaction of A(2A)Rwith ADA. We found out that A(2A)Racted as a membrane anchoring protein of ADA, more exactly, that ADA bound to A2A receptor homomers and induced a strong modification of their quaternary structure in the way it behaved as a positive allosteric ligand. In addition at the functional level, ADA markedly enhanced A2A receptor signalling, increasing the A2A receptor agonist-induced ERK 1/2 phosphorylation. This powerful regulation of A2A function exerted by ADA might have important implications in the physiology and pharmacology of neuronal A2A receptors that are implicated in the striatal motor regulation. The second aim of this thesis was to search for a compound possibly useful in the treatment Huntington´s disease (among other neurological diseases), concretely a more selective antagonist of A2A receptor for presynaptic A1-A2A receptor heteromers versus postsynaptic A2A-D2 receptor heteromers. Applying in vitro and in vivo approaches, we discovered that the A2A receptor antagonists SCH-442416 showed a presynaptically preferential profile and, on the other hand, the KW-6002 behaved as a postsynaptically preferential A(2A)Rantagonist. Other analysed compounds ZM-241385, MSX-2, SCH-420814, and SCH-58261 showed no clear presynaptic or postsynaptic preference, i.e. presented a mixed profiles. The presynaptic preference of SCH-442416 was due to a strong negative cooperativity induced by the physical presence of dopamine D2 receptor in the A2A-D2 receptor heteromer that was detected by the compound SCH-442416. This cooperativity also indicates that A2A-A2A receptor homodimers are present in the A2A-D2 receptor heteromers. In summary, on the basis of their preferential pre- versus postsynaptic actions, SCH-442416 can be used as a lead compound in the development of antidyskinetic drugs in Huntington’s disease, meanwhile KW-6002 confirms to be possibly beneficial in Parkinson’s disease. The third aim of this thesis consisted in investigation of pharmacological and functional properties of A2A receptors in the A2A-CB1 receptor heteromers and determination whether selective A2A receptor antagonists show different selectivity for A2A receptors or A2A-CB1 receptor heteromers. We observed that adenosine A2A receptor changed its G-protein coupling from stimulatory Gs to inhibitory Gi protein when it formed heteromer with CB1 receptor and a synergistic cross-talk in G-protein activation was observed when both receptors were co-activated. At the same time, we saw that CB1 receptor mainly controled the ERK 1/2 signalling under the A2A-CB1 receptor heteromer. The A2A-CB1 receptor heteromers did not show allosteric effects at the ligand binding level. The two specific A2A receptor antagonist, KW-6002 and VER-7835 lost affinity for A2A receptors when expressed in A2A-CB1 receptor heteromers. This all means that A2A-CB1 receptor heteromers constitute a singular unit for adenosine and cannabinoids signalling, introducing diversity in A2A receptor signalling that can be therapeutically relevant in neurological diseases involving striatal neurons. In summary, the results presented in this Thesis show the importance of GPCR heteromers in the brain striatum and their physiological importance for the treatment of neurological diseases. / En esta Tesis hemos estudiado las consecuencias farmacológicas y funcionales de la interacción del receptor A2A de adenosina con otras proteínas, concretamente con otros receptores de neurotransmisores localizados en el cerebro humano y la enzima ecto-adenosina desaminasa. Hemos demostrado que el A2AR actúa como una proteína de anclaje de ADA que se une a los homodímeros de este receptor y a su vez induce una fuerte modificación en su estructura cuarternaria. Esta propiedad hace de ADA un ligando alostérico de los A2AR que modula positivamente la unión de agonistas y antagonistas al sitio ortostérico de este receptor. En segundo lugar, buscamos un antagonista de A2AR potencialmente útil para el tratamiento de la enfermedad de Huntigton, concretamente un antagonista preferencialmente más selectivo para el heterodímero "presináptico" A1R-A2AR versus el heterodímero "postsináptico" A2AR-D2R. Encontramos un compuesto, SCH-442416, que mostró este perfil presináptico, cual fue debido a una fuerte cooperatividad negativa inducida por presencia física del receptor de dopamina D2 en el heterómero A2AR-D2R. Al revés, otro compuesto, KW-6002, mostró un perfil preferencial postsináptico. Por ello, SCH-442416 puede ser utilizado como un compuesto de partida para el desarrollo de fármacos antidiscinéticos para la enfermedad de Huntington, y por su parte KW-6002 comprobó ser posiblemente beneficioso en la enfermedad de Parkinson. En tercer lugar demostramos que el receptor CB1 influye al A2AR cual cambia su acoplamiento de una proteína Gs estimuladora a una Gi inhibidora en el heterodímero A2AR-CB1R y también controla la señalización de ERK 1/2 y que el KW-6002 pierde afinidad por los receptores A2A en este heterómero. En resumen esta Tesis muestra la importancia de heterómeros de receptores acoplados a proteína G en el cerebro y su relevancia fisiológica a la hora de búsqueda de tratamiento para las enfermedades neurológicas.

