Dissertations / Theses on the topic 'Class A GPCR'

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.

G protein-coupled receptors (GPCRs) are the largest family of signaling proteins in animals and represent the largest family of druggable targets in the human genome. Therefore, it is of no surprise that the molecular mechanisms of GPCR activation and signal transduction have attracted close attention for the past few decades. Several stabilizing interactions within the GPCR transmembrane (TM) domain helices regulate receptor activation. An example is a salt bridge between 2 highly conserved amino acids at the bottom of TM3 and TM6 that has been characterized for a large number of GPCRs. Through structural modeling and molecular dynamics (MD) simulations, we predicted several electrostatic interactions to be involved in metabotropic glutamate receptor 2 (mGlu2R) activation. To experimentally test these predictions, we employed a charge reversal mutagenesis approach to disrupt predicted receptor electrostatic intramolecular interactions as well as intermolecular interactions between the receptor and G proteins. Using two electrode voltage clamp in Xenopus laevis oocytes expressing mutant receptors and G-proteins, we revealed novel electrostatic interactions, mostly located around intracellular loops 2 and 3 of mGlu2R, that are critical for both receptor and G-protein activation. These studies contribute to elucidating the molecular determinants of mGluRs activation and conformational coupling to G-proteins, and can likely be extended to include other classes of GPCRs.

Les récepteurs couplés aux protéines G (RCPG) constituent l'une des plus grandes familles de gènes du génome des mammifères. Ils sont impliqués dans la plupart des processus physiologiques et physiopathologiques ce qui en a fait des cibles thérapeutiques de choix. GPR158 est un récepteur orphelin, dont on ne connaît pas de ligand, de la classe C des RCPG. Il partage 20% d'identité de séquence entre son domaine transmembranaire (TM) et celui du récepteur GABAB mais son domaine N-terminal est dépourvu du domaine Venus Flytrap (VFT), le domaine de liaison du ligand caractéristique des RCPG de la classe C, ce qui suggère que cette protéine a développé un autre mode de liaison de son ligand endogène (s'il existe). GPR158 est exprimé majoritairement au niveau du cerveau. De manière intéressante, son expression a été aussi décrite, dans des cribles à plus ou moins grande échelle, comme étant associée ou modifiée dans différentes conditions pathologiques dont 50% sont des maladies cancéreuses.À ce jour, on ne connait ni la fonction physiologique, ni les voies de signalisation de GPR158. Dans un premier temps, notre objectif était de comprendre la fonctionnalité de GPR158 en cherchant une activité constitutive de ce récepteur ou en essayant de générer des mutants, des récepteurs tronqués ou des récepteurs chimériques constitutivement actifs. Jusqu'à présent, nous n'avons pas réussi à avoir un récepteur actif, malgré les résidus des boucles intracellulaires et des domaines TM importants pour le couplage aux protéines G et pour l'activation des autres RCPG, qui sont conservés dans GPR158. Ce qui suggère que ce récepteur pourrait ne pas avoir une signalisation en lui même, et il régulerait ainsi l'activité d'autres RCPG. Dans le cas inverse, GPR158 aurait un mode de signalisation très original qui reste à découvrir. Puis nous avons cherché à comprendre le rôle physiologique lié aux trois motifs VCPWE, que nous avons identifiés au niveau du domaine C-terminal (C-ter), très long, de ce récepteur. Ces motifs sont bien conservés chez les différentes espèces et joueraient ainsi des rôles fonctionnels importants. À ce sujet, nous avons montré que le troisième motif s'associe spécifiquement avec la sous-unité Gαo, probablement activée, des protéines G. Et nous avons également identifié, un site d'interaction d'un régulateur de l'activité des protéines G, RGS7, au niveau du domaine C-ter de GPR158, en amont des motifs. Vu que Gαo est le substrat de RGS7, nous suggérons que Gαo lierait en même temps le domaine RGS de la protéine RGS7, et ils formeraient ainsi avec GPR158 un complexe de régulation de l'activité du récepteur orphelin ainsi que d'autres RCPG présents dans le nano-environnement de GPR158. Enfin, afin de mieux comprendre la fonction et les possibles voies de signalisation de