Refine
Has Fulltext
- yes (12)
Is part of the Bibliography
- yes (12)
Document Type
- Doctoral Thesis (12)
Keywords
- G-Protein gekoppelte Rezeptoren (12) (remove)
Institute
- Graduate School of Life Sciences (12) (remove)
Whereas G-protein coupled receptors (GPCRs) have been long believed to signal through cyclic AMP exclusively at cell surface, our group has previously shown that GPCRs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent cAMP production. This phenomenon, which we originally described for the thyroid stimulating hormone receptor (TSHR) in thyroid cells, has been observed also for other GPCRs. However, the intracellular compartment(s) responsible for such persistent signaling and its consequences on downstream effectors were insufficiently characterized. The aim of this study was to follow by live-cell imaging the trafficking of internalized TSHRs and other involved signaling proteins as well as to understand the consequences of signaling by internalized TSHRs on the downstream activation of protein kinase A (PKA). cAMP and PKA
activity was measured in real-time in living thyroid cells using FRET-based sensors Epac1-camp and AKAR2 respectively. The results suggest that TSH co-internalizes with its receptor and that the internalized TSH/TSHR complexes traffic retrogradely to the trans-Golgi network (TGN). This study also provides evidence that these internalized TSH/TSHR complexes meet an intracellular pool of Gs proteins in sorting endosomes and in TGN and activate it there, as visualized in real-time using a conformational biosensor nanobody, Nb37. Acute Brefeldin A-induced Golgi collapse hinders the retrograde trafficking of TSH/TSHR complexes, leading to reduced cAMP production and PKA signaling. BFA pretreatment was also able to attenuate CREB phosphorylation suggesting that an intact Golgi/TGN organisation is essential
for an efficient cAMP/PKA signaling by internalized TSH/TSHR complexes. Taken together this data provides evidence that internalized TSH/TSHR complexes meet and activate Gs proteins in sorting endosomes and at the TGN, leading to a local activation of PKA and consequently increased CREB activation. These findings suggest unexpected functions for receptor internalization, with major pathophysiological and pharmacological implications.
Der Fluoreszenz-Resonanz-Energie-Transfer ist ein Phänomen, welches erstmals 1948 von Theodor Förster beschrieben wurde. Mit der Entwicklung von Fluoreszenzproteinen konnten in Kombination mit Mikroskopietechniken Einblicke in zellbiologische Vorgänge gewonnen werden, die durch biochemische oder physiologische Experimente nicht möglich sind. Dabei spielt die hohe zeitliche und räumliche Auflösung eine wichtige Rolle. Auf dem Forschungsgebiet der GPCR, welche die größte Gruppe von Membranproteinen bei den Säugetieren darstellen, wurden insbesondere Erkenntnisse über Konformationsänderungen der Rezeptoren, die Kinetik der Rezeptoraktivierung und die Interaktion mit intrazellulären Signalproteinen gewonnen. Der µ-Opioidrezeptor gehört zur Familie der GPCR und stellt aufgrund seiner analgetischen Wirkungen eine wichtige pharmakologische Zielstruktur dar. Das Ziel dieser Arbeit war sowohl den Rezeptor als auch seine Signalwege mittels FRET-Mikroskopie zu untersuchen. Zunächst sollte ein intramolekularer FRET-Sensor des µ-Opioidrezeptors entwickelt werden, dazu wurden basierend auf den Kenntnissen über die Tertiärstruktur und dem Aufbau bereits bekannter GPCR-Sensoren verschiedene Rezeptorkonstrukte kloniert. Bei den Konstrukten wurden entweder zwei Fluoreszenzproteine oder ein Fluoreszenzprotein und ein Fluorophor-bindendes Tetracysteinmotiv kombiniert. Auch die Positionen der eingefügten Sequenzen wurden in den intrazellulären Domänen variiert, da der Rezeptor auf die Modifikationen mit beeinträchtigter Membranlokalisation reagierte. Durch die Optimierung wurden Rezeptoren konstruiert, die an der Zellmembran lokalisiert waren. Jedoch zeigte keines der Rezeptorkonstrukte Funktionalität im Hinblick auf die Rezeptoraktivierung. Im zweiten Teil wurden die pharmakologischen Effekte der Metabolite von Morphin am humanen µ-Opioidrezeptor systematisch analysiert. Dazu wurde die Fähigkeit der Metabolite, Gi-Proteine zu aktivieren und β-Arrestin2 zu rekrutieren, mittels FRET-basierter Messungen an lebenden Zellen untersucht. Außerdem wurde die Affinität der Metabolite zum humanen µ Opioidrezeptor anhand der Verdrängung eines radioaktiven Liganden analysiert. Meine Experimente identifizierten eine Gruppe mit stark agonistischen und eine mit schwach agonistischen Eigenschaften. Die starken Partialagonisten aktivieren den Rezeptor bereits bei nanomolaren Konzentrationen, während die schwachen Metabolite den Rezeptor erst bei Konzentrationen im mikromolaren Bereich aktivieren. Die Metabolite Normorphin, Morphin-6-Glucuronid und 6-Acetylmorphin zeigen geringere Potenz als Morphin bei der Gi-Aktivierung aber überraschenderweise höhere Potenz und Effizienz für die β-Arrestin-Rekrutierung. Dies deutet auf eine bevorzugte Aktivierung von β-Arrestin2 hin. Die aus diesen Studien gewonnenen Ergebnisse liefern Hinweise darauf, welche Metabolite bei der Signalverarbeitung am µ Opioidrezeptor in vivo beteiligt sind.
