CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes

© 2020 The Authors G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of β-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels

Modulators of CXCR4 and CXCR7/ACKR3 Function

Copyright © 2019 by The Author(s). The two G protein-coupled receptors (GPCRs) C-X-C chemokine receptor type 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are part of the class A chemokine GPCR family and represent important drug targets for human immunodeficiency virus (HIV) infection, cancer, and inflammation diseases. CXCR4 is one of only three chemokine receptors with a US Food and Drug Administration approved therapeutic agent, the small-molecule modulator AMD3100. In this review, known modulators of the two receptors are discussed in detail. Initially, the structural relationship between receptors and ligands is reviewed on the basis of common structural motifs and available crystal structures. To date, no atypical chemokine receptor has been crystallized, which makes ligand design and predictions for these receptors more difficult. Next, the selectivity, receptor activation, and the resulting ligand-induced signaling output of chemokines and other peptide ligands are reviewed. Binding of pepducins, a class of lipid-peptides whose basis is the internal loop of a GPCR, to CXCR4 is also discussed. Finally, small-molecule modulators of CXCR4 and ACKR3 are reviewed. These modulators have led to the development of radio- and fluorescently labeled tool compounds, enabling the visualization of ligand binding and receptor characterization both in vitro and in vivo. SIGNIFICANCE STATEMENT: To investigate the pharmacological modulation of CXCR4 and ACKR3, significant effort has been focused on the discovery and development of a range of ligands, including small-molecule modulators, pepducins, and synthetic peptides. Imaging tools, such as fluorescent probes, also play a pivotal role in the field of drug discovery. This review aims to provide an overview of the aforementioned modulators that facilitate the study of CXCR4 and ACKR3 receptors

Modulation of the Allosteric Communication between the Polo-Box Domain and the Catalytic Domain in Plk1 by Small Compounds

The Polo-like kinases (Plks) are an evolutionary conserved family of Ser/Thr protein kinases that possess, in addition to the classical kinase domain at the N-terminus, a C-terminal polo-box domain (PBD) that binds to phosphorylated proteins and modulates the kinase activity and its localization. Plk1, which regulates the formation of the mitotic spindle, has emerged as a validated drug target for the treatment of cancer, because it is required for numerous types of cancer cells but not for the cell division in noncancer cells. Here, we employed chemical biology methods to investigate the allosteric communication between the PBD and the catalytic domain of Plk1. We identified small compounds that bind to the catalytic domain and inhibit or enhance the interaction of Plk1 with the phosphorylated peptide PoloBoxtide in vitro. In cells, two new allosteric Plk1 inhibitors affected the proliferation of cancer cells in culture and the cell cycle but had distinct phenotypic effects on spindle formation. Both compounds inhibited Plk1 signaling, indicating that they specifically act on Plk1 in cultured cells.Fil: Raab, Monika. Goethe Universitat Frankfurt; AlemaniaFil: Sanhaji, Mourad. Goethe Universitat Frankfurt; AlemaniaFil: Pietsch, Larissa. German Cancer Research Center; Alemania. Goethe Universitat Frankfurt; AlemaniaFil: Béquignon, Isabelle. Goethe Universitat Frankfurt; AlemaniaFil: Herbrand, Amanda K.. Goethe Universitat Frankfurt; AlemaniaFil: Süß, Evelyn. Goethe Universitat Frankfurt; AlemaniaFil: Gande, Santosh L.. German Cancer Research Center; Alemania. Goethe Universitat Frankfurt; AlemaniaFil: Caspar, Birgit. Goethe Universitat Frankfurt; AlemaniaFil: Kudlinzki, Denis. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; AlemaniaFil: Saxena, Krishna. Goethe Universitat Frankfurt; AlemaniaFil: Sreeramulu, Sridhar. Goethe Universitat Frankfurt; AlemaniaFil: Schwalbe, Harald. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; AlemaniaFil: Strebhardt, Klaus. Goethe Universitat Frankfurt; Alemania. German Cancer Research Center; AlemaniaFil: Biondi, Ricardo Miguel. German Cancer Research Center; Alemania. Goethe Universitat Frankfurt; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentin

Small Molecule Fluorescent Ligands for the Atypical Chemokine Receptor 3 (ACKR3)

The atypical chemokine receptor 3 (ACKR3) is a receptor that induces cancer progression and metastasis in multiple cell types. Therefore, new chemical tools are required to study the role of ACKR3 in cancer and other diseases. In this study, fluorescent probes, based on a series of small molecule ACKR3 agonists, were synthesized. Three fluorescent probes, which showed specific binding to ACKR3 through a luminescence-based NanoBRET binding assay (pKd ranging from 6.8 to 7.8) are disclosed. Due to their high affinity at the ACKR3, we have shown their application in both competition binding experiments and confocal microscopy studies showing the cellular distribution of this receptor

Investigating the mode of action of intracellular loop 1 pepducins at the CXCR4 receptor

