Rhodopsin ist als Sehpigment der Photorezeptorzellen einer der am aktivsten untersuchten GPCRs. Es besteht aus dem Apoprotein Opsin und dem inversen Agonisten 11-cis-Retinal. Der inaktivierende Ligand ist in der sieben Transmembran- Helix (TM)-Struktur des Rezeptors kovalent gebunden und muss durch Licht cis/trans-isomerisiert werden, um den Rezeptor zu aktivieren. Der aktivierte Rezep-tor katalysiert den Nukleotidaustausch im G-Protein und zerfällt innerhalb von Minuten in Opsin und all-trans-Retinal. Das visuelle Pigment wird dann durch erneute Beladung des Opsins mit 11-cis-Retinal wieder hergestellt. In der vorliegenden Arbeit wird die erfolgreiche Kristallisation des nativen Opsins aus der Stäbchenzelle der Rinderretina und die Bestimmung der Proteinstruktur bei 2.9 Å Auf-lösung dargestellt. Im Vergleich zur bekannten Struktur des inaktiven Rhodopsins zeigt Opsin deutliche Strukturänderungen in den konservierten E(D)RY und NPxxY(x)5,6F Regionen und in TM5-TM7. Auf der intrazellulären Seite ist TM6 ca. 6-7 Å nach außen gekippt, während die TM5 Helix verlängert und näher zu TM6 verschoben ist. Durch die strukturellen Änderungen, von denen einige einem aktiven GPCR Zustand zugeschrieben werden können, wird die leere Retinalbindungstasche reorganisiert, um zwei Öffnungen für Aus- und Eintritt von Retinal bereitzustellen. Die Struktur von Opsin liefert neue Erkenntnisse zur Bindung von hydrophoben Liganden an GPCRs, zur GPCR-Aktivierung und zur Signalübertragung auf das G-Protein. / Rhodopsin as the visual pigment in photoreceptor cells is one of the most actively studied GPCRs. It consists of the apoprotein opsin and the inverse agonist, 11-cis-retinal. The inactivating ligand is bound in the seven-transmembrane helix (TM) bundle and cis/trans-isomerized by light to activate the receptor. The active receptor state is capable of catalyzing nucleotide exchange in the G protein and decays within minutes into opsin and all-trans-retinal. The visual pigment is then restored by reloading opsin with new 11-cis-retinal. In the present work, the successful crystallization of native opsin from bovine retinal rod cells and determination of the protein structure to 2.9 Å resolution is presented. Compared with the known structure of inactive rhodopsin, opsin displays prominent structural changes in the conserved E(D)RY and NPxxY(x)5,6F regions and TM5-TM7. At the cytoplasmic side, TM6 is tilted outwards by 6-7 Å, whereas the helix structure of TM5 is more elongated and close to TM6. These structural changes, of which some are attributed to an active GPCR state, reorganize the empty retinal binding pocket to disclose two openings for exit and entry of retinal. The opsin structure thus sheds new light on binding of hydrophobic ligands to GPCRs, GPCR activation and signal transfer to the G protein.

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.

Growth factor receptors have significant effects on various normal function of body such as cell proliferation, differentiation and apoptosis. They are also involved in neuronal function and dysfunction, cardiovascular diseases, and malignancies. Recently, multiple G protein-coupled receptors (GPCRs) have been shown to transactivate receptor tyrosine kinases (RTKs). Since both classes of receptors have complicated downstream cascades individually, understanding the signaling differences between GPCR-induced growth factor receptor transactivation and direct ligand activation is an important challenge. To clarifying this phenomenon we investigated the phosphorylation profile and downstream effectors of ligand-activated vs. transactivated PDGF?? receptors. Dopamine receptors (one of the receptors of the GPCRs family) were used to compare the PDGF?? receptor phosphorylation and activity during direct activation and transactivation. Dose-response and time-course data between these two stimuli were evaluated. Furthermore, the phosphorylation site profiles and the intracellular signaling pathways of PDGF?? receptor after direct activation and transactivation were examined. In addition, possible synergic effects between transactivation and direct activation were explored. The results of this project showed that the phosphorylation profile and downstream effectors of ligand activated receptors versus transactivated receptors are different. Our data indicated that transactivation-induced pathways are more involved in survival and proliferation effects compared to ligand activation. This research answered basic questions about transactivation phenomena and proposes that these transactivation pathways could be exploited as a therapeutic approach for neurodegenerative diseases.