<p>The chemical cues that provide an olfactory portrait of mammalian individuals are in part detected by chemosensory receptors in the vomeronasal organ (VNO). By and large, the pertinent receptor-cue combinations used for olfactory communication are unidentified. Here we identify members of the formyl peptide receptor (FPR) family of G protein coupled receptors as candidate chemosensory receptors in the VNO of mice. We demonstrate that N-formylated mitochondrially encoded peptides presented by the major histocompatibility complex (MHC) molecule H2-M3 stimulate a subset of the VNO sensory neurons (VSNs). We show that one VNO localized FPR, Fpr-rs1, is differentially activated by strain specific variants of N-formylated peptides. We show that N-formylated peptides can function as chemosignals in a strain selective pregnancy block. We propose that this link between self-recognition peptides of the immune system and chemosensory pathways provides a possible molecular means to communicate the nature of an individual's maternal lineage or strain.</p> / Dissertation

Signal transduction pathways mediated by G protein-coupled receptors (GPCRs) and their intracellular coupling partners, the heterotrimeric G proteins, are crucial for several physiological functions in eukaryotes, including humans. This thesis describes a broad genomic survey and extensive comparative phylogenetic analysis of GPCR and G protein families from a wide selection of eukaryotes. A robust mining of GPCR families in fungal genomes (Paper I) provides the first evidence that homologs of the mammalian families of GPCRs, including Rhodopsin, Adhesion, Glutamate and Frizzled are present in Fungi. These findings further support the hypothesis that all main GPCR families share a common origin. Moreover, we clarified the evolutionary hierarchy by showing for the first time that Rhodopsin family members are found outside metazoan lineages. We also characterized the GPCR superfamily in two important model organisms (Amphimedon queenslandica and Saccoglossus kowalevskii) that belong to different metazoan phyla and which differ greatly in morphological characteristics. Curation of the GPCR superfamily (Paper II) in Amphimedon queenslandica (an important model to understand evolution of animal multicellularity) reveals the presence of four of the five GRAFS families and several other GPCR gene families. However, we find that the sponge GPCR subset is divergent from GPCRs in other studied bilaterian and eumetazoan lineages. Mapping of the GPCR superfamily (Paper III) in a hemichordate Saccoglossus kowalevskii (an essential model to understand the evolution of the chordate body plan) revealed the presence of all major GPCR GRAFS families. We find that S. kowalevskii encodes local expansions of peptide and somatostatin- like GPCRs. Furthermore, we delineate the overall evolutionary hierarchy of vertebrate-like G protein families (Paper IV) and provide a comparative perspective with GPCR repertoires. The study also maps the individual gene gain/loss events of G proteins across holozoans with more expanded invertebrate taxon sampling than earlier reports. In addition, Paper V describes a broad survey of nematode chemosensory GPCR families and provides insights into the evolutionary events that shaped the GPCR mediated chemosensory system in protostomes. Overall, our findings further illustrate the evolutionary hierarchy and the diversity of the major components of the G protein-coupled receptor signaling system in eukaryotes.