GPR158, une analyse protéomique des complexes multi-protéiques bâtis autour du domaine C-ter de GPR158, a été menée. Après purification du récepteur orphelin et des protéines associées par immunoprécipitation, l'identification par spectrométrie de masse des protéines présentes a permis d'identifier 6 nouveaux partenaires potentiels. Parmi eux, quatre protéines, p53, PPM1G, Sgt1 et SIRT1, sont des régulateurs du facteur de transcription suppresseur de tumeur p53, et deux protéines, SIRT1 et TRIM58, sont impliquées dans le processus de vieillissement cellulaire. Par conséquent, une implication dans la transcription, la régulation du cycle cellulaire, la réparation de l'ADN, la prolifération, l'apoptose, la tumorigenèse et le vieillissement, du récepteur orphelin GPR158 peut être envisagée
G protein-coupled receptors (GPCR) are known to form the largest family of cell communication proteins, and to participate to all functions of the body, making them high potential therapeutic targets. However, lots of these proteins are still orphan receptors, for which no ligand, neither function have been described, although some could be of very high interest, like GPR158, a class C orphan GPCR. The seven transmembrane domain (7TM) of this orphan receptor was related to class C GPCR (GPR158 and GABAB share 20% sequence identity in the TM core region) but its N-terminal domain was not homologous to the typical Venus Flytrap (VFT) known to bind the ligands in most of class C receptors. Which suggests that GPR158 has developed different ligand binding mode. GPR158 is expressed mainly in the brain. Interestingly, the expression of this receptor has been found in many cells and tissues to be potentially regulated in pathological conditions, of which 50% are cancerous diseases. We thus intended to decipher its cellular function and partners, to understand its potential physiological and physiopathological roles. Initially, our goal was to determine the functionality of GPR158, and the possible signaling and cellular mechanisms it was involved in, by looking for some constitutive activity for this orphan GPCR, in the absensce of any ligand. Curiously, we could not detect any G protein coupling, like constitutive G protein stimulation by overexpression of wild type, mutated, truncated and chimeric receptors. This despite the residues of intracellular loops and TM domain, important for the G protein coupling and for the activation of other GPCR, which are conserved in GPR158. This suggests that GPR158 in itself might not have a signalization, and thus it would regulate the activity of other GPCR. Alternatively, GPR158 would have an original way of signaling to be discovered with more sophisticated techniques.Then, we tried to understand the role of three VCPWE specific motifs that we have identified at the long C-terminal (C-ter) domain of GPR158. These motifs are well conserved among different species and thus would play important functional roles. Therefore, we have shown that the third motif indeed binds G protein alpha o subunit, likely in active state. Interestingly, we have also shown that RGS7 that deactivated alpha o, interacts constitutively with the C-terminal domain of GPR158 upstream of VCPWE motifs. Thus, RGS7 would regulate the alpha subunit association with GPR158. Hence, GPR158 would act as a signaling regulatory platform, controlling G protein pathways by binding active alpha subunit and RGS7. This would be of great importance as a local signaling regulatory mechanism. Finally, to better understand the function and possible signaling pathways of GPR158, a proteomic analysis of multi-protein complexes built around the C-ter domain of GPR158, was conducted. After purification of the orphan receptor and its associated proteins by immunoprecipitation, the identification by mass spectrometry of GPR158 interacting proteins led to the identification of six potential new partners. Among them, four proteins, p53, PPM1G, SGT1 and SIRT1, are regulators of the p53 tumor suppressor protein widely known for its role as a transcription factor that regulates the expression of stress response genes, and two proteins, SIRT1 and TRIM58 are involved in cellular aging process. Therefore, GPR158 could be involved in transcription, cell cycle regulation, DNA repair, proliferation, apoptosis, tumorigenesis and cell aging

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.

