Chemokines are chemotactic cytokines which play an important role in the migration of immune cells to distant tissues or compartments within tissues. These proteins have also been demonstrated to play a major role in cancer metastasis. The C-C chemokine receptor type 7 (CCR7) is a member of the chemokine receptor family. CCR7 along with its ligands CCL19 and CCL21 plays an important role in innate immune response by trafficking of lymphocytes. In cancer, tumour cells expressing CCR7 migrate to lymphoid organs and thus disseminate to other organs. Neutralizing the interactions between CCL21/CCR7 would therefore be expected to inhibit the progression and metastasis of many different types of cancer to regional lymph nodes or distant organs. Our objective was to identify a potent small molecule antagonist of CCR7 as a prelude to the investigation of the role of this axis in cancer metastasis. In this study, we provided a brief description of chemokines and their role in health and disease with an emphasis on the CCR7/CCL19/CCL21 axis, as well as identification of a CCR7 antagonist “hit”. The potency of the CCR7 antagonist “hit” was optimised by synthesizing different CCR7 antagonist analogues. The “hit” optimization process has led to discover the most active compound amongst a series of different analogues which have the ability to bind and block CCR7 receptor. The efficacy of the most active compound and other analogues were evaluated in vitro using a calcium flux assay which is based on detecting fluorescent light emitted upon release of calcium ions. To identify a suitable cell line, which expresses CCR7 and capably respond to it, amongst a panel of cell lines for in vitro assessment of potency of synthesised compounds, we used Western blot assay and later by flow cytometry assay. The activity and selectivity of the most effective compound against CCR7 receptor was evaluated in vitro by other functional assays such as “configured agarose spot assay” and scratch assay. We first configured the existing under agarose assay to fulfil our requirements and then used it to assess activity and selectivity of compounds. The configured agarose spot assay also describes the application of the agarose spot for evaluation of cells chemotactic response to multiple chemokines under identical experiment conditions.

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Objectifs : Les Récepteurs Couplés aux Protéines G (RCPG) constituent une grande famille ubiquitaire. Leur structure est caractérisée par sept hélices transmembranaires. Les déformations de ces hélices jouent un rôle majeur dans l’activation de ces récepteurs. La plupart de ces déformations sont liées à la présence de prolines conservées. Cependant, les prolines de l’hélice 2 et 5 des RCPG ne sont pas systématiquement présentes. De plus, la position de la proline dans l’hélice 2 est variable (2.58, 2.59 ou 2.60). Nous nous intéressons aux rôles des prolines des hélices 2 et 5 dans l’activation de deux RCPG : le récepteur de la thyrotropine (TSHR) et le récepteur 2 de la vasopressine (V2R). Méthodes : pour le TSHR et le V2R, nous concevons et caractérisons des mutants pour chaque position conservée de la proline dans l’hélice 2 et/ou 5, ainsi que des mutants sans proline. Résultats : Les mutants du TSHR n’ont pas le même comportement en termes d’expression, de glycosylation ou d’activité. La position la mieux tolérée, 2.59, nous permet de rapprocher le TSHR des récepteurs avec une proline en position 2.59 qui possèdent un renflement dans l’hélice 2. Pour l’hélice 5, les données expérimentales couplées à l’analyse des séquences et la modélisation moléculaire suggèrent une structure non renflée. Pour le V2R, le changement de position de la proline de l’hélice 2 est plus délétère que l’absence de proline dans cette hélice. La proline de l’hélice 5 est indispensable pour l’activité de ce récepteur. Conclusion : Les données obtenues sur le TSHR permettent de proposer un modèle avec une hélice 2 renflée et une hélice 5 non renflée et d’améliorer la modélisation de la cavité interne de ce récepteur, ce qui est essentiel pour le drug design. L’étude du V2R permet de proposer un modèle évolutif de ce récepteur et met en évidence sa spécificité par rapport à des récepteurs proches. / Objectives : Class A G-Protein-coupled receptors (GPCRs) constitute a large family of transmembrane receptors. Helical distortions play a major role in the overall fold and in the activation mechanism of these receptors. Most distortions are related to the presence of conserved proline residues. However, in helices TM2 and TM5, the presence of proline is not mandatory and the correlated mutation of these proline residues is observed in several GPCR sub-families. In addition, the position of the TM2 proline is variable (2.58 to 2.60). We are interested in the role of the TM2 and TM5 proline residues in the folding and activation mechanism of two GPCRs : the thyrotropin receptor (TSHR) and the vasopressin receptor type 2 (V2R). Methods : For both receptors, we engineered and characterized mutants with proline residues at different positions in TM2 and/or at position 5.50 in TM5, and without proline. Results : The expression, the glycolysation or the activity of TSHR mutants are differentially altered by changes in the proline pattern. The “best” mutant, TSHR P2.59, is consistent with a bulged structure for TM2. Experimental data in addition to sequences analysis and modeling suggest an unbulged structure for TM5. For V2R, the absence of proline in TM2 is better tolerated than ashift in the position. The TM5 proline is mandatory for the receptor activation. Conclusion : We suggest a model for TSHR with a bulged TM2 and an unbulged TM5. This should improve the modeling of the transmembrane cavity, which is fundamental for drug design. Our results on V2R suggest an evolutionary model for this receptor and enlighten its specificity compared to nearby receptors.