Adenosine receptors that belong to the rhodopsin-like G protein-coupled receptors (GPCRs) are involved in a lot of regulatory processes and are widely distributed throughout the body which makes them an attractive target for drugs. However, pharmacological knowledge of these receptors is still limited. A big advance regarding the structural knowledge of adenosine receptors was the development of the first crystal structure of the adenosine A2A receptor in 2008. The crystal structure revealed the amino acids that form the ligand binding pocket of the receptor and depicted the endpoint of receptor movement in the ligand binding process. Within the scope of this work two members of the adenosine receptor family were investigated, namely the adenosine A1 and the A2A receptor (A1R, A2AR). A1R was generated on base of the previously developed A2AR. Receptors were tagged with fluorophores, with the cyan fluorescent protein (CFP) at the C-terminal end of receptor and the Fluorescein Arsenical Hairpin binder (FlAsH) binding sequence within the third intracellular loop of receptors. Resulting fluorescent receptor sensors
A1 Fl3 CFP and A2A Fl3 CFP were investigated with help of Fluorescence Resonance Energy Transfer (FRET) measurements within living cells. FRET experiments enable the examination of alteration in the distance of two fluorophores and thus the observation of receptor dynamical movements.
For comparison of A1R and A2AR regarding receptor dynamical movement upon ligand binding, fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were superfused with various ligands and the outcomes of FRET experiments were compared regarding signal height of FRET ratio evoked by the distinct ligand that is correlated to the conformational change of receptor upon ligand binding. Beside the different direction of FRET ratio upon ligand binding at A1R and A2AR sensor, there were differences observable when signal height and association and dissociation kinetics of the various ligands investigated were compared to each other. Differences between the adenosine receptor subtypes were especially remarkable for the A1R subtype selective agonist CPA and the A2AR subtype selective agonist CGS 21680. Another part of the project was to investigate the influence of single amino acids in the ligand binding process within the fluorescent A1R sensor. Amino acid positions were derived from the crystal structure of the A2AR forming the ligand binding pocket and these amino acids were mutated in the A1R structure. Investigation of the A1R sensor and its mutants regarding confocal analysis showed involvement
of some amino acids in receptor localization. When these amino acids were mutated receptors were not expressed in the plasma membrane of cells. Some amino acids investigated were found to be involved in the ligand binding process in general whereas other amino acids were found to have an influence on the binding of distinct structural groups of the ligands investigated. In a further step, A1R and A2AR were N-terminally tagged with SNAP or CLIP which allowed to label receptor sensors with multiple fluorophores. With this technique receptor distribution in cells could be investigated with help of confocal analysis. Furthermore, ligand binding with fluorescent adenosine receptor ligands and their competition with help of a non-fluorescent antagonist was examined at the SNAP tagged A1R and A2AR. Finally the previously developed receptor sensors were combined to the triple labeled receptor sensors SNAP A1 Fl3 CFP and SNAP A2A Fl3 CFP which were functional regarding FRET experiments and plasma membrane expression was confirmed via confocal analysis. In the future, with the help of this technique, interaction between fluorescent ligand and SNAP tagged receptor can be monitored simultaneously with the receptor movement that is indicated by the distance alteration between FlAsH and CFP. This can
lead to a better understanding of receptor function and its dynamical movement upon ligand binding which may contribute to the development of new and more specific drugs for the A1R and A2AR in the future.