Arginine vasopressin (AVP) is a hypothalamic nonapeptide which regulates the hypothalamic-pituitary-adrenal axis response to stress by stimulating the secretion of adrenocorticotropin (ACTH) from corticotroph cells of the anterior pituitary. This effect is mediated by binding of AVP to the pituitary vasopressin receptor (V1bR). The V1bR belongs to the G protein-coupled receptor (GPCR) super family. Repeated stimulation of anterior pituitary cells with AVP has been shown to produce a loss of responsiveness to subsequent AVP stimulation. This phenomenon appears to be mediated by desensitisation of the V1bR, and may be due to phosphorylation of the receptor by G protein-coupled receptor kinase 5 (GRK5). The aim of this research was to establish and validate methods that would allow the role of GRK5 in the desensitisation of V1bR to AVP stimulation to be investigated. As no isoform specific inhibitors for GRK5 were available, HEK293 cells transiently transfected with the rat V1bR were used as a model system for this research. This allowed RNA interference (RNAi) to be used to knockdown GRK5 expression. The protocol for RNAi-mediated knockdown of GRK5 was established as part of this research. Protocols for Western blotting and qRT-PCR were also established to allow the RNAi-mediated knockdown of GRK5 protein and mRNA to be measured. Transfection of HEK293 cells with 10nM GRK5-targeting small interfering RNAs (siRNAs) reduced the expression of GRK5 protein to 53.4% ± 3.4% (mean ± SEM) of that seen in untreated control cells at 84 hours after transfection, while GRK5 mRNA levels were reduced to 28.7% ± 1.9% (mean ± SEM) of that of control cells 48 hours after transfection. An experimental protocol was designed in this research that would coordinate the RNAi-mediated knockdown of GRK5 with transient transfection of the HEK293 cells with the rV1bR. Since, activated V1bRs couple to Gq/11 and stimulate the production of inositol phosphates (IPs), the responsiveness of the V1bR can be determined by measuring the accumulation of [H³]-IPs in cells labelled with [H³]-myo-inositol. In the protocol designed, the effect of GRK5 knockdown on V1bR desensitisation is determined by stimulating HEK293 cells expressing the rV1bR (and previously transfected with GRK5-targeting siRNA) with 0nM or 100nM AVP for 0, 5, 15, 30 or 60 minutes, and comparing the accumulation if IPs over time with that of cells that are not transfected with GRK5-targeting siRNA. This protocol can be used in future to investigate the role of GRK5 in V1bR desensitisation, and may be adapted to determine if other GRK isoforms are involved in V1bR desensitisation.

In humans, taste is one of the five senses, and helps in the recognition of nutritionally important and potentially harmful substances. It triggers innate behaviour to accept or reject food. Humans can sense five basic tastes, which are sweet, umami, bitter, salt and sour. The receptors that mediate bitter, sweet and umami tastes belong to the G protein-coupled receptor (GPCR) superfamily. A group of three receptors sense sweet and umami tastes, whereas bitter taste is sensed by 25 bitter taste receptors (referred as T2Rs). T2Rs are activated by structurally diverse natural and synthetic bitter compounds. Many common pharmaceutical compounds are bitter in taste and these are effective ligands for T2Rs. Recent finding of T2Rs in extra-oral tissues suggests these receptors are also involved in various physiological and pathophysiological processes. To understand the structure and function of these receptors, studies directed at elucidating their mechanisms of activation, and identification of novel ligands including bitter blockers (antagonists and inverse agonists), are required. To obtain mechanistic insights into the role of the highly conserved, and receptor specific residues, two bitter taste receptors (T2R1 and T2R4) were targeted. In this study, a combination of molecular, biochemical and pharmacological approaches were used to identify the amino acids and motifs, important for T2Rs to switch from inactive to active state. A hydrogen-bonding network between transmembrane (TM) helices 1-2-7 was identified as important for T2R activation. Alanine-scan mutagenesis of intracellular loops (ICLs) 2 and 3 identified T2R regions important for G protein binding, and receptor activation. A pharmacological method was developed, to screen potential bitter blockers for T2Rs. Using this method, three novel bitter blockers, which include two natural antagonists and one synthetic inverse agonist for T2R4, were discovered. The role of expression tags in enhancing T2R4 expression was also pursued. T2R4 expression on the cell surface was increased 2.5 fold, when its N-terminus was tagged with rhodopsin N-terminal 33 residues (Rho33- T2R4 chimera). In conclusion, work carried out provides novel insights into the mechanisms of T2R activation, and in the discovery of bitter blockers for T2R4.