La consommation excessive et chronique de sucre est un facteur de risque pour l'apparition de pathologies telles que le diabète de type II ou l'obésité. Une des solutions pour répondre à cet enjeu majeur de santé publique, tout en conservant le plaisir de la saveur sucrée, consiste en l'utilisation d'édulcorants en substitution du sucre. Actuellement, un certain nombre d'édulcorants sur le marché présentent arrière-goût amer ou sont sujets à débat quant à leurs effets sur la santé. Un des objectifs de ces travaux de thèse consiste à proposer de nouveaux édulcorants grâce à des approches rationnelles in silico. Un modèle statistique a été établi sur la base des structures chimiques et a permis d'identifier de nouveaux édulcorants d'origine naturelle. Ensuite, la reconstruction par homologie du récepteur à la saveur sucrée et l'étude des sites de liaison apportent des indices, à l'échelle atomique, qui permettront d'identifier ou même de concevoir de nouveaux édulcorants. L'étude dynamique d'un récepteur de la même famille (Récepteur Couplé aux Protéines G (RCPG) de classe C) a permis d'émettre une hypothèse sur le mécanisme d'activation, phénomène important pour la compréhension de la conversion du signal chimique en signal électrique
Sugar overconsumption is a risk factor for pathologies such as type II diabetes or obesity. Sweeteners consumption is used to overcome this public health issue. Indeed, they have low caloric value but still preserve the pleasure of sweet taste. Currently, number of sweeteners are commercially available, but they present a bitter aftertaste or there is a debate about their safety. One aim of this work was to propose new intense sweeteners using computational modeling strategies. Through a statistical approach to predict the sweetness based on the chemical structure of already known sweeteners, new natural compounds have been identified. Furthermore, the structural study of the homology model of the sweet taste receptor provides some clues to design new sweeteners. The molecular dynamic study of a class C G-protein coupled receptor gives the first molecular hypothesis of the activation process

The third intracellular loop (IL3) and cytoplasmic tail (CT), which are the most divergent regions between human D1-class dopaminergic receptors (hD1R and hD5R), have been implicated in modulating their subtype-specific functional phenotypes. The importance of the IL3 for Guanine nucleotide-binding protein (G-protein) coupling and specificity has long been acknowledged in the G-protein-coupled receptor (GPCR) field. However, the exact role the central region of the IL3, notably the N- and C-terminal moieties, plays in GPCR receptor functionality remains unclear. Studies in our laboratory indicated that the IL3/N-terminal moiety of hD1-class receptors appears to be critical for facilitating agonist-independent and dependent activation of hD1R and hD5R. Furthermore, the IL3/C-terminal portion of hD1-class receptors constrains the receptor in the inactive state and reduces receptor affinity for agonists and G-protein coupling. I put forward the following hypothesis: 1. The functional properties of hD1-class receptors are regulated via a molecular micro-switch present within the IL3 central region modulating the functional properties of the receptor distinctly, 2. The functional differences between D1R and D5R require structural elements from both N- and C-terminal halves of the IL3 central region, and 3. The molecular interplay between the N- and C-terminal halves of the IL3 central region is dependent on the amino acid chain length and content. Herein, I have employed site-directed mutagenesis, and alanine replacement approaches to analyze comprehensively the structural determinants within the N- and C-terminal moieties of the IL3 central region that regulate ligand binding and G-protein coupling properties of hD1-class receptors. Moreover, my Ph.D. research aimed to pinpoint whether the IL3 length and/or structural motif(s) regulate ligand binding and activation properties of hD1R and hD5R. The results of my study highlight the importance of structural elements from both the proximal and distal segments of the IL3/central region of hD1-class receptors for the ligand binding and receptor activation status. Additionally, my results underline the significance of preserving the length of the IL3 regardless of the amino acid content. This study also shows the pivotal role played by a phenylalanine residue, F2646.27, in the signaling properties of hD1R. Notably, mutating F2646.27 leads to a mutant hD1R with characteristics resembling those of constitutively active mutant GPCRs. Unraveling the amino acid/amino acids constraining the receptor in the inactive state will perhaps provide an attractive target for drug design. Future work aims at developing drugs that particularly bind to the intracellular face of hD1R and improving selectivity towards hD1R may prove useful in limiting the side effects associated with the conventional therapy of brain disorders such as in the case of L-DOPA induced dyskinesia (LID) seen in individuals suffering from Parkinson’s disease.

