Impact of membrane lipid polyunsaturation on dopamine D2 receptor ligand binding and signaling.

Increasing evidence supports a relationship between lipid metabolism and mental health. In particular, the biostatus of polyunsaturated fatty acids (PUFAs) correlates with some symptoms of psychiatric disorders, as well as the efficacy of pharmacological treatments. Recent findings highlight a direct association between brain PUFA levels and dopamine transmission, a major neuromodulatory system implicated in the etiology of psychiatric symptoms. However, the mechanisms underlying this relationship are still unknown. Here we demonstrate that membrane enrichment in the n-3 PUFA docosahexaenoic acid (DHA), potentiates ligand binding to the dopamine D2 receptor (D2R), suggesting that DHA acts as an allosteric modulator of this receptor. Molecular dynamics simulations confirm that DHA has a high preference for interaction with the D2R and show that membrane unsaturation selectively enhances the conformational dynamics of the receptor around its second intracellular loop. We find that membrane unsaturation spares G protein activity but potentiates the recruitment of ß-arrestin in cells. Furthermore, in vivo n-3 PUFA deficiency blunts the behavioral effects of two D2R ligands, quinpirole and aripiprazole. These results highlight the importance of membrane unsaturation for D2R activity and provide a putative mechanism for the ability of PUFAs to enhance antipsychotic efficacy.

Structural insights into recognition of chemokine receptors by Staphylococcus aureus leukotoxins.

Staphylococcus aureus (SA) leukocidin ED (LukED) belongs to a family of bicomponent pore forming toxins that play important roles in SA immune evasion and nutrient acquisition. LukED targets specific G protein-coupled chemokine receptors to lyse human erythrocytes (red blood cells) and leukocytes (white blood cells). The first recognition step of receptors is critical for specific cell targeting and lysis. The structural and molecular bases for this mechanism are not well understood but could constitute essential information to guide antibiotic development. Here, we characterized the interaction of LukE with chemokine receptors ACKR1, CCR2, and CCR5 using a combination of structural, pharmacological, and computational approaches. First, crystal structures of LukE in complex with a small molecule mimicking sulfotyrosine side chain (p-cresyl sulfate) and with peptides containing sulfotyrosines issued from receptor sequences revealed the location of receptor sulfotyrosine binding sites in the toxins. Then, by combining previous and novel experimental data with protein docking, classical and accelerated weight histogram (AWH) molecular dynamics we propose models of the ACKR1-LukE and CCR5-LukE complexes. This work provides novel insights into chemokine receptor recognition by leukotoxins and suggests that the conserved sulfotyrosine binding pocket could be a target of choice for future drug development.

Molecular insights into mechanisms of GPCR hijacking by Staphylococcus aureus.

Atypical chemokine receptor 1 (ACKR1) is a G protein-coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein-binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1-Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.

Differential Involvement of ACKR3 C-Tail in ß-Arrestin Recruitment, Trafficking and Internalization.

Background: The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits ß-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of ß-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods: We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of ß-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results: Upon CXCL12 stimulation, ACKR3 recruits both ß-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for ß-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that ß-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T352 and in part S355 are important residues for ß-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential ß-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when ß-arrestin recruitment is impaired or in the absence of ß-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced ß-arrestin recruitment, ACKR3 trafficking and internalization.

The ligand-bound state of a G protein-coupled receptor stabilizes the interaction of functional cholesterol molecules.

Cholesterol is a major component of mammalian plasma membranes that not only affects the physical properties of the lipid bilayer but also is the function of many membrane proteins including G protein-coupled receptors. The oxytocin receptor (OXTR) is involved in parturition and lactation of mammals and in their emotional and social behaviors. Cholesterol acts on OXTR as an allosteric modulator inducing a high-affinity state for orthosteric ligands through a molecular mechanism that has yet to be determined. Using the ion channel-coupled receptor technology, we developed a functional assay of cholesterol modulation of G protein-coupled receptors that is independent of intracellular signaling pathways and operational in living cells. Using this assay, we discovered a stable binding of cholesterol molecules to the receptor when it adopts an orthosteric ligand-bound state. This stable interaction preserves the cholesterol-dependent activity of the receptor in cholesterol-depleted membranes. This mechanism was confirmed using time-resolved FRET experiments on WT OXTR expressed in CHO cells. Consequently, a positive cross-regulation sequentially occurs in OXTR between cholesterol and orthosteric ligands.