RKIP reguliert Proteinkinasen der Signaltransduktionskaskaden von G Protein-gekoppelten Rezeptoren, der Raf/MEK/ERK-MAPK, des Transkriptionsfaktors NFκB und von GSK3β. Unklar war bisher, wie die spezifische Interaktion von RKIP mit seinen mannigfaltigen Interaktionspartnern ermöglicht und reguliert wird. Raf1 und GRK2 sind die einzigen bekannten direkten Interaktionspartner von RKIP und wurden deshalb gewählt, um die zugrundeliegenden molekularen Mechanismen dieser Interaktion genauer zu untersuchen. In dieser Arbeit wurde gezeigt, dass RKIP nach PKC-vermittelter Phosphorylierung von Serin153 dimerisiert und dass diese Dimerisierung für die RKIP/Raf1-Dissoziation und die RKIP/GRK2-Interaktion essentiell ist. Co-Immunpräzipitationsexperimente mit einer phosphorylierungsdefizienten Mutante zeigten, dass für diese Dimerisierung die Phosphorylierung von beiden RKIP-Molekülen notwendig ist. Als Dimerinteraktionsfläche wurden die Aminosäuren 127-146 von RKIP identifiziert, da das Peptid RKIP127-146 die Dimerisierung von RKIP spezifisch und effizient hemmte. Um die Bedeutung dieser phosphorylierungsinduzierten Dimerisierung von RKIP für seine Interaktion mit Raf1 und GRK2 zu untersuchen, wurden eine phosphomimetische Mutante (RKIPSK153/7EE) und eine Mutante von RKIP generiert, welche bereits unphosphoryliert dimerisiert (RKIP∆143-6). Folgende Ergebnisse legen nahe, dass die Dimerisierung von RKIP für die spezifische Interaktion mit Raf1 bzw. GRK2 entscheidend ist: (i) Die Dimerisierung von phosphoryliertem RKIP ging mit der Dissoziation von RKIP und Raf1 und der Assoziation von RKIP und GRK2 einher; (ii) die Mutanten RKIPSK153/7EE und RKIP∆143-6, die bereits in unstimulierten Zellen eine starke Dimerisierung zeigten, hatten eine höhere Affinität zu GRK2 als zu Raf1; (iii) die Hemmung der RKIP-Dimerisierung interferierte nur mit der RKIP/GKR2- aber nicht mit der RKIP/Raf1-Interaktion; (iv) in vitro und in Mausherzen konnte ein RKIP- und GRK2-immunreaktiver Komplex nachgewiesen werden; (v) Untersuchungen zur RKIP-vermittelten Hemmung der Kinaseaktivität von GRK2 und Raf implizierten, dass dimerisiertes RKIP nur die Aktivität von GRK2, nicht aber von Raf hemmt. Diese Arbeit zeigt, dass die phosphorylierungsinduzierte Dimerisierung von RKIP die spezifische Interaktion von RKIP mit Raf1 und GRK2 koordiniert. Die Aufklärung dieses Mechanismus erweitert unser Verständnis der spezifischen Interaktion von Kinasen mit ihren Regulatorproteinen.
