Previous studies have shown that the subtype-specific pharmacological properties of D1-class receptors (D1R and D5R) can be attributed to their third intracellular domain and C-terminal tail. However, the importance of their first and second intracellular domains (IC1 and IC2) has yet to be explored. Using mutagenesis and bioinformatics, we examine the functional and structural roles of Ser/Thr spanning IC1 and IC2—most of which are conserved not only among D1-class receptors but also among other GPCRs. Mutant receptors of human D1-class receptors (hD1R and hD5R) were constructed whereby all Ser and Thr were mutated to the respective Ala and Val in the IC1 region (termed ST1 mutant receptors) and in the IC2 region (termed ST2 mutant receptors). We found that hD1-ST2 and hD5-ST2 exhibited contrasting properties of agonist affinity, constitutive activity, and dopamine potency. On the other hand, both ST2 mutants underwent internalization as wild-type but displayed weakened desensitization abilities. Homology models, which have been refined under membrane simulations, illustrate that the conserved Ser3.55 and Thr3.65 utilize their side chains to anchor the loop regions of IC2 to cytoplasmic helices. We also found multiple functional alterations in the hD1-ST1 and hD5-ST2, but in a subtype-similar manner. Mutating the conserved Thr2.39 recapitulated the ablated basal activity and drastic decrease in dopamine potency previously witnessed in the hD1-ST1. Based on the recurring theme observed in crystal structures, the side-chain of Thr2.39 may help to position IC2 to have proper contacts with the G protein. Mutating the conserved Ser2.45 was found to be solely responsible for the elevated Emax (maximal response) of the hD1-ST1. Using single point mutagenesis, we further found that breaking the potential molecular interactions of Ser2.45 in hD1R (i.e. with Asn3.42 and Trp4.50) mimicked its elevated Emax. This elevated Emax was not found to be caused by altered abilities to undergo agonist-induced desensitization or internalization relative to hD1R. Overall, our work highlights the important functional and structural roles of IC1 and IC2 that needs to be accounted for in our current canonical models of GPCR signalling. Given the conserved nature of these Ser/Thr, our work may also be pertinent towards understanding the roles of IC1 and IC2 for other GPCRs.

acase@tulane.edu / G-protein coupled receptors (GPCRs) represent the largest family of receptor proteins in the living world, having approximately 800 human genes predicted. GPCRs are therapeutically relevant, as approximately 35% of all drugs on the market target them. However, these drugs can be associated with unwanted side effects, due to ubiquitous GPCR expression throughout the body, as well as GPCRs within the same family having common ligands, but varying or contradictory responses. Although there are approximately 800 human GPCRs predicted, the high-resolution crystal structure of only 41 GPCRs are available. The first human GPCR to be crystallized was the β2-adrenergic receptor (β2AR) in 2007. Shortly thereafter an alternate crystal form of the β2AR revealed a specific cholesterol binding site between helices I, II, III and IV. From this work a cholesterol consensus motif (CCM) was established, which defined specific interactions between cholesterol and the receptor. Utilization of this CCM predicted that as many as 25% of all class A GPCRs could have a specific interaction with cholesterol at this site, including the Adenosine A2a receptor. However, the first crystal structure of the human Adenosine A2a receptor (bound to an antagonist) revealed a lipid, not cholesterol bound at the CCM. Simulations have given insight into other potential binding sites for cholesterol on the Adenosine A2a receptor’s helices 5 and 6, based on the crystal structure bound to an antagonist. Cholesterol has been shown to modulate the activity of multiple G Protein-coupled receptors (GPCRs), yet whether cholesterol acts through specific interactions, or indirectly via