Master of Science
Department of Civil Engineering
Eric J. Fitzsimmons
The rail industry's recent shift towards larger and heavier railcars has influenced Class III / short line railroad operation and track maintenance costs. Class III railroads earn less than $38.1 million in in annual revenue and generally operate first and last leg shipping for their customers. In Kansas, Class III railroads operate approximately 40 percent of the roughly 2,800 miles (4,500 km) of rail; however, due to the current Class III track condition they move lighter railcars at lower speeds than Class I railroads. The State of Kansas statutorily allots $5 million to support rail improvement projects, primarily for Class III railroads. Therefore, the objective of this study was to conduct an inventory of Kansas’s Class III rail network to identify the track segments in need of this support that would be most beneficial to the rail system. Representatives of each railroad were contacted and received a survey requesting information regarding the operational and structural status of their systems. The data collected were organized and processed to determine the sections of track that can accommodate the heavier axle load cars that are currently being utilized by Class I railroads. This study identified that Class III railroads shipped over 155,000 carloads of freight in 2016 and 30 percent of Kansas’s Class III track can currently accommodate heavy axle cars. The increased load from the increased railcar size has also increased the risk of damage to railroad’s track structure. Railroad ballast is the free draining granular material that supports the track structure. As the track ages, small particles can fill the voids of the granular material which is a process known as fouling. Established methods for determining the fouling of a section of ballast are destructive tests that usually require the railroad to restrict or reroute traffic on its network. Ground Penetrating Radar (GPR) is a nondestructive geophysical surveying method that measures the time required for electromagnetic wave impulses to reflect off differing subsurface interfaces. Historically, GPR surveys of track structures primarily determine the depth of ballast and track geometry. The objective of this study was to determine the viability of utilizing the laboratory’s existing GPR equipment to develop a methodology of measuring ballast fouling nondestructively. A 48 x 48 x 48 in (1.2 x 1.2 x 1.2 m) test box was built. The test box was filled with 48 in (1.2 m) of clean and ballast. Tests were run on dry and partially saturated material, wetted using 6 gallons (22.7 L). GPR data were collected hourly for the first 6 hours, then at the multiples of 12 and 24 hour marks for one week. Sand was chosen as an absorbent geologic material for the second stage of testing since no fouled ballast could be acquired at the time of the study. A 27 x18 x 18 in (0.69 x 0.46 x 0.046 m) box was filled with sand and wetted with water in one gallon (7.5 L) increments. GPR scans and samples to determine the water content were collected after the addition of each gallon. The data collected were processed to determine soil properties. Preliminary results from this research indicate that the GPR set up utilized can effectively determine the dielectric constant of geologic materials including ballast, although the dielectric constant is highly dependent on the volumetric moisture content of the material.

Despite the large number of G protein-coupled receptors (GPCRs) expressed in the central nervous system (CNS), little is known about their location, organization, and dynamics in functional nanodomains at synapses. Class C GPCRs including metabotropic glutamate receptors (mGluRs) and the γ-aminobutyric acid subtype B receptor (GABABR) mediate several key functions in synaptic transmission. However, it is still insufficiently understood how these receptors function at synapses to modulate neurotransmission. One limitation is the availability of techniques to examine receptors with high spatiotemporal resolution in physiologically relevant cells. To investigate the distribution and spatiotemporal dynamics of mGluR4 and GABABR in cerebellar slices and cultured hippocampal neurons, I used advanced imaging techniques, including single-molecule imaging and superresolution microscopy with high spatial (10-20 nm) and temporal (20 ms) resolution. The presynaptic active zone (AZ) is a highly organized structure that specializes in