Background Pepducins are lipid-peptides derived from the intracellular loop sequences of a G protein-coupled receptor and have been shown to act as allosteric modulators. Pepducins have been described for the chemokine receptor, CXCR4, which can exhibit agonist activity in the absence of the endogenous ligand C-X-C-Ligand 12 (CXCL12). To date, their precise mode of action is unclear. In this study, we investigated the mechanism of action of intracellular loop 1 pepducins at the CXCR4 receptor Experimental Approach Experiments were performed in HEK293 cells stably expressing the GlosensorTM cAMP sensor (HEK293G) and human CXCR4 tagged with (a) NanoLuc on its N-terminus (NL-CXCR4), (b) C-terminus (CXCR4-NL), (c) SNAP on its N-terminus (SNAP-CXCR4), (d) human CCR5 or (e) human CXCR4 with the first internal loop swapped for the CCR5 sequence (CXCR4_CCR5il1). The binding of fluorescently labelled CXCR4 ligands and their displacement was quantified with a NanoBRET assay using NL-CXCR4 or CXCR4-NL cells. Conformational changes caused by CXCL12 and pepducins were monitored with an intramolecular biosensor and a BRET assay looking at dimerisation. Moreover, cells were tested in functional assays looking at G protein activation, cAMP inhibition, ß-arrestin recruitment and internalisation after the addition of endogenous ligand CXCL12 or pepducin. Results The affinity of fluorescent CXCL12 (CXCL12-red) was determined through NanoBRET saturation binding. Competition binding experiments showed that CXCL12-red binding was inhibited by addition of small molecules or ATI-2341. Control pepducins with no lipid tail or modified sequences were unable to displace CXCL12-red at concentrations up to 10 µM. ATI-2341f, a fluorescent version of ATI-2341, with an additional TAMRA tagged lysine on the N-terminal end of the sequence showed a displaceable increase in BRET ratio in CXCR4-NL, but only a small change in NL-CXCR4 membranes. An intramolecular biosensor showed activation of CXCR4 by CXCL12 and ATI-2341. However, activation by ATI-2341 was delayed by 30 s. Dimers measured via BRET from one receptor to another showed an increase in BRET with CXCL12 and a decrease in BRET with ATI-2341. Functional assays showed similar activation of CXCR4 by CXCL12 and ATI-2341. An ATI-2341 threonine to alanine mutant showed reduced potencies in all tested assays. Key Conclusion These data suggest that ATI-2341 follows the previously proposed interaction mechanism of pepducins. In a first step, the lipid tail interacts with the membrane and the pepducin is flipped into the cell as supported by the 30 s activation delay observed with ATI-2341 in comparison to the endogenous ligand. Then, the interaction of ATI-2341 and CXCR4 takes place at the intracellular part of the receptor as suggested by the BRET binding studies. However, this interaction impacts the endogenous binding pocket of CXCL12. Furthermore, the functional activation of CXCR4 by CXCL12 is similar to the one observed with ATI-2341. The only difference can be observed in dimerisation and ß-arrestin recruitment experiments. Mutations of the pepducin identified the threonine as an important amino acid

Investigating the mode of action of intracellular loop 1 pepducins at the CXCR4 receptor

Background Pepducins are lipid-peptides derived from the intracellular loop sequences of a G protein-coupled receptor and have been shown to act as allosteric modulators. Pepducins have been described for the chemokine receptor, CXCR4, which can exhibit agonist activity in the absence of the endogenous ligand C-X-C-Ligand 12 (CXCL12). To date, their precise mode of action is unclear. In this study, we investigated the mechanism of action of intracellular loop 1 pepducins at the CXCR4 receptor Experimental Approach Experiments were performed in HEK293 cells stably expressing the GlosensorTM cAMP sensor (HEK293G) and human CXCR4 tagged with (a) NanoLuc on its N-terminus (NL-CXCR4), (b) C-terminus (CXCR4-NL), (c) SNAP on its N-terminus (SNAP-CXCR4), (d) human CCR5 or (e) human CXCR4 with the first internal loop swapped for the CCR5 sequence (CXCR4_CCR5il1). The binding of fluorescently labelled CXCR4 ligands and their displacement was quantified with a NanoBRET assay using NL-CXCR4 or CXCR4-NL cells. Conformational changes caused by CXCL12 and pepducins were monitored with an intramolecular biosensor and a BRET assay looking at dimerisation. Moreover, cells were tested in functional assays looking at G protein activation, cAMP inhibition, ß-arrestin recruitment and internalisation after the addition of endogenous ligand CXCL12 or pepducin. Results The affinity of fluorescent CXCL12 (CXCL12-red) was determined through NanoBRET saturation binding. Competition binding experiments showed that CXCL12-red binding was inhibited by addition of small molecules or ATI-2341. Control pepducins with no lipid tail or modified sequences were unable to displace CXCL12-red at concentrations up to 10 µM. ATI-2341f, a fluorescent version of ATI-2341, with an additional TAMRA tagged lysine on the N-terminal end of the sequence showed a displaceable increase in BRET ratio in CXCR4-NL, but only a small change in NL-CXCR4 membranes. An intramolecular biosensor showed activation of CXCR4 by CXCL12 and ATI-2341. However, activation by ATI-2341 was delayed by 30 s. Dimers measured via BRET from one receptor to another showed an increase in BRET with CXCL12 and a decrease in BRET with ATI-2341. Functional assays showed similar activation of CXCR4 by CXCL12 and ATI-2341. An ATI-2341 threonine to alanine mutant showed reduced potencies in all tested assays. Key Conclusion These data suggest that ATI-2341 follows the previously proposed interaction mechanism of pepducins. In a first step, the lipid tail interacts with the membrane and the pepducin is flipped into the cell as supported by the 30 s activation delay observed with ATI-2341 in comparison to the endogenous ligand. Then, the interaction of ATI-2341 and CXCR4 takes place at the intracellular part of the receptor as suggested by the BRET binding studies. However, this interaction impacts the endogenous binding pocket of CXCL12. Furthermore, the functional activation of CXCR4 by CXCL12 is similar to the one observed with ATI-2341. The only difference can be observed in dimerisation and ß-arrestin recruitment experiments. Mutations of the pepducin identified the threonine as an important amino acid