modifications to the membrane is not well understood. Here we report that the activity of the adenosine A2a receptor (A2aR) is dependent on cholesterol as determined by membrane cholesterol depletion with methyl-beta-cyclodextrin (MβCD). We also tested whether a specific interaction occurs between A2aR and cholesterol, by testing the impact of mutational changes to predicted cholesterol binding sites on functional consequences. Understanding how cholesterol modulates GPCRs could help in the design of superior drugs to target different cell types with varying membrane cholesterol concentrations, or in disease states where cholesterol homeostasis is disrupted. Additionally, the widespread use of cholesterol reducing drugs such as statins, has posed the new question of how plasma membrane cholesterol concentrations and activation of membrane embedded proteins are affected by these drugs. / 1 / Claire McGraw

Sleep is an evolutionary conserved behaviour which in most species is essential for survival. However, the mechanisms involved in the genetic regulation of sleep remain poorly understood. C. elegans exhibit a number of sleep-like behaviours: a) lethargus which is a developmental period between moults, b) satiety which occurs after feeding and c) after long durations of thrashing in liquid media. We have isolated a C. elegans G-protein coupled receptor, named NPR-14,that is most similar to the human orexin receptor 2 and is involved in the above behaviours and functions through a protein kinase G - dependent and independent pathways. NPR-14 works together with other signalling pathways that have been implicated in sleep regulation including Notch, GABA, and insulin signalling. A null mutation in npr-14 results in anachronistic quiescence especially in the presence of food. It also results in egg-laying defects, episodic swimming behaviour, asynchronicity during development and despite feeding less these mutants appear to accumulate more fat than N2 worms. / Thesis (Master, Biology) -- Queen's University, 2014-02-26 22:24:32.243

G protein-coupled receptors (GPCRs) are an important class of drug targets due to their involvement in various signaling pathways. GPCRs transmit signals from the extracellular environment to the interior of cells, regulating numerous physiological processes. GPCRs are known to engage in protein-protein interactions (PPIs) with a variety of intracellular signaling molecules, including G proteins, β-arrestins, and other adaptor proteins. Understanding these interactions is crucial for unraveling the complex signaling networks mediated by GPCRs and developing targeted therapeutics. Assay development plays a pivotal role in studying GPCR PPIs, allowing researchers to investigate the binding partners, functional consequences, and dynamics of these interactions. Developing assays for GPCR PPIs requires careful consideration of the specific interaction being studied, the choice of experimental techniques, and the suitability for the desired readout. These assays provide valuable insights into the molecular mechanisms underlying GPCR signaling and can aid in the discovery and development of novel therapeutic strategies targeting GPCR associated diseases. Considering that PPIs are highly dynamic processes, cellular assays are often essential for their study because they closely mimic the biological complexities of cellular environments. The present thesis aimed to develop an in cellulo proteomic assay by developing an intracellular nanobody targeting the V5-peptide tag. Functionalizing this anti-V5 intrabody would allow it to be implemented in various cell-based assays with any protein carrying the V5-tag. Therefore, the NbV5:V5 tag system presents itself as a versatile tool for live-cell imaging and a befitting adaptation to existing cellular assays dedicated to probing PPIs. The NbV5:V5 tag system has been applied to interrogate G protein-coupled receptor signaling with the ultimate goal of performing cellular studies of a broader and systematic nature using the genome-wide V5-tagged ORF library.