The superfamily of G protein-coupled receptors (GPCR) regulates numerous physiological and pathophysiological processes. Hence GPCRs are of significant interest for pharmacological therapy. Embedded into cytoplasmic membranes, GPCRs represent the core of large signaling complexes, which are critical for transduction of exogenous stimuli towards activation of downstream signaling pathways. As a member of the GPCR family B, the parathyroid hormone receptor (PTHR) activates adenylyl cyclases, phospholipases C β as well as mitogen-activated protein kinase-dependent signaling pathways, thereby mediating endocrine and paracrine effects of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), respectively. This regulates, calcium homeostasis, bone metabolism and bone development. Paradoxically, PTH is able to induce both catabolic and anabolic bone metabolism. The anabolic effect of PTH is successfully applied in the therapy of severe osteoporosis. Domination of anabolic or catabolic bone-metabolism is entailed by temporal and cell-type specific determinants. The molecular bases are presumably differential arrangements of adaptor proteins within large signaling complexes that may lead to differential activation of signaling pathways, thereby regulating physiological effects. The molecular mechanisms are largely unclear; thus, there is significant interest in revealing a better understanding of PTHR-related adaptor proteins. To identify novel adaptor proteins which direct PTHR signaling pathways, a proteomic screening approach was developed. In this screening, vav2, a guanine-nucleotide exchange factor (GEF) for small GTPases which regulates cytoskeleton reorganization, was found to interact with intracellular domains of PTHR. Evidence is provided that vav2 impairs PTH-mediated phospholipase C β (PLCβ) signaling pathways by competitive interactions with G protein αq subunits. Vice versa, PTH was shown to regulate phosphorylation and subsequent GEF activity of vav2. These findings may thus shed new light on the molecular mechanisms underlying the effects of PTH on bone metabolism by PLC-signaling, cell migration and cytoskeleton organization. In addition to the understanding of intracellular molecular signaling processes, screening for ligands is a fundamental and demanding prerequisite for modern drug development. To this end, ligand binding assays represent a fundamental technique. As a substitution for expensive and potentially harmful radioligand binding, fluorescence-based ligand-binding assays for PTHR were developed in this work. Based on time-resolved fluorescence, several assay variants were established to facilitate drug development for the PTHR.
Differenzierte β-Arrestin2 Rekrutierung am μ-Opioid Rezeptor durch klinisch eingesetzte Opioide
(2021)
Opioide gehören zu den potentesten Analgetika für die Behandlung akuter und chronischer Schmerzen, werden jedoch in ihrer Anwendung durch analgetische Toleranz aber auch Nebenwirkungen wie Abhängigkeit, Atemdepression und Obstipation limitiert. Opioid-Analgetika vermitteln dabei nahezu alle klinisch relevanten Wirkungen durch Stimulation des μ-Opioidrezeptors, einem G- Protein-gekoppelten Rezeptor. Die „klassische“ Signaltransduktion durch Aktivierung inhibitorischer Gi/0-Proteine kann durch G-Protein gekoppelte Rezeptorkinasen (GRKs) und β-Arrestine negativ reguliert werden. Zusätzlich können durch β-Arrestin-Bindung an den Rezeptor G-Protein-unabhängige Signalwege aktiviert werden. Die genauen Mechanismen wie β-Arrestin- assoziierte Rezeptordesensibilisierung, -internalisierung und G-Protein- unabhängige Signalwege an der physiologischen Antwort und insbesondere an Toleranzentwicklung und Abhängigkeit von Opioid-Analgetika beteiligt sind, können bislang nicht ausreichend erklärt werden.