Schizophrenia is a severe mental disorder affecting ~1% of world population. Two G protein coupled receptors (GPCRs): Gi-coupled dopamine D2 receptor (D2R), and Gq-coupled serotonin 2A receptor (2AR), are targeted by the typical and atypical antipsychotic drugs to treat schizophrenia. These two receptors have been shown to co-localize in brain regions relevant to schizophrenia, including the ventral tegmental area (VTA), striatum, and prefrontal cortex (PFC). Studies in our lab characterized the integrated signaling of the D2R-2AR heteromer and found that both the Gi activity of D2R and the Gq activity of 2AR were potentiated in response to dopamine (DA) and serotonin (5-HT), whereas the potency of the typical antipsychotic drug (APD) haloperidol antagonizing Gi and Gq signaling was also enhanced. Using a peptide mimicking the transmembrane (TM) domain 5 of D2R, we showed disruption of the formation and function of the D2R-2AR heteromer in heterologous systems and ex vivo brain slices. Our functional and mutagenesis data suggested that D2R and 2AR heteromerize though a symmetric TM5,6-TM5,6 interface, and a network of Pi-Pi stacking interaction among eight conserved aromatic residues of D2R and 2AR may underlie the mechanism for the functional cross-talk between D2R and 2AR. Based on these results, we built a structural model for the D2R-2AR heteromer recapitulating its functional cross-talk characteristics. We are presently pursuing behavioral experiments to investigate the effectiveness of antipsychotic drugs on the function of the D2R-2AR heteromer in animal models of psychosis. Our overall study shows a dual role of the D2R-2AR heteromer in schizophrenia-associated psychosis and sheds light on the development of future therapeutic drugs for schizophrenia and other psychotic diseases.

Les canaux potassiques sensibles à l'ATP (KATP) jouent un rôle primordial dans la sécrétion pancréatique d'insuline et participent au contrôle du tonus vasculaire ainsi que de l'excitabilité des cellules musculaires cardiaques et neuronales. Constitués de l'assemblage unique d'un récepteur membranaire de la famille des transporteurs ABC, le récepteur des sulphonylurées SUR, et d'un canal potassique rectifiant entrant, Kir6.2, ces canaux couplent le métabolisme cellulaire au potentiel membranaire et constituent en ce sens un modèle naturel de biocapteur.<br />Le caractère unique de cet assemblage tient au fait que SUR est capable de réguler l'activité de Kir6.2 suite à la fixation de ligands: nucléotides, activateurs ou inhibiteurs pharmacologiques. Nous nous sommes intéressés aux déterminants moléculaires intervenant dans le couplage fonctionnel de SUR au canal Kir6.2. Utilisant une stratégie chimérique, nous avons identifié une région C-terminale de l'isoforme SUR2A essentielle aux mécanismes d'activation du canal, assurant le lien entre la fixation de ligands à SUR et l'ouverture de Kir6.2.<br />Nous avons également utilisé notre connaissance du modèle du canal KATP pour développer un nouveau type de biocapteur électrique, les Ion Channel Coupled Receptors (ICCR), fondé sur le couplage fonctionnel artificiel entre Kir6.2 et des récepteurs couplés aux protéines G (GPCR). Par ingénierie protéique, nous avons créé deux modèles d'ICCR impliquant respectivement les récepteurs muscarinique M2 et dopaminergique D2: la fixation d'agonistes ou antagonistes spécifiques sur ces récepteurs entraîne une activation ou une inhibition du canal mesurables électriquement en temps réel, jetant les bases prometteuses d'une nouvelle génération de biocapteurs acellulaires.

<p>The melanocortin (MC) receptors are G-protein coupled receptors thatparticipate in several important physiological functions such as the regulation of the energy balance. This thesis focuses on the evolutionary aspect of the MC receptors and their pharmacology.</p><p>One MC4 receptor and two MC5 receptor subtypes were found in a teleost fish, zebrafish. This indicates that the MC receptor subtypes arose very early in vertebrate evolution. Important pharmacological and functional properties, as well as gene structure and syntenic relationships have been highly conserved over a period of more than 400 million years implying that these receptors participate in vital physiological functions. Moreover, we found a MC4 receptor from a shark, spiny dogfish that represents the most distant MC receptor gene cloned to date. We also characterized the pharmacology of a MC4 receptor in goldfish. The conserved central expression pattern and physiological role in regulation of food intake of the MC4 receptor suggests that neuronal pathways of the melanocortin system may be important for regulation of energy homeostasis in most vertebrates. We determined the chromosomal position of the chicken MC receptors genes and found conserved synteny of the MC2, MC5, and MC4 receptor genes. These results suggest that there exist a clustering of these genes that is ancient. Analysis of conserved synteny with mammalian genomes and paralogon segments prompted us to predict an ancestral gene organization that may explain how this family has been formed through both local duplication and tetraploidization processes.</p><p>There are several common point mutations in the human MC1 receptor that are over represented in North European red-heads, and in individuals with pale skin. We pharmacologically characterised four naturally occurring human MC1 receptor variants providing molecular explanation to the respective phenotype.</p><p>The MC receptor subtypes have highly diverse physiological functions despite having relative high similarities in their primary structure. Our studies on the structural and functional properties of the MC receptor subtypes have provided insight into the molecular mechanism of how the specification of these receptors may have occurred.</p>