A near-infrared fluorogenic dimer enables background-free imaging of endogenous GPCRs in living mice.

Fluorescent probes are commonly used in studying G protein-coupled receptors in living cells; however their application to the whole animal receptor imaging is still challenging. To address this problem, we report the design and the synthesis of the first near-infrared emitting fluorogenic dimer with environment-sensitive folding. Due to the formation of non-fluorescent H-aggregates in an aqueous medium, the near-infrared fluorogenic dimer displays a strong turn-on response (up to 140-fold) in an apolar environment and exceptional brightness: 56% quantum yield and ≈444 000 M-1 cm-1 extinction coefficient. Grafted on a ligand of the oxytocin receptor, it allows the unprecedented background-free and target-specific imaging of the naturally expressed receptor in living mice.

The atypical chemokine receptor 3 interacts with Connexin 43 inhibiting astrocytic gap junctional intercellular communication.

The atypical chemokine receptor 3 (ACKR3) plays a pivotal role in directing the migration of various cellular populations and its over-expression in tumors promotes cell proliferation and invasiveness. The intracellular signaling pathways transducing ACKR3-dependent effects remain poorly characterized, an issue we addressed by identifying the interactome of ACKR3. Here, we report that recombinant ACKR3 expressed in HEK293T cells recruits the gap junction protein Connexin 43 (Cx43). Cx43 and ACKR3 are co-expressed in mouse brain astrocytes and human glioblastoma cells and form a complex in embryonic mouse brain. Functional in vitro studies show enhanced ACKR3 interaction with Cx43 upon ACKR3 agonist stimulation. Furthermore, ACKR3 activation promotes ß-arrestin2- and dynamin-dependent Cx43 internalization to inhibit gap junctional intercellular communication in primary astrocytes. These results demonstrate a functional link between ACKR3 and gap junctions that might be of pathophysiological relevance.

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 physiologic and pathologic conditions, and their signaling can be intricate. Many factors influence their signaling behavior, 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, CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3), both members of the GPCR superfamily, are important and established therapeutic targets in relation to cancer, human immunodeficiency virus infection, and inflammatory diseases. Therefore, it is crucial to understand how the signaling of these receptors works to be able to specifically target them. In this review, we discuss how the signaling pathways activated by CXCR4 and ACKR3 can vary in different situations. G protein signaling 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 signaling spectrum upon heteromerization with other receptors, such as CXCR4, endothelial growth factor receptor, or the α 1-adrenergic receptor (α 1-AR). Also, CXCR4 forms heteromers with CC chemokine receptor (CCR) 2, CCR5, the Na+/H+ exchanger regulatory factor 1, CXCR3, α 1-AR, and the opioid receptors, which results in differential signaling from 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 signaling properties. In this review, we also provide an overview of the currently known critical amino acids involved in CXCR4 and ACKR3 signaling.

Chemoselective Acylation of Hydrazinopeptides to Access Fluorescent Probes for Time-Resolved FRET Assays on GPCRs.

Fluorescence techniques represent a powerful tool to investigate dynamic and functional architecture of GPCRs. Thus, fluorescent GPCR ligands have found various applications in cellular imaging, in the development of binding assays as replacements for radioligands in the study of ligand-receptor but also in receptor-receptor interactions at the cell surface or in native tissues. To extend the applicability of these techniques, the design and the synthesis of fluorescent probes are critical steps. As there are numerous peptide receptors in the GPCR family, fluorescent peptide-based probes are of importance. Herein, we present a convenient method to facilitate the solution-phase fluorescent labeling of peptides which is based on the chemoselective acylation of α-hydrazinopeptides. This approach combines the advantages to use commercially available amine-reactive dyes and very mild conditions that are fully compatible with the chemical sensitivity of the dyes. It gives a rapid access to fluorescent peptidic probes compatible with the time-resolved fluorescence resonance energy transfer (TR-FRET) techniques.

Time-Resolved FRET-Based Assays to Characterize G Protein-Coupled Receptor Hetero-oligomer Pharmacology.

