G protein-coupled receptor (GPCR) genes are large gene families in every animal, sometimes making up to 1-2% of the animal's genome. Of all insect GPCRs, the neurohormone (neuropeptide, protein hormone, biogenic amine) GPCRs are especially important, because they, together with their ligands, occupy a high hierarchic position in the physiology of insects and steer crucial processes such as development, reproduction, and behavior. In this paper, we give a review of our current knowledge on Drosophila melanogaster GPCRs and use this information to annotate the neurohormone GPCR genes present in the recently sequenced genome from the honey bee Apis mellifera. We found 35 neuropeptide receptor genes in the honey bee (44 in Drosophila) and two genes, coding for leucine-rich repeats-containing protein hormone GPCRs (4 in Drosophila). In addition, the honey bee has 19 biogenic amine receptor genes (21 in Drosophila). The larger numbers of neurohormone receptors in Drosophila are probably due to gene duplications that occurred during recent evolution of the fly. Our analyses also yielded the likely ligands for 40 of the 56 honey bee neurohormone GPCRs identified in this study. In addition, we made some interesting observations on neurohormone GPCR evolution and the evolution and co-evolution of their ligands. For neuropeptide and protein hormone GPCRs, there appears to be a general co-evolution between receptors and their ligands. This is in contrast to biogenic amine GPCRs, where evolutionarily unrelated GPCRs often bind to the same biogenic amine, suggesting frequent ligand exchanges ("ligand hops") during GPCR evolution. (c) 2006 Elsevier Ltd. All rights reserved.

The superfamily of G protein-coupled receptors (GPCRs) is one of the largest protein families of mammalian genomes and can be divided into five main families; Glutamate, Rhodopsin, Adhesion, Frizzled, and Secretin. GPCRs participate in most major physiological functions, contributing to the fact that they are important targets in drug discovery. In paper I we mined the human and mouse genomes for new Adhesion GPCR genes. We found two new human genes (GPR133 and GPR144) and 17 mouse Adhesion genes, bringing the number up to 33 human and 31 mouse genes. In paper II we describe 53 new splice variants for human Adhesion receptors supported by expressed sequence tags (EST) data. 29 of these variants seem to code for functional proteins, several of which lack one or more functional domains in the N-termini. Lack of certain domains is likely to affect ligand binding or interaction with other proteins. Paper III describes the Glutamate GPCR in human, mouse, Fugu, and zebrafish. We gathered a total of 22 human, 79 mouse, 30 Fugu, and 32 zebrafish sequences and grouped these into eight clans using phylogenetic methods. The report provides an overview of the expansion or deletions among the different branches of the Glutamate receptor family. Paper IV focuses on the trace amine (TA) clan of Rhodopsin GPCRs. We identified 18 new rodent genes, 57 zebrafish genes, and eight Fugu genes belonging to the clan. Chromosomal mapping together with phylogenetic relationships suggests that the family arose through several mechanisms involving tetraploidisation, block duplications, and local duplication events. Paper V provides a comprehensive dataset of the GPCR superfamily of human and mouse containing 495 mouse and 400 human non-olfactory GPCRs. Phylogenetic analyses showed that 329 of the receptors are found in one-to-one orthologous pairs, whereas other receptors may have originated from species-specific expansions.

