The CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3) are seven transmembrane receptors that are involved in numerous pathologies, including several types of cancers. Both receptors bind the same chemokine, CXCL12, leading to significantly different outcomes. While CXCR4 activation generally leads to canonical GPCR signaling, involving Gi proteins and β‐arrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via β‐arrestin‐dependent signaling pathways. Understanding the dynamics and kinetics of their activation in response to their ligands is of importance to understand how signaling proceeds via these two receptors. In this thesis, different Förster resonance energy transfer (FRET)‐based approaches have been combined to individually investigate the early events of their signaling cascades. In order to investigate receptor activation, intramolecular FRET sensors for CXCR4 and ACKR3 were developed by using the pair of fluorophores cyan fluorescence protein and fluorescence arsenical hairpin binder. The sensors, which exhibited similar functional properties to their wild‐type counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RET‐based receptor sensors in the field of chemokine receptors. Additional FRET‐based settings were also established to investigate the coupling of receptors with G proteins, rearrangements within dimers, as well as G protein activation. On one hand, CXCR4 showed a complex activation mechanism in response to CXCL12 that involved rearrangements in the transmembrane domain of the receptor followed by rearrangements between the receptor and the G protein as well as rearrangements between CXCR4 protomers, suggesting a role of homodimers in the activation course of this receptor. This was followed by a prolonged activation of Gi proteins, but not Gq activation, via the axis CXCL12/CXCR4. In contrast, the structural rearrangements at each step of the signaling cascade in response to macrophage migration inhibitory factor (MIF) were dynamically and kinetically different and no Gi protein activation via this axis was detected. These findings suggest distinct mechanisms of action of CXCL12 and MIF on CXCR4 and provide evidence for a new type of sequential signaling events of a GPCR. Importantly, evidence in this work revealed that CXCR4 exhibits some degree of constitutive activity, a potentially important feature for drug development. On the other hand, by cotransfecting the ACKR3 sensor with K44A dynamin, it was possible to increase its presence in the plasma membrane and measure the ligand‐induced activation of this receptor. Different kinetics of ACKR3 activation were observed in response to CXCL12 and three other agonists by means of using the receptor sensor developed in this thesis, showing that it is a valuable tool to study the activation of this atypical receptor and pharmacologically characterize ligands. No CXCL12‐induced G protein activation via ACKR3 was observed even when the receptor was re-localized to the plasma membrane by means of using the mutant dynamin. Altogether, this thesis work provides the temporal resolution of signaling patterns of two chemokine receptors for the first time as well as valuable tools that can be applied to characterize their activation in response to pharmacologically relevant ligands. / Der CXC Chemokin‐Rezeptor 4 (CXCR4) und der atypische Chemokin‐Rezeptor 3 (ACKR3) sind heptatransmembranäre Rezeptoren, die in zahlreichen Krankheitsbildern eine Rolle spielen, wie in einigen Krebsarten. Beide Rezeptoren werden zwar von dem gleichen Chemokin CXCL12 aktiviert, allerdings mit unterschiedlichen Signalweiterleitungsmustern. Die Aktivierung von CXCR4 führt zu kanonischer GPCR Signaltransduktion über Gi‐Proteine und β‐Arrestine. Die Signalweiterleitung des Rezeptors ACKR3 hingegen, welcher hauptsächlich in intrazellulären Vesikeln vorliegt, erfolgt über ß‐Arrestinabhängige Signalwege. Es ist von großer Wichtigkeit die Dynamik und Kinetik dieser beiden Rezeptoren hinsichtlich der Aktivierung durch ihre Liganden und der Signalweiterleitung zu verstehen. In dieser Arbeit wurden verschiedene Förster‐Resonanzenergietransfer (FRET) Anwendungen kombiniert, um die frühen Phasen der Signal‐Kaskade von CXCR4 und ACKR3 zu untersuchen. Zur genaueren Aufklärung der Rezeptoraktivierung wurden intramolekulare FRET‐Sensoren entwickelt, hierzu wurden die Fluorophore Cyan‐fluoreszierendes Protein und engl. fluorescence arsenical hairpin binder verwendet. Die generierten Sensoren zeigten ähnliche funktionelle Eigenschaften wie die unveränderten Rezeptoren. Liganden‐induzierte Änderungen der Rezeptorkonformation können mittels dieser Sensoren beobachtet werden und