Although G protein-coupled receptor (GPCR) oligomerization is a matter of debate, it has been shown that the nature of the GPCR partners within the oligomers can influence the pharmacological properties of the receptors. Therefore, finding specific ligands for homo- or hetero-oligomers opens new perspectives for drug discovery. However, no efficient experimental strategy to screen for such ligands existed yet. Indeed, conventional binding strategies do not discriminate ligand binding on GPCR monomers, homo- or hetero-oligomers. To address this issue, we recently developed a new assay based on a time-resolved FRET method that is easy to implement and that can focus on ligand binding specifically on the hetero-oligomer.

LIT-001, the First Nonpeptide Oxytocin Receptor Agonist that Improves Social Interaction in a Mouse Model of Autism.

Oxytocin (OT) and its receptor (OT-R) are implicated in the etiology of autism spectrum disorders (ASD), and OT-R is a potential target for therapeutic intervention. Very few nonpeptide oxytocin agonists have currently been reported. Their molecular and in vivo pharmacology remain to be clarified, and none of them has been shown to be efficient in improving social interaction in animal models relevant to ASD. In an attempt to rationalize the design of centrally active nonpeptide full agonists, we studied in a systematic way the structural determinants of the affinity and efficacy of representative ligands of the V1a and V2 vasopressin receptor subtypes (V1a-R and V2-R) and of the oxytocin receptor. Our results confirm the subtlety of the structure-affinity and structure-efficacy relationships around vasopressin/oxytocin receptor ligands and lead however to the first nonpeptide OT receptor agonist active in a mouse model of ASD after peripheral ip administration.

Profiling of orthosteric and allosteric group-III metabotropic glutamate receptor ligands on various G protein-coupled receptors with Tag-lite® assays.

Group-III metabotropic glutamate (mGlu) receptors are important synaptic regulators and are potential druggable targets for Parkinson disease, autism and pain. Potential drugs include orthosteric agonists in the glutamate binding extracellular domain and positive allosteric modulators interacting with seven-pass transmembrane domains. Orthosteric agonists are rarely completely specific for an individual group-III mGlu subtype. Furthermore they often fail to pass the blood-brain barrier and they constitutively activate their target receptor. These properties limit the potential therapeutic use of orthosteric agonists. Allosteric modulators are more specific and maintain the biological activity of the targeted receptor. However, they bind in a hydrophobic pocket and this limits their bio-availability and increases possible off-target action. It is therefore important to characterize the action of potential drug targets with a multifaceted and deeply informative assay. Here we aimed at multifaceted deep profiling of the effect of seven different agonists, and seven positive allosteric modulators on 34 different G protein-coupled receptors by a Tag-lite® assay. Our results did not reveal off-target activity of mGlu orthosteric agonists. However, five allosteric modulators had either positive or negative effects on non-cognate G protein-coupled receptors. In conclusion, we demonstrate the power of the Tag-lite® assay for potential drug ligand profiling on G protein-coupled receptors and its potential to identify positive allosteric compounds.

Oligomerization of a G protein-coupled receptor in neurons controlled by its structural dynamics.

G protein coupled receptors (GPCRs) play essential roles in intercellular communication. Although reported two decades ago, the assembly of GPCRs into dimer and larger oligomers in their native environment is still a matter of intense debate. Here, using number and brightness analysis of fluorescently labeled receptors in cultured hippocampal neurons, we confirm that the metabotropic glutamate receptor type 2 (mGlu2) is a homodimer at expression levels in the physiological range, while heterodimeric GABAB receptors form larger complexes. Surprisingly, we observed the formation of larger mGlu2 oligomers upon both activation and inhibition of the receptor. Stabilizing the receptor in its inactive conformation using biochemical constraints also led to the observation of oligomers. Following our recent observation that mGlu receptors are in constant and rapid equilibrium between several states under basal conditions, we propose that this structural heterogeneity limits receptor oligomerization. Such assemblies are expected to stabilize either the active or the inactive state of the receptor.

V1b vasopressin receptor trafficking and signaling: Role of arrestins, G proteins and Src kinase.