La rodopsina (Rho) és el receptor visual responsable de la visió a baixa intensitat lumínica. Aquest receptor, que es troba en les cèl·lules bastó de la retina, està constituït per 7 hèlices transmembrana i és el prototip dels receptors acoblats a proteïna G (GPCR), degut a que és l'únic del qual s'ha resolt l'estructura per difracció de raig X. Els GPCR tenen interès farmacològic ja que es troben involucrats en un gran nombre de processos fisiològics i patofisiològics. L'estudi estructural i funcional de la Rho ens ha de permetre determinar motius estructurals comuns en els GPCR, així com, elucidar el possible mecanisme molecular comú d'activació. A més, l'estudi de mutacions de Rho associades a malalties com la retinosi pigmentària (RP) ens ha d'apropar a les bases moleculars de la patologia. L'objectiu principal d'aquesta tesi ha estat l'estudi del mecanisme molecular d'activació de la Rho i la seva interacció amb la transducina. Per això, s'ha analitzat el paper estructural i funcional de les hèlices I i II de la Rho, així com el paper del retinal en el procés de fotoactivació del receptor. Per altra banda, també s'ha abordat la tasca de determinar quins aminoàcids poden estar implicats en l'especificitat de reconeixement de proteïna G. S'han estudiat les mutacions d'adRP G51A, G51V i G89D, així com mutacions no naturals en aquestes posicions, per tal de determinar el paper de les hèlices I i II en l'estructura i la funció de la Rho. La caracterització dels mutants de la posició 51 mostra que l'augment de la cadena lateral de l'aminoàcid introduït provoca una disminució de l'estabilitat de la conformació inactiva i també de la conformació activa (MetaII), que es pot relacionar amb la capacitat del receptor d'activar la transducina. L'estudi dels mutants de la posició 89 suggereix que en aquesta posició el que tindria importància seria la càrrega de la cadena lateral de l'aminoàcid. A més, els mutants G51V i G89D presenten un procés de fotoactivació anòmal, amb la formació d'un fotointermediari alterat que es troba en equilibri amb la MetaII. El mutants G51V i G89D presentarien un desplaçament en l'equilibri entre les conformacions MetaIa, MetaIb i MetaII cap al fotointermediari MetaIb, i aquest fet conjuntament amb la inestabilitat de la MetaII podrien ser els defectes moleculars que causarien RP. Per altra banda, la proteïna Wt va ser regenerada amb 11-cis-7-metilretinal (7 metil-Rho), amb la finalitat de determinar el paper del retinal en la fotoactivació del receptor. La proteïna 7-metil-Rho forma cromòfor de forma similar a Rho però presenta un procés de fotoactivació anòmal, amb la formació d'un fotointermediari alterat que es troba en equilibri amb la MetaII. La introducció d'un grup metil en el C7 del retinal provocaria canvis estructurals que afectarien la Met-207 i que farien que el receptor quedés atrapat en el fotointermediari MetaIa. L'equilibri existent entre les conformacions MetaIa, MetaIb i MetaII estaria controlat per una xarxa d'interaccions complexa entre les hèlices I, II i VII de la Rho i també per les interaccions entre la opsina i el retinal. També es van construir i expressar els mutants rodopsina-M3 de les nanses C-II i C-III. Aquests mutants presenten formació de cromòfor i un comportament en front la il·luminació similar al de la rodopsina Wt. Els mutants simples de la nansa C-II presenten la mateixa capacitat d'activar la transducina que el receptor Wt a excepció del mutant V138S que presenta una activació de transducina reduïda a l'igual que els mutants de la nansa C-III i que els mutants conjugats d'aquestes nanses. Cap dels mutants d'estudi presenta, però, un augment en l'activació de proteïna Giαq respecte al Wt. Els conjunt de resultats obtinguts suggereixen que els aminoàcids Val-138, Val-227, Val250, Val254 i Ile-255 de la Rho participarien en el reconeixement i/o activació de la transducina, i indicarien que les interaccions hidrofòbiques serien claus per la interacció Rho-transducina. / Rhodopsin (Rho) is the visual photoreceptor responsible for dim light vision. This receptor, that is located in the rod cell of the retina, has seven transmembrane helices and is a prototypical member of the G protein coupled receptors (GPCR) superfamily, being the only member of this superfamily whose structure has been resolved by X-ray chrystallography. The study of GPCR is of outstanding pharmacological interest because they are involved in a wide variety of physiological and pathophysiological processes. Structural and functional studies of Rho should provide clues about common structural motifs in GPCR and allow us to elucidate the molecular bases of a proposed common activation mechanism. Besides, the study of Rho mutants associated with retinal diseases, such as retinitis pigmentosa (RP) provides information about the molecular mechanism of this pathology. The main goal of this work is unraveling the structural details underlying the molecular mechanism of Rho activation and its interaction with transducin. To this aim we have analyzed the structural and functional role of helices I and II of Rho, as well as the role of retinal in the photoactivation process of the receptor. We have also tried to determine the amino acids involved in the specificity of G protein recognition. We have studied the adRP mutants G51A, G51V and G89D, as well as non-natural mutations at these positions, in order to determine the role of helices I and II in Rho structure and conformational changes upon light activation. The detailed characterization of mutations at 51 position shows that this position is very sensitive to the size of the introduced side chain. The increase volume of side chain of the introduced amino acid can be correlated with dark and active conformation (MetaII) stability and eventually with the capacity to activate the G-protein transducin. The study of mutations at 89 position suggests that the charge effect is more important for mutations in this position. Additionally, G51V and G89D mutants showed an abnormal photobleaching, with formation of an altered photointermediate which is in equilibrium to MetaII. We suggest that G51V and G89D mutants alter the proposed equilibrium between MetaIa, MetaIb and MetaII to favour the MetaIb photointermediate, and this fact together with the observed MetaII instability could be the molecular defects that caused RP. Wt Rho was regenerated with 11-cis-7 methylretinal (7-methyl-Rho), to gain insights into the opsin-retinal coupling process and to determine the role of retinal in the photoactivation process. 7-methyl-Rho showed chromophore formation similar to Rho but an abnormal photobleaching behaviour with formation of an altered photointermediate which is in equilibrium to MetaII photointermediate. The methyl group at C7 of retinal would introduce a structural change at the vicinity of Met-207 that would result in receptor trapping at the MetaIa conformation during the photoactivation process. MetaIa, MetaIb and MetaII equilibrium would be controlled by a network of complex interactions between helices I, II and VII of rhodopsin and also by opsin and retinal interactions. In another approach, we constructed and expressed Rho-m3 mutants at intracellular loops C-II and C-III. These mutants showed chromophore formation and photobleaching behaviour similar to Wt Rho. Single point mutants at the C-II loop showed transducin activation like Wt Rho with the exception of V138S mutation that caused a reduction in transducin activation like mutants at C-III loop and conjugated mutants at C-II and C-III loops. None of these mutants showed Giαq activation with regard to Wt protein. The results suggest that Val-138, Val-227, Val-250, Val-254 and Ile-255 amino acids of Rho participate in transducin binding and/or activation, and that hydrophobic interactions involving these residues would be an important factor mediating Rho-transducin interaction.