stellen die ersten RET‐basierten Sensoren auf dem Forschungsgebiet der Chemokin‐Rezeptoren dar. Weitere FRET‐basierte Methoden wurden zur Untersuchung von Interaktionen zwischen Rezeptor und G‐Protein, Neuanordnung von Dimeren, sowie der G‐Protein Aktivierung eingesetzt und für beide Chemokin‐Rezeptoren etabliert. CXCR4 zeigte einen komplexen Aktivierungsmechanismus nach Stimulation durch CXCL12, bei welchem zunächst eine Neuordnung der Rezeptor‐Transmembrandomäne gefolgt von Neuordnungen zwischen Rezeptor und G‐Protein und zuletzt eine Neuordnung zwischen CXCR4 Protomeren erfolgte. Dies impliziert, dass im Aktivierungsprozess des Rezeptors Homodimere eine Rolle spielen. Zudem wurde eine verlängerte Gi ‐Protein Aktivierung gegenüber der Gq‐Protein Aktivierung bei CXCL12 stimuliertem CXCR4 beobachtet. Hingegen zeigte eine Stimulierung mit dem Macrophage Migration Inhibitory Factor (MIF) bei jedem Schritt der frühen Singal‐Kaskade veränderte Dynamiken und Kinetiken im Vergleich zu CXCL12. Darüber hinaus konnte keine Gi ‐Protein Aktivierung festgestellt werden. Dieser Befund zeigt individuelle Mechanismen für MIF und CXCL12 am CXCR4‐Rezeptor und liefert Belege für eine neuer Art von sequenziellen Signalweiterleitungen an GPCRs. Eine wichtige Beobachtung dieser Arbeit für eine potentielle Medikamentenentwicklung ist das CXCR4 ligandenunabhängige Aktivität zeigt. Um die Aktivierung des ACKR3 Sensors messen zu können wurde durch eine Co‐Transfektion mit K44A Dynamin eine höhere Membranständigkeit erreicht. CXCL12 und drei weiteren Agonisten zeigten am hier entwickelten ACKR3‐Sensor unterscheidbare Kinetiken. Mit diesem wertvollen Werkzeug können Liganden an diesem atypischen Rezeptor pharmakologisch charakterisiert werden. Es konnte keine CXCL12‐induzierte G‐Protein Aktivierung gemessen werden, trotz der stärkeren Präsenz an der Plasmamembran mit Hilfe der Dynamin‐Mutante. In Summe liefert diese Arbeit zum ersten Mal eine zeitliche Auflösung von Signalweiterleitungsmustern von zwei Chemokin‐Rezeptoren sowie wertvolle Werkzeuge zur Charakterisierung der frühen Phase der Signal‐Kaskade durch andere pharmakologisch relevanten Liganden.

Metabolite-sensing G protein-coupled receptors (msGPCRs) are GPCRs that are activated by metabolites originating from various sources. Several of the known msGPCRs are expressed on immune cells and adipocytes as well as the gut epithelium and metabolic tissues like the pancreas. Some of their known agonists are produced endogenously while others are of exogenous origin. Examples for agonists of exogenous origin are metabolites produced by (intestinal) bacteria. The expression profile and the nature of their agonists link msGPCRs to functions in the regulation of metabolic processes and immune cell responses. Both receptors investigated in my thesis project, hydroxycarboxylic acid receptor 3 (HCA3) and GPR84, are msGPCRs highly expressed on cells of the innate immune system such as neutrophils, monocytes, and macrophages. HCA3 is a member of the HCA family consisting of three receptors, which show high sequence homology. Especially HCA2 and HCA3 only differ in a few positions resulting in an amino acid sequence differing in 17 amino acids and the extended C-terminus of HCA3. While HCA1 and HCA2 are present in the genome of all mammals, HCA3 is only present in higher primates such as chimpanzee, orangutan, and human. As a result, there is a lack of accessible animal models and the receptor is still insufficiently studied. 3 hydroxyoctanoic acid (3HO), 3 hydroxydecanoic acid (3HDec) and aromatic D-amino acids D-phenylalanine (D-Phe) and D-tryptophan (D-Trp) are previously reported agonists of HCA3 with 3HO being its endogenous agonist. Regarding its physiological function, it is known that the receptor is involved in a negative feedback loop in the regulation of lipolysis and fatty acid oxidation under prolonged fasting conditions in adipocytes. Further, it has been shown that aromatic D-amino acids induce chemotaxis in human neutrophils. GPR84 is a receptor for medium chain fatty acids (MCFAs) with a chain length of 9 to 14 carbon atoms (C9 - C14) and their hydroxylated derivatives. Thus, GPR84 and HCA3 share 3HDec as a common agonist. Further, both receptors couple to Gαi proteins resulting in the inhibition of adenylyl cyclase and subsequent decrease of intracellular cyclic AMP (cAMP) levels, and induce the phosphorylation and activation of extracellular signal regulated kinase1/2 (ERK1/2). As opposed to HCA3, GPR84 is present in the genome of most mammals and various studies have linked GPR84 to pro-inflammatory functions and processes like phagocytosis, chemotaxis and upregulation