Made available in DSpace on 2014-06-12T15:55:34Z (GMT). No. of bitstreams: 2 arquivo6307_1.pdf: 824660 bytes, checksum: 1953df07244da37349b76a1e19dc4b06 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A 6 [(E) estiril] piran 2 - ona (pirona-198) é uma estiril-pirona natural isolada a partir do extrato clorofórmico dos frutos de Aniba panurensis, espécie constituinte da família Lauraceae. Com a ausência de estudos sobre os efeitos da 6 [(E) estiril] piran 2 - ona (pirona-198) no sistema vascular, objetivou-se então investigar os efeitos desta pirona sobre anéis de artéria mesentérica superior isolada de rato Wistar. Em anéis pré-contraídos com fenilefrina (FEN), pirona-198 foi capaz de induzir vasorelaxamento dependente de concentração (CE50 = 1,1 ± 0,69 x 10-5 M) e esse efeito não foi alterado após a remoção do endotélio funcional (CE50 = 1,57 ± 0,35 x 10-5M). Este efeito vasorelaxante induzido pela pirona-198 não foi significativamente alterado em soluções contendo KCl 20 mM. Em anéis précontraídos com KCl 80 mM, pirona-198 induziu um efeito relaxante significante (p < 0,001; CE50 = 1,2 ± 0,15 x 10-4 M (endotélio intacto); CE50 = 2,0 ± 0,38 x 10-4 M (endotélio removido)), que foi menos potente do que em anéis pré-contraídos com FEN. Os efeitos da pirona sobre o influxo de cálcio foi avaliado, em que concentrações de pirona-198 (10-7 10-4 M) não foram capazes de inibir a curva concentração resposta para o CaCl2, entretanto, na maior concentração (10-3 M), ela foi capaz de diminuir significativamente a resposta máxima (p<0,001). Em uma solução despolarizante livre de cálcio, pirona-198 (10-8 - 10-3 M) antagonizou as contrações transientes induzidas por FEN (10 &#956;M; p<0,001). Pirona-198 (10-4 -10-3 M) antagonizou as contrações transientes induzidas por 20 mM de cafeína (p<0,001). As contrações induzidas por ortovanadato de sódio (10-5 - 3 x 10-3 M) foram significativamente inibidas por altas concentrações de pirona-198 (10-4 e 10-3 M). Esses resultados sugerem que a pirona-198 exerce um efeito relaxante independente do endotélio funcional que parece ser decorrente da possível interação com a via de sinalização estimulada a partir da ativação de receptores acoplados a proteína G (GPCRs), que envolve a mobilização de cálcio pelos receptores sensíveis a IP3 e interação com a via da RhoA Rho cinase. Outro mecanismo de ação sugerido para a atividade vasorelaxante da pirona-198 que se adiciona aos anteriores, é o bloqueio do influxo transmembranar de cálcio através de canais de cálcio dependentes de voltagem, adicionado a inibição da mobilização de cálcio intracelular mediada pelos receptores de rianodina

No description available.