Les récepteurs couplés aux protéines G (GPCRs) représentent une famille génique parmi les plus nombreuses du génome humain avec plus de 1000 représentants identifiés. Ils ont été classés en sous-familles en fonction de leurs homologies de séquence, la structure de leurs ligands et leur rôle physiologique. Ils régulent un très grand nombre de fonctions physiologiques comme la tension artérielle, le métabolisme, la plupart des actions hormonales et de très nombreuses fonctions cérébrales, et constituent de ce fait des cibles privilégiées pour les agents thérapeutiques. Récemment, deux nouveaux ligands de GPCRs ont pu être identifiés au laboratoire. La chémérine, ligand de haute affinité pour le récepteur ChemR23 et F2L, identifié comme le ligand endogène de FPRL2. Suite à leur identification respective, différents projets ont été initiés afin d’avancer dans la compréhension de leur rôle physiologique. C’est dans cette optique que mon travail de thèse a pu commencer. Dans un premier temps, nous nous sommes attachés à déterminer, parmi les récepteurs FPRs murins, quel récepteur pouvait répondre à F2L, et par extrapolation, être considéré comme l’orthologue murin de FPRL2, l’étude d’homologie de séquence n’ayant pas permis de déterminer un candidat clair entre les deux espèces. Nous avons tout d’abord réalisé un criblage fonctionnel (test de libération de calcium intracellulaire et d’inhibition de AMPc) afin d’identifier, parmi les huit récepteurs cibles, le récepteur répondant à F2L. Fpr2 s’est avéré être le seul candidat répondant à F2L. Nous avons ensuite étudié l’activité fonctionnelle de F2L sur des cellules immunes exprimant Fpr2 (les neutrophiles, les DCs et les macrophages) et enfin nous avons pu effectuer un test de chimiotactisme in vivo prouvant que F2L pouvait recruter ces différents types cellulaires. A l’aide d’une collaboration, nous avons pu également établir que cette activité de F2L sur les neutrophiles ne pouvait être induite que par Fpr2 au vu des résultats négatifs obtenus sur des neutrophiles issus de souris invalidées du gène codant pour Fpr2. Dans un deuxième temps, nous avons entrepris de comprendre le mécanisme de régulation protéolytique de la chémérine, ligand de ChemR23. Sécrétée en tant que précurseur inactif, la prochémérine nécessite le clivage protéolytique de ces 6 ou 7 acides aminés carboxy-terminaux pour devenir le ligand affin de ChemR23, la chémérine. Dans une précédente étude, nous avions identifié la cathepsine G et l’élastase comme responsable de ce clivage protéolytique, respectivement la chémérine-156 et la chémérine-157. Suite à cette identification, nous sommes attachés à identifier les protéases extracellulaires responsables de la génération des formes de chémérine-155 et chémérine-154, préexistant in vivo et ne présentant pas d’activité fonctionnelle sur le récepteur ChemR23. Ainsi, nous avons démontré que la protéinase 3, conjointement présente dans les granules azurophiles des neutrophiles avec l’élastase et la cathepsine G, clive la prochémérine en un variant inactif, spécifiquement la chémérine-155, comme nous l’avons montré en spectrométrie de masse. Enfin, contrairement aux autres protéases, nous avons identifié la chymase comme la protéase permettant de réguler la chémérine bioactive, en générant à partir de cette dernière un autre variant inactif, la chémérine-154. A côté des récepteurs caractérisés fonctionnellement, existent également des récepteurs dits « orphelins », dont les ligands et la fonction sont inconnus à ce jour. La caractérisation de ces récepteurs représente un enjeu important puisqu’elle devrait permettre l’identification de nouveaux systèmes de communication intracellulaire constituant des cibles potentielles pour le développement d’agents thérapeutiques. Nous nous sommes également attachés à identifier de potentielles activités biologiques liées à la présence d’un ligand endogène de GPCRs orphelins testés. Pour ce faire, nous avons réalisé deux études indépendantes mais complémentaires dans ce but. La première a consisté en la détermination de l’expression génomique de divers GPCRs sur des populations leucocytaires purifiées à partir de sang. Via l’approche de q-PCR à haut débit, nous avons pu obtenir une liste de GPCRs orphelins montrant une expression d’intérêt sur une ou plusieurs populations leucocytaires. Enfin, en parallèle, différentes stratégies de criblage par chromatographie ont été développées afin de permettre la rétention et la séparation d’un grand nombre de molécules diverses et variées pouvant potentiellement activées un GPCR orphelin testé. Ce travail n’a pas encore permis de mettre à jour de nouvelles activités fonctionnelles, mais restent l’objectif majeur de notre étude.