neurotransmitter release. mGluR4 is a prototypical presynaptic class C GPCR. mGluR4 mediates an inhibitory effect on presynaptic glutamate release mainly via the inhibition of P/Q type voltage dependent calcium channels (CaV2.1). In this study, I analyzed the organization of mGluR4 at the synapse between parallel fibers and Purkinje cells in the mouse cerebellum with near-molecular resolution using two-color direct stochastic optical reconstruction microscopy (dSTORM). Quantitative analyses revealed a four-fold mGluR4 enrichment at parallel fiber AZs. I found that an AZ contains 29 mGluR4 nanoclusters on average. Each nanocluster contains one or two mGluR4s, with few nanoclusters containing three or more receptors. To assess the spatial distribution of mGluR4 relative to functional active zone elements such as CaV2.1 and Munc 18-1 (an essential component of the synaptic secretory machinery), a distance-based colocalization analysis was used. The analysis revealed positive correlation between mGluR4 and both proteins at a distance of 40 nm. Interestingly, mGluR4 showed a higher positive correlation to Munc 18-1 in comparison to CaV2.1. These results suggest that mGluR4 might directly inhibit the exocytotic machinery to reduce glutamate release from the synaptic vesicles in addition to its role in the inhibition of presynaptic calcium influx. The revealed high degree of mGluR4 organization may provide a new ultrastructural basis to explain the depressive effect of mGluR4 on the neurotransmission. Moreover, I directly imaged GABABR dynamic behavior with high spatiotemporal resolution in living hippocampal neurons utilizing single-molecule total internal reflection fluorescence microscopy (TIRFM). To this purpose, the GABAB1 subunit was engineered with an N-terminal SNAP-tag to enable specific labeling with bright organic fluorophores. On the plasma membrane surface, immobile and mobile GABABRs were detected at both synaptic and extrasynaptic compartments. A mean square displacement analysis (MSD) revealed characteristic dynamic patterns of GABABR depending on receptor location inside or outside of the synapses. The majority of receptors belonging to the extrasynaptic pool displayed rapid and free diffusion. In contrast, approximately 80% of receptors residing at the synaptic compartments were immobile or confined within limited regions. Receptors located at pre- and post-synaptic sites showed a similar behavior. GABABR lateral diffusion patterns inside and outside synapses might be important for the regulation of efficacy of synaptic inhibition. Altogether, this study puts forward previously unknown GPCR nanoscopic details in functional nanodomains. GPCR spatial organization might be important for the efficiency, fidelity, and rapid signaling required for synaptic transmission
Trotz der großen Anzahl an G Protein-gekoppelten Rezeptoren (GPCRs) die im zentralen Nervensystem (ZNS) exprimiert werden, ist deren Lokalisierung, Anordnung und Dynamik in funktionellen Nanodomänen an Synapsen gegenwärtig weitgehend unbekannt. Klasse C GPCRs, einschließlich metabotroper Glutamatrezeptoren (mGluRs) und des γ- Aminobuttersäure-B-Rezeptors (GABABR), vermitteln einige Schlüsselfunktionen der synaptischen Übertragung. Grundlegende Prinzipien wie diese Rezeptoren an Synapsen funktionieren, um die Neurotransmission zu modulieren, sind jedoch nur unvollständig verstanden. Eine Schwierigkeit ist die Verfügbarkeit von Techniken zur Untersuchung von Rezeptoren mit hoher raum-zeitlicher Auflösung in physiologisch relevanten Zellen. Um die Anordnung und die raum-zeitliche Dynamik von mGluR4 und GABABR in Kleinhirnschnitten und kultivierten hippocampalen Neuronen zu untersuchen, verwendete ich neue optische Verfahren wie Einzelmolekül- und hochauflösende Mikroskopie (dSTORM) mit hoher räumlicher (10-20 nm) und zeitlicher Auflösung (20 ms). Die präsynaptische aktive Zone (AZ) ist eine hoch organisierte Struktur, die auf die Transmitterausschüttung spezialisiert ist. Der mGluR4 ist ein prototypischer, präsynaptischer Klasse C GPCR. Hauptsächlich durch die Inhibierung spannungsgesteuerter Calciumkanäle des P/Q-Typs (CaV2.1) vermittelt mGluR4 eine inhibitorische Wirkung auf die Glutamatfreisetzung.In dieser Arbeit analysierte ich die Organisation des mGluR4 an der Synapse zwischen parallelen Fasern und Purkinje-Zellen im Kleinhirn der Maus unter Verwendung der Zweifarben direkten stochastischen optischen Rekonstruktionsmikroskopie (dSTORM). Quantitative Analysen