Una de las principales cuestiones en farmacología molecular de los GPCR es entender los mecanismos estructurales de las siete hélices transmembrana (TM) que se producen para estabilizar ya sea Rg o los diferentes estados R*. Para entender el mecanismo que cambia el equilibrio del conjunto a un estado activo R* se construyeron tres de los receptores de la serotonina (5-HT4, 5-HT6, y 5 HT7) sobre la base de su información más reciente de cristalografía de rayos X. Dando lugar a dos modelos de cada receptor: una inactiva y otra activa. Los modelos, mejorados y evaluados con la ayuda de datos farmacológicos y químicos se utilizaron principalmente para comprender la interacción entre un ligando y su receptor y su mecanismo de acción. Estos hallazgos estructurales pueden a su vez resultar útiles para el diseño de nuevos fármacos más eficaces y selectivos. / One of the main questions in G protein coupled receptors (GPCRs) molecular pharmacology is to understand the structural arrangements of the seven transmembrane (TM) helices that occur to stabilize either the ground state (Rg) or different active states (R*) of the receptors. In order to understand the mechanism that shift the equilibrium of the ensemble to an active R* state models of the inactive and the active state of three serotonin receptors (5-HT4, 5-HT6, and 5-HT7) were built based on the latest information from X-ray crystallography. The resulting models were mainly used to understand the interaction between a ligand and its receptor and the mechanism of action. With the help of pharmacological and chemical data these models and complexes were improved and evaluated. These findings may prove valuable for structural based drug discovery efforts and facilitate the design of more effective and selective pharmaceuticals.

In meiner Dissertation klärte ich wesentliche Fragen zu 7-Transmembrandomänen-unabhängigen Funktionen von Adhäsions-G-protein-gekoppelten Rezeptoren auf. Diese einzigartigen Zelloberflächenmoleküle sind essentiell für die Physiologie des Menschen, jedoch auf molekularer Ebene erst in Grundzügen verstanden. Unter anderem haben sie als G-protein-gekoppelte Rezeptoren die Fähigkeit, Signale aus der zellulären Umgebung mittels ihrer 7-Transmembrandomäne in die Zelle weiterzuleiten. Jedoch sind sie auch im Stande unabhängig von ihrer 7-Transmembrandomäne agieren. In meiner Arbeit zeigte ich wie solche Funktionen in vivo implementiert werden können und präsentierte weiterhin, dass die Rezeptoren in diesem Kontext entgegen ihrer 'Natur' im Stande sind, Signale auf benachbarten Zellen auszulösen. Meine Arbeit bildet somit die Grundlage zur weiteren Erforschung der komplexen Signalmechanismen von Adhäsions-GPCRs.

Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes.

<p>Molecular interactions lie at the heart of myriad biological processes. Knowledge of molecular recognition processes and the ability to model and predict interactions of any biological molecule to any chemical compound are the key for better understanding of cell functions and discovery of more efficacious medicines.</p><p>This thesis presents contributions to the development of a novel chemo-bioinformatics approach called proteochemometrics; a general method for interaction space analysis of biological macromolecules and their ligands. In this work we explore proteochemometrics-based interaction models over broad groups of protein families, evaluate their validity and scope, and compare proteochemometrics to traditional modeling approaches.</p><p>Through the proteochemometric analysis of large interaction data sets of multiple retroviral proteases from various viral species we investigate complex mechanisms of drug resistance in HIV-1 and discover general physicochemical determinants of substrate cleavage efficiency and binding in retroviral proteases. We further demonstrate how global proteochemometric models can be used for design of protease inhibitors with broad activity on drug-resistant viral mutants, for monitoring drug resistance mechanisms in the physicochemical sense and prediction of potential HIV-1 evolution trajectories. We provide novel insights into the complexity of HIV-1 protease specificity by constructing a generalized IF-THEN rule model based on bioinformatics analysis of the largest set of HIV-1 protease substrates and non-substrates.</p><p>We discuss how proteochemometrics can be used to map recognition sites of entire protein families in great detail and demonstrate how it can incorporate target variability into drug discovery process. Finally, we assess the utility of the proteochemometric approach in evaluation of ADMET properties of drug candidates with a special focus on inhibition of cytochrome P450 enzymes and investigate application of the approach in the pharmacogenomics field.</p>