Notre travail a permis tout d’abord d’investiguer les effets des nucléotides extracellulaires sur les cellules dendritiques (DCs) qui sont des cellules présentatrices d’antigènes capables d’initier et de réguler la réponse immunitaire. Afin d’avoir une vue globale de l’action des nucléotides extracellulaires sur les DCs, un profil d’expression génique de l’ATPgS – dérivé stable de l’ATP - a été réalisé par microarray dans les cellules dendritiques dérivées de monocytes (MoDCs). Notre groupe a préalablement montré que malgré que l’ATP est considéré comme un signal de danger, il confère des propriétés immunosuppressives aux DCs (Marteau et al, 2005). Nous nous sommes focalisés sur des régulations géniques pouvant être mises en relation avec un action anti-inflammatoire de l’ATP. Nous avons ainsi démontré que l’ATP était capable d’inhiber la sécrétion des chimiokines MCP-1 et MIP- 1a initiée par l’action du LPS, ce qui a pour conséquence de diminuer la capacité des DCs à recruter des monocytes ou d’autres DCs. Ce travail a fait l’objet d’une publication en tant que premier auteur (Horckmans et al, 2005). Un grand nombre d’autres gènes régulés liés à la réponse immune et à l’inflammation a été identifiée dans le profil microarray de l’ATPgS. Nous avions notamment pu identifier une augmentation de la sécrétion de VEGF-A en réponse à l’ATP, amplifiée en présence de LPS. Cette régulation est extrêmement intéressante au vu de l’action immunosuppressive du VEGF sur les DCs. Par ailleurs, cette régulation pourrait constituer un lien entre les DCs et l’angiogénese. Ce travail a fait l’objet d’une publication en tant que premier co-auteur dans la revue Journal of Immunology (Bles et al, 2007). En conclusion, nos données nous ont ainsi permis de montrer que les nucléotides adényliques peuvent avoir par leur action sur les cellules dendritiques une action anti-inflammatoire voire pro-angiogénique, en inhibant le recrutement leucocytaire et une action immunosuppressive en stimulant la sécrétion de VEGF.

Chemokines are important mediators of leukocyte migration. Chemokines bind to G protein–coupled receptors (GPCR) and cause conformational changes that trigger intracellular signaling pathways involved in inflammation, injury healing, cancer, metastasis, and HIV infections. No direct structural information about any chemokine receptor is available, but the structure of chemokines has been well studied. Structural studies of chemokines coupled with cell-biological investigations may lead to a better understanding of the mechanisms of chemokine-receptor interactions. In this Ph.D. project, I studied the structural and functional relationship between chemokines and chemokine receptors using NMR, X-ray crystallography, and mutagenesis approaches, coupled with several different cell-biology assays. We found that the conserved “chemokine fold” can support different dimerization types in the chemokines family, although changing the dimers from CC- to CXC-type fold is not readily accomplished. I also used an engineered covalently-bound dimer of the MIP-1β mutant, MIP-1β-A10C, to study the relationship between dimerization of chemokines and their interaction with the CCR5 receptor. My results suggest that MIP-1β dimer neither bind nor activate the CCR5 receptor. I also studied the biophysical properties of one N-terminal awkward mutant of P2-RANTES, which was originally selected by others from a phage display using CCR5-expressing cells. Although the NMR and X-ray crystal studies revealed that the wild type RANTES is a tight homodimer, analytical ultracentrifugation reveals that P2-RANTES is a monomer in solution, the 1.7 Å resolution X-ray crystal structure of P2-RANTES was found to be a packed tetramer. The mutated N-terminal residues play a very important role in the tetramerization in the X-ray crystal structure. Finally I used the HIV-1 env mediated cell-cell fusion assay to study the combination of chemokines or chemokine variants with anti-HIV peptides C37 or/and T-20. A surprisingly synergistic effect was found between P2-RANTES and C37 or T-20. This combination stratagem may lead to further useful drug combinations or drug delivery for more potent anti-HIV treatments.