The neuropeptides orexin-A and -B act as endogenous ligands for G-protein-coupled receptors (GPCRs) called OX1 and OX2 receptors. Previous observations have established that orexin receptors have an ability to couple to different G-proteins and signaling pathways and induce Ca2+ elevations via both receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs). This thesis further elucidates the intracellular signaling mechanisms of orexin receptors. Orexin receptors were shown to activate ERK (extracellular signal-regulated kinase) via Ras, protein kinase C, phosphatidylinositol-3 kinase and Src. Ca2+ influx was shown to be obligatory for the activation of ERK and adenylyl cyclase, wherewith a hypothesis was formed that submembrane Ca2+ elevation is of central importance for the regulation of orexin receptors' coupling to different signaling pathways. This was further investigated with respect to OX1R-mediated activation of phospholipase C (PLC) showing that ROC influx was of more central importance for the OX1R signaling, but also SOCs amplified PLC activity. A technique to block OX1R-induced IP3 increase and subsequent Ca2+ release was devised, leaving ROC influx as the only source of Ca2+ elevation upon OX1R activation. This block had no effect on OX1R-mediated activation of ERK, showing that ROC-dependent influx is the most central Ca2+ elevating process in OX1R signaling. OX1Rs' coupling to PLC was further investigated by measuring the metabolites generated, inositol phosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities, which results in, at low orexin-A concentrations, DAG production without concomitant production of IP3. At even lower orexin-A concentrations, OX1Rs generate DAG by activating phospholipase D. In conclusion, the results strengthen the hypothesis that ROCs have a central role in orexin receptor signaling and DAG may be the signal of preference.

The human melanocortin receptors (hMCRs) play a fundamental role in human behavior such as satiety, feeding, sexual and more. A set of SHU9119 peptide derivatives were studied for their structure-activity relationships. These peptides contained a sequential a-N-methylation amino acid scan.A second set of peptide derivatives intended to be used to create TNF-a; inhibition, via the melanocortin receptors. These peptides were shown to bind to all of the hMCR receptors, and only exhibit cAMP stimulation at hMC1R/hMC5R.The data from both of the sets of compounds illustrate that small changes in the stereochemistry of the SH9119 and TNF-a; derivatives cause drastic changes in the binding and the agonistic/antagonist properties of the compounds.This thesis determined the effect that hMC1R mutations have on the binding and cAMP response of well characterized ligands. This study ruled out 9 different residues for being the required for the cAMP response of the hMC1R.

Activity of the NMDA receptor is crucial for CA1 plasticity. Functional modification of the receptor is one way to modulate synaptic plasticity and affect hippocampus dependent behaviours. Two GPCRs, the dopamine receptor D1 and the PACAP38 receptor PAC1, have been shown to enhance NMDA activity via Gq and Gs signaling pathways respectively. Enhancement of NMDAR activity by the D1/Gs pathway depends on phosphorylation of the NR2B subunit by Fyn kinase. Conversely, enhancement by the PAC1/Gq pathway depends on phosphorylation of the NR2A subunit by Src kinase. SKF81297, a D1 agonist, was shown to enhance LTD whereas PACAP38, through the PAC1 pathway, was shown to lower the threshold for LTP. Both effects were blocked by specific antagonists and shown to be dependent on NR2 subunit phosphorylation. Ultimately, physiological metaplasticity at the CA1 synapse may be mediated by the relative activation of many GPCR signaling pathways via modification of the NR2 subunit.

Activity of the NMDA receptor is crucial for CA1 plasticity. Functional modification of the receptor is one way to modulate synaptic plasticity and affect hippocampus dependent behaviours. Two GPCRs, the dopamine receptor D1 and the PACAP38 receptor PAC1, have been shown to enhance NMDA activity via Gq and Gs signaling pathways respectively. Enhancement of NMDAR activity by the D1/Gs pathway depends on phosphorylation of the NR2B subunit by Fyn kinase. Conversely, enhancement by the PAC1/Gq pathway depends on phosphorylation of the NR2A subunit by Src kinase. SKF81297, a D1 agonist, was shown to enhance LTD whereas PACAP38, through the PAC1 pathway, was shown to lower the threshold for LTP. Both effects were blocked by specific antagonists and shown to be dependent on NR2 subunit phosphorylation. Ultimately, physiological metaplasticity at the CA1 synapse may be mediated by the relative activation of many GPCR signaling pathways via modification of the NR2 subunit.