In dieser Arbeit konnte in HEK293-Zellen mit Lebendzell-Konfokalmikroskopie und Luciferase-Komplementierung für 17 Opioide eine differenzierte β-Arrestin2- Rekrutierung zum μ-Opioidrezeptor gezeigt werden. Von den untersuchten Opioiden sind 13 häufig eingesetzte Opioid-Analgetika. Durch die Erstellung detaillierter pharmakologischer Profile ließen sich die Opioide bezüglich ihres β- Arrestin2-Rekrutierungsvermögens in Voll-, Partial und Antagonisten eingruppieren. Bemerkenswert war die fehlende β-Arrestin2-Rekrutierung für Buprenorphin, Tramadol und Tilidin, sodass diese interessante Substanzen für weitere Untersuchungen in physiologischerem Kontext sind. Durch Überexpression von GRK2 konnte die β-Arrestin2-Rekrutierung insbesondere für Partialagonisten gesteigert werden, was die Abhängigkeit der β-Arrestin- Rekrutierung vom GRK-Expressionslevel, das in verschiedenen Assays und Gewebetypen variieren kann, zeigt. Außerdem konnte ein heterogenes Bild der Rezeptorregulierung demonstriert werden, welches indirekt durch Endozytosehemmung unter Verwendung von Dynamin-Inhibitoren erfasst wurde. Die erhobenen Daten dienen als Anknüpfungspunkt für weiteren Arbeiten auf dem Gebiet der μ-Opioidrezeptorregulation. Ein besseres Verständnis der molekularen Mechanismen ist nötig, um sichere und nebenwirkungsärmere Opioid-Analgetika entwickeln zu können.
G protein-coupled receptors (GPCRs) form the biggest receptor family that is encoded in the human genome and represent the most druggable target structure for modern therapeutics respectively future drug development. Belonging to aminergic class A GPCRs muscarinic Acetylcholine receptors (mAChRs) are already now of clinical relevance and are also seen as promising future drug targets for treating neurodegenerative diseases like Alzheimer or Parkinson. The mAChR family consist of five subtypes showing high sequence identity for the endogenous ligand binding region and thus it is challenging until now to selectively activate a single receptor subtype. A well accepted method to study ligand binding, dynamic receptor activation and downstream signaling is the fluorescence resonance energy transfer (FRET) application. Here, there relative distance between two fluorophores in close proximity (<10 nm) can be monitored in a dynamic manner. The perquisite for that is the spectral overlap of the emission spectrum of the first fluorophore with the excitation spectrum of the second fluorophore. By inserting two fluorophores into the molecular receptor structure receptor FRET sensors can serve as a powerful tool to study dynamic receptor pharmacology.
Dualsteric Ligands consist of two different pharmacophoric entities and are regarded as a promising ligand design for future drug development. The orthosteric part interacts with high affinity with the endogenous ligand binding region whereas the allosteric part binds to a different receptor region mostly located in the extracellular vestibule. Both moieties are covalently linked. Dualsteric ligands exhibit a dynamic ligand binding. The dualsteric binding position is characterized by a simultaneous binding of the orthosteric and allosteric moiety to the receptor and thus by receptor activation. In the purely allosteric binding position no receptor activation can be monitored.
In the present work the first receptor FRET sensor for the muscarinic subtype 1 (M1) was generated and characterized. The M1-I3N-CFP sensor showed an unaltered physiological behavior as well as ligand and concentration dependent responses. The sensor was used to characterize different sets of dualsteric ligands concerning their pharmacological properties like receptor activation. It was shown that the hybrids consisting of the synthetic full agonist iperoxo and the positive allosteric modulator of BQCA type is very promising. Furthermore, it was shown for orthosteric as well as dualsteric ligands that the degree of receptor activation is highly dependent on the length of and the chemical properties of the linker moiety. For dualsteric ligands a bell-shaped activation characteristic was reported for the first time, suggesting that there is an optimal linker length for dualsteric ligands. The gained knowledge about hybrid design was then used to generate and characterize the first photo-switchable dualsteric ligand. The resulting hybrids were characterized with the M1-I3N-CFP sensor and were described as photo-inactivatable and dimmable. In addition to the ligand characterization the ligand application methodology was further developed and improved. Thus, a fragment-based screening approach for dualsteric ligands was reported in this study for the first time. With this approach it is possible to investigate dualsteric ligands in greater detail by applying either single ligand fragments alone or in a mixture of building blocks. These studies revealed the insights that the effect of dualsteric ligands on a GPCR can be rebuild by applying the single building blocks simultaneously. The fragment-based screening provides high potential for the molecular understanding of dualsteric ligands and for future screening approaches. Next, a further development of the standard procedure for measuring FRET by sensitized emission was performed. Under normal conditions single cell FRET is measured on glass coverslips. After coating the coverslips surface with a 20 nm thick gold layer an increased FRET efficiency up to 60 % could be reported. This finding was validated in different approaches und in different configurations. This FRET enhancement by plasmonic surfaces was until yet unreported in the literature for physiological systems and make FRET for future projects even more powerful.