BAI2 gehört zu den Adhesion-G-Protein-gekoppelten Rezeptoren (aGPCR). Diese bisher wenig untersuchte Klasse von ca. 30 GPCR ist charakterisiert durch eine komplexe genomische Struktur, sehr große extrazelluläre Domänen und eine Vielzahl von Splicevarianten. Bisher ist bei den meisten aGPCR, wie auch bei BAI2, wenig über ihre Signaltransduktion und Funktion bekannt. Zum Verständnis der physiologischen Relevanz und zur Suche nach dem endogenen Agonist sind Kenntnisse über Proteinstruktur, Splicevarianten und Signaltransduktion essentiell. Ziel dieser Arbeit war es, mittels verschiedener in vitro-Methoden die Proteinstruktur des BAI2 in den transmembranären und intrazellulären Domänen näher zu untersuchen, sowie die natürlichen Splicevarianten in diesem Bereich, deren evolutionäre Konservierung, Gewebespezifität und Quantität zu erfassen. Für beide gefundenen Splicevarianten, eine im dritten intrazellulären Loop (ICL3) und eine im C-Terminus, konnte eine evolutionäre Konservierung auf Aminosäure- und genomischer Organisationsebene, sowie ihre Entstehung durch Exonskipping nachgewiesen werden. Nachfolgend wurden die Splicevarianten auf mögliche Interaktionen mit intrazellulären Komponenten untersucht. In dieser Arbeit konnte gezeigt werden, dass beide ICL3-Splicevarianten natürlicherweise in einem definierten Verhältnis auftreten. Außerdem konnte gezeigt werden, dass die lange ICL3-Variante des BAI2 nicht zu einer Änderung der Membrantopologie des Rezeptors, einer Homodimerisierung über die zusätzliche Aminosäuresequenz oder zu einer Interaktion mit dem C-Terminus führt. Die Splicevariante im humanen C-Terminus des BAI2 konnte als eine variable, durch Exonskipping entstandene Calcium-unabhängige Calmodulin-Bindungsstelle identifiziert werden. Diese Arbeit belegt die Existenz mehrerer BAI2-Isoformen in vivo. Die Struktur dieser Isoformen lässt unterschiedliche Funktionalitäten vermuten. Auch wenn erste Untersuchungen zwischen den beiden ICL3-Varianten keinen Unterschied ergaben, sind diese Erkenntnisse für die weitere Analyse der Signaltransduktion und Ligandensuche bedeutend. Es ist z.B. denkbar, dass sich die beiden ICL3-Varianten in der G-Protein-Kopplung oder bei der Rekrutierung von intrazellulären Interaktionspartnern unterscheiden oder dass die Splicevariante im C-Terminus zu einer Scaffold- Funktion des Calmodulins führt und/oder die Signaltransduktion durch eine permanente Bindung des Calmodulins an einer Isoform moduliert wird.

archives@tulane.edu / G protein-coupled receptors (GPCRs) constitute the largest family of human proteins with approximately around 800 genes and, are therapeutic targets for more than 35% of the FDA-approved drugs with around 180 billion dollars in sales. The therapeutic potential of GPCRs is still not completely utilized. Structure-guided drug discovery will definitely aid in identifying novel compounds with therapeutic responses while minimizing adverse effects in patients. Multiple protein engineering strategies including deletions, truncations, insertion of stable soluble protein or thermostabilizing mutations have been utilized to obtain the structural information of these membrane proteins. Yet to date, the high-resolution structure of only 63 GPCRs have been determined, with only a few of these structures being of active protein. Heterologous expression of most GPCRs results in low yields of active receptors and additional strategies are required to improve active receptor yields for these “hard-to-express” GPCRs. Yeast provides a unique heterologous expression platform to produce mammalian GPCRs, as it also allows us to measure downstream signaling activity with an easy and inexpensive high-throughput assay via its MAPK response pathway. The adenosine A2A receptor (A2AR) shows exceptional expression and trafficking to the plasma membrane in yeast; however, this is not the case for other adenosine receptors. A2AR has a longer C-terminus than the other adenosine receptor subtypes, which may contribute to its exceptional trafficking to the plasma membrane. To test the possibility to improve trafficking of the adenosine A1 receptor (A1R), chimeric receptors containing the seven transmembrane domains of A1R and the full-length or truncated A2AR C-terminus were constructed. The chimeric receptor showed improved localization to the plasma membrane and was capable of binding radioligand with native-like A1R affinity. Functionally active A1R receptor variants were produced at a theoretical yield of 95 pmol/mg total membrane