G proteins coupled receptors (GPCRs) play essential developmental roles with functions in all of the immune, olfactory sensory systems amongst other systems as well as exhibiting essential roles in the central and peripheral nervous system. GPCRs are also major targets of pharmaceutical drugs currently used to treat a vast number of conditions. Despite their clear importance, the function of many GPCRs is still obscure. Identifying the physiological role of more GPCRs provides a niche for more drugs to be developed and thus more conditions to be treated. The C.elegans lat-1 gene encodes the latrophilin vertebrate homolog; it is a member of the adhesion GPCR family and is structurally related to the flamingo/CELSR, an essential component of planar cell polarity pathway. This study aims to phenotypically characterise lat-1 mutants in C.elegans to provide insights into the physiological role of this important member of adhesion GPCRs. lat-1 mutants exhibit several morphological defects throughout development and during vulva development. Analysing the embryonic development of such mutants also identified an anterior-posterior polarity defect. The results implicate a second evolutionary conserved subfamily of adhesion GPCRs in the control of tissue polarity and morphogenesis.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Le GABA est le principal neurotransmetteur inhibiteur du SNC et est impliqué dans le développement du cerveau, la plasticité synaptique et la pathogénèse de maladies telles que l’épilepsie, les troubles de l’anxiété et la douleur chronique. Le modèle actuel de fonctionnement du récepteur GABA-B implique l’hétérodimérisation GABA-B1/B2, laquelle est requise au ciblage à la surface membranaire et au couplage des effecteurs. Il y est cependant des régions du cerveau, des types cellulaires et des périodes du développement cérébral où la sous-unité GABA-B1 est exprimée en plus grande quantité que GABA-B2, ce qui suggère qu’elle puisse être fonctionnelle seule ou en association avec des partenaires inconnus, à la surface cellulaire ou sur la membrane réticulaire. Dans le cadre de cette thèse, nous montrons la capacité des récepteurs GABA-B1 endogènes à activer la voie MAPK-ERK1/2 dans la lignée dérivée de la glie DI-TNC1, qui n’exprime pas GABA-B2. Les mécanismes qui sous-tendent ce couplage demeurent mal définis mais dépendent de Gi/o et PKC. L’immunohistochimie de récepteurs endogènes montre par ailleurs que des anticorps GABA-B1 dirigés contre la partie N-terminale reconnaissent des protéines localisées au RE tandis des anticorps C-terminaux (CT) marquent une protéine intranucléaire. Ces données suggèrent que le domaine CT de GABA-B1 pourrait être relâché par protéolyse. L’intensité des fragments potentiels est affectée par le traitement agoniste tant en immunohistochimie qu’en immunobuvardage de type western. Nous avons ensuite examiné la régulation du clivage par le protéasome en traitant les cellules avec l’inhibiteur epoxomicine pendant 12 h. Cela a résulté en l’augmentation du marquage intranucléaire de GABA-B1-CT et d’un interacteur connu, le facteur de transcription pro-survie ATF-4. Dans des cellules surexprimant GABA-B1-CT, l’induction et la translocation nucléaire d’ATF-4, qui suit le traitement epoxomicine, a complètement été abolie. Cette observation est associée à une forte diminution du décompte cellulaire. Étant donné que les trois derniers résidus de GABA-B1-CT (LYK) codent un ligand pseudo-PDZ et que les protéines à domaines PDZ sont impliquées dans la régulation du ciblage nucléaire et de la stabilité de protéines, en complément de leur rôle d’échaffaud à la surface cellulaire, nous avons muté les trois derniers résidus de GABA-B1-CT en alanines. Cette mutation a complètement annulé les effets de GABA-B1-CT sur l’induction d’ATF-4 et le décompte cellulaire. Cette deuxième série d’expériences suggère l’existence possible de fragments GABA-B1 intranucléaires régulés par le traitement agoniste et le protéasome dans les cellules DI-TNC1. Cette régulation