CXCR4/AckR3 phosphorylation and recruitment of interacting proteins: Key mechanisms regulating their functional status

The C-X-C motif chemokine receptor type 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3/CXCR7) are class A G protein-coupled receptors (GPCRs). Accumulating evidence indicates that GPCR subcellular localization, trafficking, transduction properties, and ultimately their pathophysiological functions are regulated by both interacting proteins and post-translational modifications. This has encouraged the development of novel techniques to characterize the GPCR interactome and to identify residues subjected to post-translational modifications, with a special focus on phosphorylation. This review first describes state-of-the-art methods for the identification of GPCR-interacting proteins and GPCR phosphorylated sites. In addition, we provide an overview of the current knowledge of CXCR4 and ACKR3 post-translational modifications and an exhaustive list of previously identified CXCR4- or ACKR3-interacting proteins. We then describe studies highlighting the importance of the reciprocal influence of CXCR4/ACKR3 interactomes and phosphorylation states. We also discuss their impact on the functional status of each receptor. These studies suggest that deeper knowledge of the CXCR4/ACKR3 interactomes along with their phosphorylation and ubiquitination status would shed new light on their regulation and pathophysiological functions.European Union’s Horizon2020 MSCA European Union’s Horizon2020 MSCA Program [Grant agreement 641833 (ONCORNET)]; A.F. and P.M. are also supported by CNRS, INSERM, Université de Montpellier and Fondation pour la Recherche Médicale (FRM); F.M. laboratory is also supported by grants from Ministerio de Economía; Industria y Competitividad (MINECO) of Spain [Grant SAF2017-84125-R]; CIBERCV-Instituto de Salud Carlos III, Spain [Grant CB16/11/00278] to F.M., cofunded with European FEDER contribution); Comunidad de Madrid-B2017/BMD-3671-INFLAMUNE; and Fundación Ramón Areces. Program [Grant agreement 641833 (ONCORNET)]; A.F. and P.M. are also supported by CNRS, INSERM, Université de Montpellier and Fondation pour la Recherche Médicale (FRM); F.M. laboratory is also supported by grants from Ministerio de Economía; Industria y Competitividad (MINECO) of Spain [Grant SAF2017-84125-R]; CIBERCV-Instituto de Salud Carlos III, Spain [Grant CB16/11/00278] to F.M., cofunded with European FEDER contribution); Comunidad de Madrid-B2017/BMD-3671-INFLAMUNE; and Fundación Ramón Areces

CXCR4/ACKR3 phosphorylation and recruitment of interacting proteins: key mechanisms regulating their functional status

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Context-dependent signaling of CXC chemokine receptor 4 and atypical chemokine receptor 3

G protein-coupled receptors (GPCRs) are regulated by complex molecular mechanisms, both in physiological and pathological conditions, and their signalling can be intricate. Many factors influence their signalling behaviour, including the type of ligand that activates the GPCR, the presence of interacting partners, the kinetics involved or their location. The two CXC type chemokine receptors CXCR4 and ACKR3, both members of the GPCR superfamily, are important and established therapeutic targets in relation to cancer, HIV infection and inflammatory diseases. Therefore, it is crucial to understand how the signalling of these receptors works to be able to specifically target them. In this review, we discuss how the signalling pathways activated by CXCR4 and ACKR3 can vary in different situations. G protein signalling of CXCR4 depends on the cellular context and discrepancies exist depending on the cell lines used. ACKR3, as an atypical chemokine receptor, is generally reported to not activate G proteins, but can broaden its signalling spectrum upon heteromerisation with other receptors, such as CXCR4, endothelial growth factor receptor (EGFR) or the α1-adrenergic receptor (α1-AR). Also, CXCR4 forms heteromers with CCR2, CCR5, the Na+/H+ exchanger regulatory factor 1 (NHERF1), CXCR3, α1-AR and the opioid receptors, which results in differential signalling to that of the monomeric subunits. In addition, CXCR4 is present on membrane rafts, but can go into the nucleus during cancer progression, probably acquiring different signalling properties. In this review, we also provide an overview of the currently known critical amino acids involved in CXCR4 and ACKR3 signalling