protein, estimated using radioligand binding data, which is greater than three-fold higher than previously reported yields from other heterologous expression systems, and should facilitate biophysical characterization and drug discovery efforts. Recent efforts to determine the high-resolution crystal structures for the adenosine A1 and A2A receptors have utilized modifications to the native receptors in order to facilitate receptor crystallization and structure determination. One common modification is a truncation of the unstructured C-terminus, which has been employed for all the adenosine crystal structures obtained to date. Here, the presence of the full-length C-terminus is shown to affect downstream signaling using a yeast MAPK response-based fluorescence assay. Upon ligand binding, the A1d291R or A2Ad316R variants were unable to couple to human-yeast chimeric G-protein chimeras to generate a downstream signal in yeast, though full-length receptors showed native-like G-protein coupling. Further, constructs transfected into HEK-293 cells showed similar behavior – i.e. the variants with C-terminal truncations lacked cAMP-linked signaling compared to the full-length receptors. Although the C-terminus was essential for Ga protein- associated signaling, chimeras of A1R with a C-terminus of A2AR coupled to the A1R-specific Ga (i.e. Gai1 versus Gas). This surprising result suggests that the C-terminus is important in signaling, but not specificity, for the interaction with Ga protein. Based on these results for chimeric A1R variants, chimeric variants for adenosine A3R and tachykinin 2 receptor (NK2R) were constructed. The A3/A2AR variant was constructed using an approach similar to that for A1/A2AR; the resulting construct showed a two-fold increased expression as compared to the wild-type A3R. This chimeric variant showed native-like signaling activity in engineered yeast strains with a modified MAPK pathway, whereas the wild-type receptor showed no activity. In order to improve the functional expression of human NK2R required for high-resolution structural studies, NK2R chimeras utilizing sequences from rat NK2R previously shown to have a higher functional expression in yeast than human NK2R were utilized. Herein, we show that NK2R chimeras incorporating the rat NK2R C-terminus demonstrated improved expression, ligand binding, and downstream signaling in engineered yeast strains. Taken together, these results highlight the utility of the chimeric receptor engineering strategy to facilitate the expression of active receptors. / 0 / Abhinav Rabindra Jain

Opioids are powerful and effective drugs used for pain management, but their therapeutic usage is limited due to their side effects. Therefore, obtaining an extensive understanding of the pharmacological properties that underlie the actions of these drugs is much needed. Efficacy is the extent to which an agonist can stimulate the activity of the receptor it interacts with, and many studies have claimed to determine the efficacy of a wide range of opioid agonists. However, these opioids reportedly appear to be full agonists in some studies but seem to be partial agonists in others. Discrepancies from previous findings hamper the determination of accurate measurements of the efficacy of these drugs. As such, several assays focus on different aspects of opioid receptor signaling to deduce how efficacious these drugs may be. In this study, we focus on the μ-opioid receptor (MOR) as agonists that act on it represent the majority of clinically used opioids. We take advantage of a unique cellular model that captures the differential activation of each Gαi/o/z protein on top of measuring the relative efficacies of each tested opioid agonist. Using various cell-based assays, we demonstrate that these can be tools used to directly look at the interaction between the receptor and its effectors through the coupling of inhibitory heterotrimeric G proteins. The distinctions between each functional readout reveal insights about the nature of each established system, highlighting their advantages as well as their limitations. Key details about the mechanistic basis of inhibitory G protein activation are also uncovered. Precise determination of the efficacy of opioids could ultimately impact the understanding of opioid-mediated neuromodulation, as further links can be made between this important pharmacological parameter and the extent in which it induces analgesia and limits the side effects typically associated with opioid intake.