zeigten eine vierfache höhere Anreicherung von mGluR4 an den Parallelfaser-AZs. Ich fand heraus, dass eine AZ im Durchschnitt 29 mGluR4- Nanocluster enthält. Jeder Nanocluster enthält ein oder zwei mGluR4s, wobei wenige Nanocluster drei oder mehr Rezeptoren enthalten. Um die räumliche Verteilung von mGluR4 relativ zu funktionellen Elementen aktiver Zonen, wie CaV2.1 und Munc 18-1( ein wichtiges Protein der exozytotischen Maschinerie), zu bestimmen, wurde die Abstandsbasierte-Co- Lokalisierung-Analyse verwendet. Co-Lokalisierung wurde zwischen mGluR4 und beiden Proteinen in einem Abstand von 40 nm detektiert. Interessanterweise wurde für Munc 18-1 eine höhere positive Korrelation zu mGluR4 identifiziert. Dies deutet darauf hin, dass mGluR4, zusätzlich zu seiner Rolle bei der Hemmung des präsynaptischen Calciumeinstroms, die exozytotische Maschinerie zur Verringerung der Freisetzung von Glutamat aus sekretorischen Organellen direkt hemmen könnte. Der gezeigte hohe Grad der mGluR4-Organisation könnte eine neue ultrastrukturelle Grundlage zur Erklärung der depressiven Wirkung von mGluR4 auf die synaptische Übertragung liefern. Außerdem habe ich das dynamische Verhalten von GABABR direkt mit hoher räumlicher und zeitlicher Auflösung in lebenden hippocampalen Neuronen durch Einzelmolekül-TIRFM visualisiert. Zu diesem Zweck wurde die GABAB1-Untereinheit mit einem N-terminalen SNAP- Tag konstruiert, um eine spezifische Markierung mit hellen organischen Fluorophoren zu ermöglichen. Auf der Oberfläche der Plasmamembran wurden immobile und mobile GABABRs in synaptischen und extrasynaptischen Kompartimenten nachgewiesen. Die mittlere quadratische Verschiebung (mean square displacment analysis (MSD)) zeigte charakteristische dynamische Muster von GABABR in Abhängigkeit der Position der Rezeptoren innerhalb oder außerhalb der Synapsen. Die Mehrheit der Rezeptoren im extrasynaptischen Pool zeigte schnelle und freie Diffusion. Im Gegensatz dazu waren ungefähr 80% der synaptischen Rezeptoren immobile oder auf begrenzte Regionen beschränkt. Rezeptoren an prä- und postsynaptischen Stellen zeigten ein ähnliches Verhalten. GABABR- Diffusionsmuster innerhalb und außerhalb von Synapsen könnten außerdem für die Regulierung der Wirksamkeit der synaptischen Hemmung von Bedeutung sein. Insgesamt zeigen diese Ergebnisse bisher unbekannte Erkenntnisse zu nanoskopischen Einzelheiten von GPCRs in funktionellen Nanodomänen. Die räumliche Organisation von GPCR kann für die Effizienz, Genauigkeit und schnelle Signalisierung der Neurotransmission wichtig sein

In this work, two projects were pursued. In the first project, I investigated two different subtypes of opioid receptors, which play a key role as target for analgesia. A set of subtype specific fluorescent ligands for μ opioid receptor (MOR) and δ opioid receptor (DOR) was characterised and used to gain insights into the diffusion behaviour of those receptors. It was shown that the novel ligands hold photophysical and pharmacological properties making them suitable for single-molecule microscopy. Applying them to wild-type receptors expressed in living cells revealed that both sub-types possess a heterogeneous diffusion behaviour. Further- more, the fluorescent ligands for the MOR were used to investigate homodomerisation, a highly debated topic. The results reveal that only ≈ 5 % of the receptors are present as homodimers, and thus the majority is monomeric. G-protein coupled receptors (GPCRs) play a major role as drug targets. Accordingly, understanding the activation process is very important. For a long time GPCRs have been believed to be either active or inactive. In recent years several studies have shown, that the reality is more complex, involving more substates. [1, 2, 3, 4] In this work the α 2A AR was chosen to investigate the activation process on a single-molecule level, thus being able to distinguish also rare or short-lived events that are hidden in ensemble mea- surements. With this aim, the receptor was labelled intracellular with two fluorophores using supported membranes. Thus it was possible to acquire movies showing qualita- tively smFRET events. Unfortunately, the functionality of the used construct could not be demonstrated. To recover the functionality the CLIP-tag in the third intracellular loop was replaced successfully with an amber codon. This stop codon was used to insert an unnatural amino acid. Five different mutants were created and tested and the most promising candidate could be identified. First ensemble FRET measurements indicated that the functionality might be recovered but further improvements would be needed. Overall, I could show that single-molecule microscopy is a versatile tool to investigate the behaviour of typical class A GPCRs. I was able to show that MOR are mostly monomeric under physiological expression levels. Furthermore, I could establish intra- cellular labelling with supported membranes and acquire qualitative smFRET events
