4 research outputs found
Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists
Funding: This research was funded by European Unionâs Horizon2020 Marie SkĆodowska-Curie Actions (MSCA) Program under Grant Agreement 641833 (ONCORNET) and European Cooperation in Science and Technology (COST) Action CA18133 European Research Network on Signal Transduction (ERNEST). A. Inoue was funded by the Leading Advanced Projects for Medical Innovation (LEAP) JP19gm0010004 from the Japan Agency for Medical Research and Development.Although class A G proteinâcoupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomerâdimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.Publisher PDFPeer reviewe
Untersuchung zum Aktivierungsmechanismus des CXC ChemokinâRezeptor 4 (CXCR4) und des atypischen ChemokinâRezeptor 3 (ACKR3)
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 ÎČâarrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via ÎČâarrestinâdependent 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)âbased 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 wildâtype counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RETâbased receptor sensors in the field of chemokine receptors. Additional FRETâbased 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 ligandâinduced 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 CXCL12âinduced 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 ChemokinâRezeptor 4 (CXCR4) und der atypische ChemokinâRezeptor 3 (ACKR3) sind heptatransmembranĂ€re
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 fĂŒhrt zu kanonischer GPCR Signaltransduktion ĂŒber GiâProteine und ÎČâArrestine. Die Signalweiterleitung des Rezeptors ACKR3 hingegen, welcher hauptsĂ€chlich in intrazellulĂ€ren Vesikeln vorliegt, erfolgt ĂŒber ĂâArrestinabhĂ€ngige Signalwege. Es ist von groĂer Wichtigkeit die Dynamik und Kinetik dieser beiden Rezeptoren hinsichtlich der Aktivierung durch ihre Liganden und der Signalweiterleitung zu verstehen. In dieser Arbeit wurden verschiedene FörsterâResonanzenergietransfer (FRET) Anwendungen kombiniert, um
die frĂŒhen Phasen der SignalâKaskade von CXCR4 und ACKR3 zu untersuchen. Zur genaueren AufklĂ€rung der Rezeptoraktivierung wurden intramolekulare FRETâSensoren entwickelt, hierzu wurden die Fluorophore Cyanâfluoreszierendes Protein und engl. fluorescence arsenical hairpin binder verwendet. Die generierten Sensoren zeigten Ă€hnliche funktionelle Eigenschaften wie die
unverĂ€nderten Rezeptoren. Ligandenâinduzierte Ănderungen der Rezeptorkonformation können mittels dieser Sensoren beobachtet werden und stellen die ersten RETâbasierten Sensoren auf dem Forschungsgebiet der ChemokinâRezeptoren dar. Weitere FRETâbasierte Methoden wurden zur
Untersuchung von Interaktionen zwischen Rezeptor und GâProtein, Neuanordnung von Dimeren, sowie der GâProtein Aktivierung eingesetzt und fĂŒr beide ChemokinâRezeptoren etabliert. CXCR4 zeigte einen komplexen Aktivierungsmechanismus nach Stimulation durch CXCL12, bei welchem zunĂ€chst eine Neuordnung der RezeptorâTransmembrandomĂ€ne gefolgt von Neuordnungen zwischen
Rezeptor und GâProtein und zuletzt eine Neuordnung zwischen CXCR4 Protomeren erfolgte. Dies
impliziert, dass im Aktivierungsprozess des Rezeptors Homodimere eine Rolle spielen. Zudem wurde
eine verlĂ€ngerte Gi âProtein Aktivierung gegenĂŒber der GqâProtein Aktivierung bei CXCL12 stimuliertem CXCR4 beobachtet. Hingegen zeigte eine Stimulierung mit dem Macrophage Migration Inhibitory Factor (MIF) bei jedem Schritt der frĂŒhen SingalâKaskade verĂ€nderte Dynamiken und
Kinetiken im Vergleich zu CXCL12. DarĂŒber hinaus konnte keine Gi âProtein Aktivierung festgestellt werden. Dieser Befund zeigt individuelle Mechanismen fĂŒr MIF und CXCL12 am CXCR4âRezeptor und liefert Belege fĂŒr eine neuer Art von sequenziellen Signalweiterleitungen an GPCRs. Eine wichtige Beobachtung dieser Arbeit fĂŒr eine potentielle Medikamentenentwicklung ist das CXCR4 ligandenunabhĂ€ngige
AktivitĂ€t zeigt. Um die Aktivierung des ACKR3 Sensors messen zu können wurde durch eine CoâTransfektion mit K44A Dynamin eine höhere MembranstĂ€ndigkeit erreicht. CXCL12 und drei weiteren Agonisten zeigten am hier entwickelten ACKR3âSensor unterscheidbare Kinetiken. Mit diesem wertvollen Werkzeug können Liganden an diesem atypischen Rezeptor pharmakologisch charakterisiert werden. Es konnte keine CXCL12âinduzierte GâProtein Aktivierung gemessen werden, trotz der stĂ€rkeren PrĂ€senz an der Plasmamembran mit Hilfe der DynaminâMutante. In Summe liefert
diese Arbeit zum ersten Mal eine zeitliche Auflösung von Signalweiterleitungsmustern von zwei
ChemokinâRezeptoren sowie wertvolle Werkzeuge zur Charakterisierung der frĂŒhen Phase der SignalâKaskade durch andere pharmakologisch relevanten Liganden
Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists
Although class A G protein-coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer-dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4