d’ATF-4 dépend des résidus LYK de GABA-B1-CT, qui modulent la stabilité de GABA-B1-CT et favorisent peut-être la formation d’un complexe multiprotéique incluant GABA-B1-CT, ATF-4, de même qu’une protéine d’échaffaudage inconnue. En somme, nous démontrons que les sous-unités GABA-B1 localisées au RE, lorsque non-hétérodimérisées avec GABA-B2, demeurent capables de moduler les voies de signalisation de la prolifération, la différentiation et de la survie cellulaire, via le couplage de protéines G et possiblement la protéolyse régulée. Les mécanismes de signalisation proposés pourraient servir de nouvelle plate-forme dans la compréhension des actions retardées résultant de l’activation des récepteurs 7-TMs. / GABA is the principal inhibitory neurotransmitter in the CNS and is implicated in brain development, synaptic plasticity and the pathogenesis of diseases such as epilepsy, anxiety disorders and chronic pain. In the current model of GABA-B function, there is a requirement for GABA-B1/B2 dimerization for targetting to the cell surface and effector coupling. However, there are certain brain regions (putamen), cell types (glial cells) and times during brain development where GABA-B1 is expressed in higher amounts than GABA-B2, suggesting that GABA-B1 might be functional alone or in association with unidentified partners, either at the cell surface or on the ER membranes. In this thesis, we first show the capacity of endogenous GABA-B1 receptors to activate the MAPK-ERK1/2 pathway in the DI-TNC1 glial-derived cell line which does not express GABA-B2. The underlying mechanisms remain incompletely defined but depend on Gi/o and PKC. Immunohistochemistry of endogenous receptors shows that GABA-B1 N-terminal antibodies recognize ER-localized proteins and that C-terminal (CT) antibody shows intranuclear distribution. This data suggests that fragments of the GABA-B1 receptor are generated by proteolysis and indeed we show that agonist treatment affects the intensity of certain C-terminal GABA-B1 fragments both in immunohistochemistry and western blots suggesting that the GABA-B1 receptor is subjected to regulated proteolysis. Since a 13-residue potential PEST sequence was localized immediately distal to the ER retention motif in the GABA-B1 CT, we examined proteasome regulation of the cleavage event. Following a 12h treatment with the proteasome inhibitor, epoxomicin, we detected increases in intranuclear staining for both GABA-B1 and a known interactor, the pro-survival transcription factor ATF-4, using confocal microscopy and by western blotting of nuclear extracts. These increases are due either to proteasome inhibition or activation of the ER stress pathway. In cells overexpressing GABA-B1-CT, ATF-4 induction and nuclear translocation, which normally follows epoxomicin treatment, was completely abolished. This observation was associated to a strong decrease in cell number. Since the last three residues of GABA-B1-CT (LYK) encode a pseudo-PDZ ligand and that PDZ domain protein regulate nuclear targeting and protein stability, in complement to their role in scaffolding at the cell surface, we mutated the last three residues of GABA-B1-CT to alanines. This mutation completely reversed the effect of GABA-B1-CT on ATF-4 induction and on cell number. This second set of data suggests the existence of agonist and proteasome-regulated intranuclear GABA-B1 fragments in DI-TNC1 cells. Further, the GABA-B1-CT pseudo-PDZ ligand appears to be critically important in regulating ATF-4 induction by modulating GABA-B1-CT stability and perhaps by favoring the formation of a multiprotein complex with ATF-4, ATF-4 interactors and an unknown scaffolding protein. Overall, we show that ER-localised GABA-B1 subunits, when not dimerized with GABA-B2, can still modulate proliferation, differentiation and survival pathways, both through G-protein coupling and regulated proteolysis. The signalling mechanisms which we propose could serve as a new platform in understanding the long term effects of 7-TM receptor activation.