Les récepteurs aux hormones glycoprotéiques (rGpHs) rTSH, rFSH et rLH/CG appartiennent<p>à la classe A des GPCRs. Les récepteurs da la classe A des GPCRs sont caractérisés par la<p>similitude de séquence de leur domaine transmembranaire avec la rhodopsine. Outre ce<p>domaine dit « serpentin », qu’ils partagent avec tous les GPCRs, les rGpHs offrent la<p>particularité de présenter un grand domaine extracellulaire (ECD) responsable de la liaison et<p>de l’affinité de leurs ligands respectifs.<p>Les récepteurs couplés aux protéines G (GPCRs) sont les cibles de 50% des médicaments<p>actuellement sur le marché pharmaceutique. La compréhension de leur mode de<p>fonctionnement est donc essentielle au développement de nouvelles molécules capables de<p>cibler spécifiquement ces récepteurs. Ces dernières années, il est apparu clairement que les<p>GPCRs étaient présent à la surface cellulaire sous forme d’oligomères. Le but de ce travail<p>était d’explorer de manière approfondie, le mécanisme d’activation et de dimérisation du<p>rTSH en identifiant les résidus du domaine transmembranaire des récepteurs aux hormones<p>glycoprotéiques impliqués dans le processus d’activation et de dimérisation.<p>Au cours de ce travail de thèse, nous avons dans premier temps généré une banque de mutants<p>aléatoires du rTSH. Ces mutants ont ensuite été criblés par une approche HTRF pour mettre<p>en évidence des mutants dont l’activité constitutive est augmentée ou ayant perdu la capacité<p>de dimériser.<p>Nous avons ainsi déterminé de nouveaux résidus importants pour le mécanisme d’activation<p>du rTSH. Les résultats que nous avons obtenus permettent d’apporter des éléments de réponse<p>et une base de travail sur le mécanisme d’activation. Cependant une description détaillée de<p>l’activation reste toutefois indéterminée. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Appartenant à la sous-famille des récepteurs couplés aux protéines G (RCPGs) apparentés àla rhodopsine et identifiés comme des cibles à fort potentiel thérapeutique, les récepteurs MT, et MT2à la mélatonine, 5-HT2C à la sérotonine et CB2 aux cannabinoïdes ont été étudiés par des approches insilico afin de mettre en évidence les déterminants structuraux critiques pour l'affinité, la sélectivité etl'activité pharmacologique de leurs ligands. Bénéficiant de données cristallographiques récentes,plusieurs états conformationnels de ces quatre récepteurs ont été modélisés en fonction du profilpharmacologique recherché. L'étude comparative de ces différents états conformationnels par dessimulations de dynamique moléculaire a permis de caractériser le rôle prépondérant joué par la boucleextracellulaire E2 et l'hélice 6 dans les mécanismes d'activation de ces RCPGs. Sur la base deméthodes chémoinformatiques, le criblage virtuel de ligands ciblant ces modèles tridimensionnels apermis de caractériser un modèle du récepteur 5-HT2C très spécifique de ligands agonistes inverses etd'identifier des touches pharmacologiques sur les récepteurs MTi et CB2. / Identified as highly relevant therapeutical targets, the MT, and MT2 melatonin receptors, the5-HT2C serotonin and the CB2 cannabinoid receptors, which belong to the rhodopsin-like G proteincoupledreceptors (GPCRs) subfamily, have been studied by in silico approaches in order to identifycritical structural features for the binding, the selectivity and the pharmacological activity of theirligands. Gaining by sottie recent crystallographic data, various conformational states of these fourreceptors have been modeled according to the expected pharmacological profile. The comparativestudy of these various conformational states by molecular dynamics simulations has led to emphasizethe crucial rôle of the E2 extracellular loop and hélix 6 in the activation mechanisms of these GPCRs.On the basis of chemoinformatic methods, the virtual ligand screening targeting these threedimensionalmodels has promoted the characterization of a 5-HT2C receptor model able to bindspecifically inverse agonist ligands and the identification of pharmacological hits targeting the MTiand CB2 receptors.

No description available.