Transmembrane proteins are gatekeepers of the cells; controlling the transport of substrates as well as communicating signals among cells and between the organelles and cytosol. Solute carriers (SLC) and G protein-coupled receptors (GPCR) are the largest family of membrane transporters and membrane receptors respectively. The overall aim of this thesis was to provide a basic understanding of some of the novel SLCs and GPCRs with emphasis on expression, transport property, evolution and probable function. The first part of the thesis directs towards the study of some novel solute carriers. In an initial study, we provided an overall picture of the sequence relationship and tissue expression of 14 diverse atypical SLCs confirming some of their evolutionary conservation and highly specific expression pattern. The focus then was on the SLC17 family (mainly vesicular proteins) and a novel member named Slc17a9. This study revealed that SLC17 family could be divided into four main phylogenetic clades which were all present before the divergence of the insect lineage with Slc17a9 having the most restricted evolutionary history. Detailed expression study of Slc17a9 in the mouse brain suggests that it is also expressed in some regions important for purinergic neurotransmission. Further, we deorphanised an aminoacid transporter Slc38a7 which was expressed in a majority of neurons in the CNS and showed that it preferably mediate transport of L–glutamine and L–histidine. The second part of the thesis focuses on the study of two GPCRs belonging to the Rhodopsin superfamily, Gpr162 and Gpr153. A phylogenetic analysis revealed that both Gpr153 and Gpr162 originated from a common ancestor before the radiation of the mammalian lineage. Expression study revealed that Gpr162 had a predominant expression in the CNS and relatively lower expression in the other tissue tested whereas Gpr153 had a more widespread and similar expression pattern in both CNS and peripheral tissues. The functional studies of the two GPCRs were done using the antisense oligodeoxynucleotide knockdown rat model. These studies provided evidence linking the orphan Gpr162 gene with the regulation of food intake– related behaviour whereas Gpr153 gene caused only a slight reduction in food intake.

This thesis describes the expression of three human seven transmembrane receptors: the M2 Muscarinic; H1 Histamine and 5HT2A Serotonin receptors, in the baculovirus/insect cell expression system. Purification trials werre conducted on the M2 Muscarinic and H1 Histamine receptors. Preliminary crystallisation attempts were made with the H1 receptor.

La mélatonine est une hormone synthétisée et sécrétée uniquement la nuit par la glande pinéale. Son rôle principal est son implication dans la synchronisation de la saison de reproduction. La mélatonine se lie aux récepteurs, MT1 et MT,, membres de la famille des récepteurs à sept domaines transmembranaires couplés aux protéines G (RCPG).Le clonage récent du récepteur ovin MT2a remis en cause toutes les données connues. La pharmacologie et les voies de signalisation du récepteur oMT2ont été étudiées et sont communes à celles des récepteurs des autres espèces. En revanche, oMT2possède une originalité de séquence avec la présence du motif DRY, fortement impliqué dans l’établissement de l’activité constitutive des RCPG. D’une part, nous avons montré que l’ensemble des récepteurs MT possèdent une activité constitutive. D’autre part, nous avons identifié deux agonistes inverses pour les récepteurs hMT2, initialement décrits comme antagonistes. Dans l’optique de discriminer les rôles respectifs des récepteurs MT in vivo, le développement d’une approche par ARN interférents a été validée dans un modèle cellulaire, la lignée CHO-KI exprimant les récepteurs ovins et de rat. / Melatonin is a hormone synthesized and secreted only during night by pineal gland. A main role of melatoninconcerns its implication in the synchronization of reproductive seasonality. Binding sites of melatonin are MT1and MT2 receptors which belong to the superfamily of seven-transmembrane-spanning G protein-coupledreceptors (GPCRs).Recent cloning of ovine MT2 receptor has challenged the knowledge about melatonin receptors. Wedemonstrated that its pharmacology and signalling pathways were similar to subtype 2 receptor of othersspecies (human and rat). Nevertheless, oMT2 receptor possesses a particularity of sequence, with the presenceof DRY motif which is known to be involved in the establishment of constitutive activity of GPCRs. In ourstudy, we demonstrated the existence of constitutive activity for ail the melatonin receptors. In addition, weidentified two inverse agonists for human MT2 receptors, previously described as antagonists. To describe therespective roles of each subtype of melatonin receptors in vivo, siRNA approach was developed in cell line,CI-10-K Iexpressing ovine and rat melatonin receptors