of pro-inflammatory cytokine release. Most of these studies on GPR84 were performed using surrogate agonists. Because HCA3 is still poorly understood, the aim of my project was to shed some light on its function by evolutionary and functional analyses of HCA3 orthologs. Moreover, detailed analyses of its signal transduction and components involved in receptor-mediated downstream signaling events were performed as part of the present dissertation. Since HCA3 and GPR84 share at least one agonist and are co-expressed in different types of immune cells, we studied signaling of both receptors simultaneously. Our functional analyses of human and great ape HCA3 orthologs using cAMP inhibition assays revealed the evolutionary conservation of the endogenous agonist 3HO. By further functionally analyzing the primate HCA3 orthologs, we found both aromatic D-amino acids, D-Phe and D-Trp, to activate human HCA3 with the highest potency. Although D-Phe and D-Trp were previously described to induce HCA3-mediated chemotaxis in neutrophils a link to where the two D-amino acids would originate from in sufficiently high concentrations in a physiological context was missing. After extensive review of literature, we found that some intestinal bacteria and bacteria used to ferment food and beverages produce and secrete D-amino acids. This led us to investigate whether other structurally related D-amino acid metabolites produced by bacteria also activate HCA3. These investigations resulted in the discovery of lactic acid bacteria (LAB)-derived metabolites as highly potent agonists of HCA3. We tested both the Phe-metabolites D-phenyllactic acid (D-PLA) and L-PLA as well as the racemic mixture of the Trp-metabolite indole-3-lactic acid (ILA). All three compounds specifically induced activation of HCA3, but we found D-PLA to be a 35-fold more potent agonist than the L-enantiomer. Further, D-PLA proved to be 10-fold more potent than 3HO and 240-fold more potent than D Phe. Since D-PLA is known to be present in LAB-fermented foods such as Sauerkraut, we investigated whether D-PLA is absorbed and enters the blood circulation after oral ingestion of 100 mg D-PLA or Sauerkraut (5-6 g per kg body weight), respectively. Both, ingestion of the pure compound and of Sauerkraut resulted in a significant increase of D-PLA in the plasma post-prandial resulting in concentrations sufficiently high to activate HCA3. To examine HCA3 signaling and the involved components in more detail, we used several inhibitors of internalization and signaling components. The goal was to examine whether there are differences in the activation of signaling pathways, recruitment of signaling components, internalization behavior and endocytic routes of HCA3 and GPR84 in response to 3HO vs 3HDec and decanoic acid (C10) vs 3HDec respectively. Initial analyses of the signaling kinetics of the two receptors, using dynamic mass redistribution (DMR) measurements, indicated differences between the two respective agonists for both HCA3 and GPR84. DMR measurements allow for a time-resolved recording of the activated signaling cascades, independent of the activated pathways. Additional use of pertussis toxin to inhibit Gαi proteins and dynasore to block dynamin-2 function revealed that signaling of both receptors induced by both respective agonists was completely dependent on G protein activation, but differentially dependent on dynamin-2 function suggesting differences in desensitization and internalization mechanisms. Using cAMP inhibition and ERK1/2 activation assays, we further investigated the role of internalization for HCA3 and GPR84 signal transduction. Interestingly, ERK1/2 activation downstream of both receptors was strongly reduced when internalization was inhibited, while cAMP inhibitory signaling of HCA3 induced by both 3HO and 3HDec and GPR84 signaling induced by C10 were significantly reduced by blocking dynamin-2 function but not internalization in general. 3HDec-induced cAMP inhibition downstream of GPR84 was completely insensitive to inhibition of both. Further experiments using dominant negative dynamin-2 mutants verified dependence of HCA3-mediated Gαi and ERK1/2 signaling on dynamin-2 function. Moreover, qualitative analysis of confocal images revealed that subcellular distribution of HCA3 but not GPR84 is altered when dynamin-2 function is impaired. Interestingly, analysis of β-arrestin 2 recruitment by a luminescence-based assay (DiscoverX PathHunter) and imaging of HEK293T cells expressing mRuby-tagged receptor and YFP-tagged β-arrestin 2, showed that only 3HO- but not 3HDec-induced activation of HCA3 leads to recruitment of β-arrestin 2 and subsequent