The members of the mammalian neuropeptide Y family, i.e. the peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), are all involved in regulation of food intake. In human and most other mammals they act via receptors Y1, Y2, Y4 and Y5. NPY is released in the hypothalamus and is one of the strongest appetite-stimulating neurotransmitters whereas PP and PYY are secreted from gut endocrine cells after meals and function as appetite-reducing hormones. This thesis describes studies of the NPY system at both the molecular and the physiological level. The first part describes two investigations of receptor-ligand interactions with the human Y1 and Y2 receptors. The results clarify the importance of several amino-acid residues of the human Y1 receptor. Three amino acids previously suggested by others to form a binding pocket for the carboxy-terminus of the peptide were confirmed to be crucial for interaction with peptide ligands. However, they were found to be too distantly located from each other to be able to form a binding pocket. Further investigation of the three corresponding positions in the human Y2 receptor showed that only one of the positions was important for interaction with full-length peptides. The results indicate overlapping but, surprisingly, non-identical binding of the different peptides to human Y1 and Y2 receptors, despite the fact that the two receptors share a common ancestor. The second part of the thesis describes an investigation of the effect of PP on food intake in six beagle dogs and a test for personality characteristics in dogs (TFPC). Treatment with physiological doses of PP decreased both the appetitive and the consummatory drive but had no effect on the amount food consumed. The TFPC protocol was used to map individual behavioral differences in a population of sixteen beagle dogs. The test, which included several situations that may appear in an experimental study, revealed considerable inter-individual differences in behavioral responses despite the fact that the dogs were born and housed in the same animal facility in constant controlled conditions. These results demonstrate that PP can influence food intake in distantly related mammals and emphasize the importance of considering differences in personality in experimental animals.

The melanocortin (MC) receptors are G-protein coupled receptors thatparticipate in several important physiological functions such as the regulation of the energy balance. This thesis focuses on the evolutionary aspect of the MC receptors and their pharmacology. One MC4 receptor and two MC5 receptor subtypes were found in a teleost fish, zebrafish. This indicates that the MC receptor subtypes arose very early in vertebrate evolution. Important pharmacological and functional properties, as well as gene structure and syntenic relationships have been highly conserved over a period of more than 400 million years implying that these receptors participate in vital physiological functions. Moreover, we found a MC4 receptor from a shark, spiny dogfish that represents the most distant MC receptor gene cloned to date. We also characterized the pharmacology of a MC4 receptor in goldfish. The conserved central expression pattern and physiological role in regulation of food intake of the MC4 receptor suggests that neuronal pathways of the melanocortin system may be important for regulation of energy homeostasis in most vertebrates. We determined the chromosomal position of the chicken MC receptors genes and found conserved synteny of the MC2, MC5, and MC4 receptor genes. These results suggest that there exist a clustering of these genes that is ancient. Analysis of conserved synteny with mammalian genomes and paralogon segments prompted us to predict an ancestral gene organization that may explain how this family has been formed through both local duplication and tetraploidization processes. There are several common point mutations in the human MC1 receptor that are over represented in North European red-heads, and in individuals with pale skin. We pharmacologically characterised four naturally occurring human MC1 receptor variants providing molecular explanation to the respective phenotype. The MC receptor subtypes have highly diverse physiological functions despite having relative high similarities in their primary structure. Our studies on the structural and functional properties of the MC receptor subtypes have provided insight into the molecular mechanism of how the specification of these receptors may have occurred.

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.

The D1-class receptors (D1R, D5R) each possess distinct signaling characteristics; however, pharmacological selectivity between them remains elusive. The third intracellular loops (IL3) of D1R and D5R harbour divergent residues that may contribute to their individual signalling phenotypes. Here we probe the function of central region of IL3 of D1R and D5R using deletion mutagenesis. Radioligand binding and whole cell cAMP assays suggest that the N-terminal and C-terminal moieties of the central IL3 oppositely contribute to the constitutive and agonist-dependant activity of D1-Class receptors. Whereas the N-terminal deletions ablated constitutive activity and decreased DA-induced activation, C-terminal deletions induced robust increases. These data, interpreted in concert with structural predictions generated from homology modeling implicate the central IL3 as playing an important role in the activation and subtype-specific characteristics of the D1-class receptors. This study may serve as a basis for the development of novel drugs targeting the central IL3 region.