In dieser Arbeit wurden zwei Projekte verfolgt. Im ersten Projekt wurden zwei Subtypen der Opioidrezeptoren untersucht, die eine wichtige Rolle für die Wirksamkeit von Analgetika spielen. Ein Set von subtypspezifischen fluoreszierenden Liganden für den MOR und den DOR wurde charakterisiert und eingesetzt, um Einblicke in das Diffuionsverhalten der Rezeptoren zu gewinnen. Es konnte gezeigt werden, dass die neuartigen Liganden sowohl photophysikalische als auch pharmakologische Eigenschaften besitzen, die sie für die Einzelmolekülmikroskopie interessant machen. Versuche mit Opioidrezeptoren, die in lebenden Zellen exprimiert werden, zeigten, dass beide Subtypen heterogenes Diffuionsverhalten aufweisen. Des Weiteren wurden die fluoreszierenden Liganden für den MOR genutzt um Homodimerisierung zu untersuchen, da dies ein kontrovers diskutiertes Thema ist. Die Ergebnisse zeigen, dass lediglich ≈ 5% der Rezeptoren als Homodimere vorliegen und der Großteil monomerisch ist. GPCRs sind besonderem Interesse, weil sie Angriffspunkt vieler Medikamente sind. Deshalb ist es wichtig ihren Aktivierungsmechanismus besser zu verstehen. Lange Zeit wurde angenommen, dass GPCRs entweder aktiv oder inaktiv sind. Neuere Studien zeigten jedoch, dass die Realität komplexer ist und der Prozess Zwischenschritte involviert. [1, 2, 3, 4] In dieser Arbeit wurde der α 2A Adrenorezeptor als prototypischer Klasse A GPCR gewählt, um den Aktivierungsprozess auf Einzelmoleküllevel zu untersuchen. Durch die Betrachtung einzelner Rezeptoren ist es möglich auch seltene oder sehr kurzlebige Ereignisse zu unterscheiden, die in Kollektivmessungen untergehen. Um dies zu erreichen wurde der Rezeptor erfolgreich intrazellulär mit zwei Fluorophoren markiert. Dies gelang durch die Herstellung von „supported membranes", also Zellmembranen die auf einem Objektträger fixiert wurden. Dadurch war es möglich Videos aufzunehmen, die Einzelmolekül-FRET-Ereignisse zeigen. Jedoch gelang es nicht zu zeigen, dass der Rezeptor als Ganzes noch funktional war. Um einen funktionalen Rezeptor zu erhalten, wurde das CLIP-Tag in der dritten intrazellulären Schleife erfolgreich durch ein Stopcodon ersetzt, welches für eine nicht kanonische Aminosäure kodierte. Fünf verschiedene Mutanten wurden kloniert und getestet, wobei der vielversprechendste Mutant identifiziert werden konnte. Erste FRET-Kollektivmessungen deuten darauf hin, dass dieser Mutant funktional sein könnte. Jedoch sind weitere Verbesserungen nötig. Insgesamt konnte ich zeigen, dass Einzelmolekülmikroskopie vielseitige Möglichkeiten bietet um das Verhalten von GPCRs zu untersuchen. Ich konnte nachweisen, dass MOR unter physiologischen Bedingungen hauptsächlich als Monomere vorliegen. Des Weiteren konnte ich Dank supported membranes die Markierung durch Farbstoffe im Intrazellularbereich etablieren und qualitative smFRET Ereignisse aufnehmen

G-protein coupled receptors (GPCRs) have been shown to act as part of GPCR associated protein complexes (GAPCs) which are required to appropriately transduce downstream signaling pathways leading to specific cellular actions. I hypothesize that there are distinct molecular effectors that couple to the glucagon superfamily of B-class GPCRs (glucagon, GLP-1, GLP-2, GIP receptors) to effect the myriad of reported actions in numerous target cells including regulation of insulin secretion, intestinal growth and appetite suppression. GLP-1R, GIPR, GLP-2R and GCGR were screened using a newly developed membrane-based split-ubiquitin yeast two-hybrid (MYTH) system to reveal 181 novel candidate protein interactors associated with signal transduction, transport, metabolism and cell survival. Each candidate was validated using yeast two-hybrid prey retransformation tests and by co-purification to confirm coupling to each receptors. The present work is the first demonstration of a split-ubiquitin interaction screen using in situ membrane expressed GPCRs of the secretin-like B class.

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.

Dissertation