TY  - THES
A1  - Perpiñá Viciano, Cristina
T1  - Study of the activation mechanisms of the CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3)
T1  - Untersuchung zum Aktivierungsmechanismus des CXC Chemokin‐Rezeptor 4 (CXCR4) und des atypischen Chemokin‐Rezeptor 3 (ACKR3)
N2  - 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.
N2  - 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.
KW  - G protein-coupled receptors
KW  - Chemokine receptors
KW  - GPCR signaling
KW  - Förster Resonance Energy Transfer
KW  - FRET sensors
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-192371
ER  - 
TY  - JOUR
A1  - Butt, Elke
A1  - Stempfle, Katrin
A1  - Lister, Lorenz
A1  - Wolf, Felix
A1  - Kraft, Marcella
A1  - Herrmann, Andreas B.
A1  - Viciano, Cristina Perpina
A1  - Weber, Christian
A1  - Hochhaus, Andreas
A1  - Ernst, Thomas
A1  - Hoffmann, Carsten
A1  - Zernecke, Alma
A1  - Frietsch, Jochen J.
T1  - Phosphorylation-dependent differences in CXCR4-LASP1-AKT1 interaction between breast cancer and chronic myeloid leukemia
JF  - Cells
N2  - The serine/threonine protein kinase AKT1 is a downstream target of the chemokine receptor 4 (CXCR4), and both proteins play a central role in the modulation of diverse cellular processes, including proliferation and cell survival. While in chronic myeloid leukemia (CML) the CXCR4 is downregulated, thereby promoting the mobilization of progenitor cells into blood, the receptor is highly expressed in breast cancer cells, favoring the migratory capacity of these cells. Recently, the LIM and SH3 domain protein 1 (LASP1) has been described as a novel CXCR4 binding partner and as a promoter of the PI3K/AKT pathway. In this study, we uncovered a direct binding of LASP1, phosphorylated at S146, to both CXCR4 and AKT1, as shown by immunoprecipitation assays, pull-down experiments, and immunohistochemistry data. In contrast, phosphorylation of LASP1 at Y171 abrogated these interactions, suggesting that both LASP1 phospho-forms interact. Finally, findings demonstrating different phosphorylation patterns of LASP1 in breast cancer and chronic myeloid leukemia may have implications for CXCR4 function and tyrosine kinase inhibitor treatment.
KW  - LASP1
KW  - CXCR4
KW  - AKT1
KW  - CML
KW  - breast cancer
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200638
SN  - 2073-4409
VL  - 9
IS  - 2
ER  - 
TY  - JOUR
A1  - Oehler, Beatrice
A1  - Mohammadi, Milad
A1  - Perpina Viciano, Cristina
A1  - Hackel, Dagmar
A1  - Hoffmann, Carsten
A1  - Brack, Alexander
A1  - Rittner, Heike L.
T1  - Peripheral interaction of Resolvin D1 and E1 with opioid receptor antagonists for antinociception in inflammatory pain in rats
JF  - Frontiers in Molecular Neuroscience
N2  - Antinociceptive pathways are activated in the periphery in inflammatory pain, for instance resolvins and opioid peptides. Resolvins are biosynthesized from omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Resolvin D1 (RvD1) and resolvin E1 (RvE1) initiate the resolution of inflammation and control of hypersensitivity via induction of anti-inflammatory signaling cascades. RvD1 binds to lipoxin A4/annexin-A1 receptor/formyl-peptide receptor 2 (ALX/FPR2), RvE1 to chemerin receptor 23 (ChemR23). Antinociception of RvD1 is mediated by interaction with transient receptor potential channels ankyrin 1 (TRPA1). Endogenous opioid peptides are synthesized and released from leukocytes in the tissue and bind to opioid receptors on nociceptor terminals. Here, we further explored peripheral mechanisms of RvD1 and chemerin (Chem), the ligand of ChemR23, in complete Freund’s adjuvant (CFA)-induced hindpaw inflammation in male Wistar rats. RvD1 and Chem ameliorated CFA-induced hypersensitivity in early and late inflammatory phases. This was prevented by peripheral blockade of the μ-opioid peptide receptor (MOR) using low dose local naloxone or by local injection of anti-β-endorphin and anti-met-enkephalin (anti-ENK) antibodies. Naloxone also hindered antinociception by the TRPA1 inhibitor HC-030031. RvD1 did not stimulate the release of β-endorphin from macrophages and neutrophils, nor did RvD1 itself activate G-proteins coupled MOR or initiate β-arrestin recruitment to the membrane. TRPA1 blockade by HC-030031 in inflammation in vivo as well as inhibition of the TRPA1-mediated calcium influx in dorsal root ganglia neurons in vitro was hampered by naloxone. Peripheral application of naloxone alone in vivo already lowered mechanical nociceptive thresholds. Therefore, either a perturbation of the balance of endogenous pro- and antinociceptive mechanisms in early and late inflammation, or an interaction of TRPA1 and opioid receptors weaken the antinociceptive potency of RvD1 and TRPA1 blockers.
KW  - transient receptor potential channels
KW  - pain behavior
KW  - resolvin
KW  - opioid receptors
KW  - opioid peptides
KW  - inflammation
KW  - animals
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158642
VL  - 10
IS  - 242
ER  -