co-localization in intracellular vesicles. GPR84 data suggests that the receptor does not interact with β-arrestin 2 at all. Finally, we also analyzed HCA3 signaling kinetics and β-arrestin 2 recruitment in response to the newly identified agonists D-PLA and L-PLA and the previously known agonist D-Phe. We found signaling kinetics of L-PLA and D-Phe to be similar to 3HDec-induced kinetics, while D-PLA appears to be similar to 3HO. This was further supported by the fact that D-PLA also induced β-arrestin 2 recruitment, while L-PLA and D-Phe did not. Taken together, these findings advanced our understanding of HCA3 and GPR84. The work provides evidence that HCA3 evolved as a signaling system to communicate uptake of fermented food (together with fermenting bacteria) to the immune system. Our data in combination with literature reporting positive, anti-inflammatory properties of D-PLA and LAB in general suggests that at least some of the described positive effects are mediated by HCA3. Furthermore, we showed for the first time biased signaling of these two receptors in response to their natural agonists. Our work increases the knowledge about specific signaling components involved in downstream signaling of the respective receptor in response to the different agonists, potentially linking e.g. activation of HCA3 by 3HO and D-PLA, but not 3HDec and D-Phe, to the inhibition of pro-inflammatory cytokine release through β-arrestin 2 dependent mechanisms. Moreover, 3HDec-induced signaling downstream of GPR84 is very different from that downstream of HCA3. This suggests that 3HDec also triggers different physiological responses in immune cells depending on its local concentration and the expression levels of the two receptors. However, these findings still need to be validated in cells of innate immunity like neutrophils and macrophages that endogenously express both receptors. At last, the physiological consequences such as increased ROS-production, pro-/anti-inflammatory cytokine release, migration, and phagocytosis need to be addressed in future studies to get a better understanding of the function as well as interplay of HCA3 and GPR84 in innate immunity and their suitability as drug targets.

Recently, C-reactive protein (CRP) was shown to affect intracellular calcium signaling and blood pressure in vitro and in vivo, respectively. The aim of the present study was to further investigate if a direct effect on G-protein coupled receptor (GPCR) signaling by CRP can be observed by using CRP in combination with different GPCR agonists on spontaneously beating cultured neonatal rat cardiomyocytes. All used agonists (isoprenaline, clenbuterol, phenylephrine, angiotensin II and endothelin 1) affected the beat rate of cardiomyocytes significantly and after washing them out and re-stimulation the cells developed a pronounced desensitization of the corresponding receptors. CRP did not affect the basal beating-rate nor the initial increase/decrease in beat-rate triggered by different agonists. However, CRP co-incubated cells did not exhibit desensitization of the respective GPCRs after the stimulation with the different agonists. This lack of desensitization was independent of the GPCR type, but it was dependent on the CRP concentration. Therefore, CRP interferes with the desensitization of GPCRs and has to be considered as a novel regulator of adrenergic, angiotensin-1 and endothelin receptors.

No description available.

<p>The following thesis will focus on understanding the downstream behavioral effects of δORmediated β-arrestinsignaling. δORagonists have been implicated as effective targets for a variety of diseases, however detrimental side effects of opioid-targeting agonists limit their clinical use. δORagonists specifically can induce seizures, however the underlying mechanism contributing to this  behavior  is  unknown.  We  review  this  phenomenon  in  more  detail,  highlighting  current agonists known to induce seizures and potential circuits and pathways involved. Our work suggests β-arrestinsignaling  is  involved,  specifically β-arrestin2  mediated  signaling  may  be  largely contributing  to δORagonist-induced  seizure  behavior.  As  it  is  possible  the β-arrestinisoforms have unique roles in seizure behavior, we also analyzed methods in which to provoke β-arrestinisoform bias of δORtargeting compounds. Though the full mechanism relating δORagonists with seizures remains unknown, our work provides foundational detail of this behavior, implicating the importance of β-arrestinisoform signaling through δOR; allowing for future studies to full define this seizure pathway and develop δORsafer agonists.  </p>