To this day, opioids represent the most effective class of drugs for the treatment of severe pain. On a molecular level, all opioids in use today are agonists at the μ-opioid receptor (μ receptor). The μ receptor is a class A G protein-coupled receptor (GPCR). GPCRs are among the biological structures most frequently targeted by pharmaceuticals. They are membrane bound receptors, which confer their signals into the cell primarily by activating a variety of GTPases called G proteins. In the course of the signaling process, the μ receptor will be phosphorylated by GRKs, increasing its affinity for another entity of signaling proteins called β-arrestins (β-arrs). The binding of a β-arr to the activated μ receptor will end the G protein signal and cause the receptor to be internalized into the cell. Past research showed that the μ receptor’s G protein signal puts into effect the desired pain relieving properties of opioid drugs, whereas β-arr recruitment is more often linked to adverse effects like obstipation, tolerance, and respiratory depression. Recent work in academic and industrial research picked up on these findings and looked into the possibility of enhancing G protein signaling while suppressing β-arr recruitment. The conceptual groundwork of such approaches is the phenomenon of biased agonism. It appreciates the fact that different ligands can change the relative contribution of any given pathway to the overall downstream signaling, thus enabling not only receptor-specific but even pathway-specific signaling.
This work examined the ability of a variety of common opioid drugs to specifically activate the different signaling pathways and quantify it by means of resonance energy transfer and protein complementation experiments in living cells. Phosphorylation of the activated receptor is a central step in the canonical GPCR signaling process. Therefore, in a second step, expression levels of the phosphorylating GRKs were enhanced in search for possible effects on receptor signaling and ligand bias.
In short, detailed pharmacological profiles of 17 opioid ligands were recorded. Comparison with known clinical properties of the compounds showed robust correlation of G protein activation efficacy and analgesic potency. Ligand bias (i.e. significant preference of any path- way over another by a given agonist) was found for a number of opioids in native HEK293 cells overexpressing μ receptor and β-arrs. Furthermore, overexpression of GRK2 was shown to fundamentally
change β-arr pharmacodynamics of nearly all opioids. As a consequence, any ligand bias as detected earlier was abolished with GRK2 overexpression, with the exception of buprenorhin. In summary, the following key findings stand out: (1) Common opioid drugs exert biased agonism at the μ receptor to a small extent. (2) Ligand bias is influenced by expression levels of GRK2, which may vary between individuals, target tissues or even over time. (3) One of the opioids, buprenorhin, did not change its signaling properties with the overexpression of GRK2. This might serve as a starting point for the development of new opioids which could lack the ability of β-arr recruitment altogether and thus might help reduce adverse side effects in the treatment of severe pain.
G-protein-coupled receptors (GPCRs) regulate diverse physiological processes in the human body and represent prime targets in modern drug discovery. Engagement of different ligands to these membrane-embedded proteins evokes distinct receptor conformational rearrangements that facilitate subsequent receptor-mediated signalling and, ultimately, enable cellular adaptation to altered environmental conditions. Since the early 2000s, the technology of resonance energy transfer (RET) has been exploited to assess these conformational receptor dynamics in living cells and real time. However, to date, these conformational GPCR studies are restricted to single-cell microscopic setups, slowing down the discovery of novel GPCR-directed therapeutics. In this work, we present the development of a novel generalizable high-throughput compatible assay for the direct measurement of GPCR activation and deactivation. By screening a variety of energy partners for fluorescence (FRET) and bioluminescence resonance energy transfer (BRET), we identified a highly sensitive design for an α2A-adrenergic receptor conformational biosensor. This biosensor reports the receptor’s conformational change upon ligand binding in a 96-well plate reader format with the highest signal amplitude obtained so far. We demonstrate the capacity of this sensor prototype to faithfully quantify efficacy and potency of GPCR ligands in intact cells and real time. Furthermore, we confirm its universal applicability by cloning and validating five further equivalent GPCR biosensors. To prove the suitability of this new GPCR assay for screening purposes, we measured the well-accepted Z-factor as a parameter for the assay quality. All tested biosensors show excellent Z-factors indicating outstanding assay quality. Furthermore, we demonstrate that this assay provides excellent throughput and presents low rates of erroneous hit identification (false positives and false negatives). Following this phase of assay development, we utilized these biosensors to understand the mechanism and consequences of the postulated modulation of parathyroid hormone receptor 1 (PTHR1) through receptor activity-modifying protein 2 (RAMP2). We found that RAMP2 desensitizes PTHR1, but not the β2-adrenergic receptor (β2AR), for agonist-induced structural changes. This generalizable sensor design offers the first possibility to upscale conformational GPCR studies, which represents the most direct and unbiased approach to monitor receptor activation and deactivation. Therefore, this novel technology provides substantial advantages over currently established methods for GPCR ligand screening. We feel confident that this technology will aid the discovery of novel types of GPCR ligands, help to identify the endogenous ligands of so-called orphan GPCRs and deepen our understanding of the physiological regulation of GPCR function.
Fluorescence microscopy has become one of the most important techniques for the imaging of biological cells and tissue, since the technique allows for selective labeling with fluorescent molecules and is highly suitable for low-light applications down to the single molecule regime. The methodological requirements are well-defined for studying membrane receptors within a highly localized nanometer-thin membrane. For example, G-protein-coupled receptors (GPCRs) are an extensively studied class of membrane receptors that represent one of the most important pharmaceutical targets. Ligand binding and GPCR activation dynamics are suspected to take place at the millisecond scale and may even be far faster. Thus, techniques that are fast, selective, and live-cell compatible are required to monitor GPCR dynamics. Fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence microscopy (TIRF-M) are methods of choice to monitor the dynamics of GPCRs selectively within the cell membrane.
Despite the remarkable success of these modalities, there are limitations. Most importantly, inhomogeneous illumination can induce imaging artifacts, rendering spectroscopic evaluation difficult. Background signal due to scattering processes or imperfect labeling can hamper the signal-to-noise, thus limiting image contrast and acquisition speed. Careful consideration of the internal physiology is required for FRET sensor design, so that ligand binding and cell compatibility are well-preserved despite the fluorescence labeling procedures. This limitation of labeling positions leads to very low signal changes in FRET-based GPCR analysis. In addition, microscopy of these systems becomes even more challenging in single molecule or low-light applications where the accuracy and temporal resolution may become dramatically low. Fluorescent labels should therefore be brighter, protected from photobleaching, and as small as possible to avoid interference with the binding kinetics. The development of new fluorescent molecules and labeling methods is an ongoing process. However, a complete characterization of new labels and sensors takes time. So far, the perfect dye system for GPCR studies has not been found, even though there is high demand.
Thus, this thesis explores and applies a different approach based on improved illumination schemes for TIRF-M as well as metal-coated coverslips to enhance fluorescence and FRET efficiency. First, it is demonstrated that a 360° illumination scheme reduces typical TIRF artifacts and produces a much more homogenously illuminated field of view. Second, membrane imaging and FRET spectroscopy are improved by metal coatings that are used to modulate the fluorescent properties of common fluorescent dyes. Computer simulation methods are used to understand the underlying photophysics and to design the coatings. Third, this thesis explores the operational regime and limitations of plasmonic approaches with high sectioning capabilities. The findings are summarized by three publications that are presented in the results section of this work. In addition, the theory of fluorescence and FRET is explained, with particular attention to its emission modulations in the vicinity of metal-dielectric layers. Details of the instrumentation, computer simulations, and cell culture are described in the method section. The work concludes with a discussion of the findings within the framework of recent technological developments as well as perspectives and suggestions for future approaches complete the presented work.