The signaling pathway of G protein-coupled receptors is strongly linked to their trafficking profile. Little is known about the molecular mechanisms involved in the vasopressin receptor V1b subtype (V1b R) trafficking and its impact on receptor signaling and regulation. For this purpose, we investigated the role of ß-arrestins in receptor desensitization, internalization and recycling and attempted to dissect the V1b R-mediated MAP kinase pathway. Using MEF cells Knocked-out for ß-arrestins 1 and 2, we demonstrated that both ß-arrestins 1 and 2 play a fundamental role in internalization and recycling of V1b R with a rapid and transient V1b R-ß-arrestin interaction in contrast to a slow and long-lasting ß-arrestin recruitment of the V2 vasopressin receptor subtype (V2 R). Using V1b R-V2 R chimeras and V1b R C-terminus truncations, we demonstrated the critical role of the V1b R C-terminus in its interaction with ß-arrestins thereby regulating the receptor internalization and recycling kinetics in a phosphorylation-independent manner. In parallel, V1b R MAP kinase activation was dependent on arrestins and Src-kinase but independent on G proteins. Interestingly, Src interacted with hV1b R at basal state and dissociated when receptor internalization occurred. Altogether, our data describe for the first time the trafficking profile and MAP kinase pathway of V1b R involving both arrestins and Src kinase family.

From the Promiscuous Asenapine to Potent Fluorescent Ligands Acting at a Series of Aminergic G-Protein-Coupled Receptors.

Monoamine neurotransmitters such as serotonin, dopamine, histamine, and noradrenaline have important and varied physiological functions and similar chemical structures. Representing important pharmaceutical drug targets, the corresponding G-protein-coupled receptors (termed aminergic GPCRs) belong to the class of cell membrane receptors and share many levels of similarity as well. Given their pharmacological and structural closeness, one could hypothesize the possibility to derivatize a ubiquitous ligand to afford rapidly fluorescent probes for a large set of GPCRs to be used for instance in FRET-based binding assays. Here we report fluorescent derivatives of the nonselective agent asenapine which were designed, synthesized, and evaluated as ligands of 34 serotonin, dopamine, histamine, melatonin, acetylcholine, and adrenergic receptors. It appears that this strategy led rapidly to the discovery and development of nanomolar affinity fluorescent probes for 14 aminergic GPCRs. Selected probes were tested in competition binding assays with unlabeled competitors in order to demonstrate their suitability for drug discovery purposes.

Pharmacological evidence for a metabotropic glutamate receptor heterodimer in neuronal cells.

Metabotropic glutamate receptors (mGluRs) are mandatory dimers playing important roles in regulating CNS function. Although assumed to form exclusive homodimers, 16 possible heterodimeric mGluRs have been proposed but their existence in native cells remains elusive. Here, we set up two assays to specifically identify the pharmacological properties of rat mGlu heterodimers composed of mGlu2 and 4 subunits. We used either a heterodimer-specific conformational LRET-based biosensor or a system that guarantees the cell surface targeting of the heterodimer only. We identified mGlu2-4 specific pharmacological fingerprints that were also observed in a neuronal cell line and in lateral perforant path terminals naturally expressing mGlu2 and mGlu4. These results bring strong evidence for the existence of mGlu2-4 heterodimers in native cells. In addition to reporting a general approach to characterize heterodimeric mGluRs, our study opens new avenues to understanding the pathophysiological roles of mGlu heterodimers.

Natural amines inhibit activation of human plasmacytoid dendritic cells through CXCR4 engagement.

Plasmacytoid dendritic cells (pDC) are specialized in secretion of type I interferon in response to pathogens. Here we show that natural monoamines and synthetic amines inhibit pDC activation by RNA viruses. Furthermore, a synthetic analogue of histamine reduces type I interferon production in a mouse model of influenza infection. We identify CXC chemokine receptor 4 (CXCR4) as a receptor used by amines to inhibit pDC. Our study establishes a functional link between natural amines and the innate immune system and identifies CXCR4 as a potential 'on-off' switch of pDC activity with therapeutic potential.

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

Design and validation of a homogeneous time-resolved fluorescence cell-based assay targeting the ligand-gated ion channel 5-HT3A.

Ligand-gated ion channels (LGICs) are considered as attractive protein targets in the search for new therapeutic agents. Nowadays, this strategy involves the capability to screen large chemical libraries. We present a new Tag-lite ligand binding assay targeting LGICs on living cells. This technology combines the use of suicide enzyme tags fused to channels of interest with homogeneous time-resolved fluorescence (HTRF) as the detection readout. Using the 5-HT3 receptor as system model, we showed that the pharmacology of the HALO-5HT3 receptor was identical to that of the native receptor. After validation of the assay by using 5-HT3 agonists and antagonists of reference, a pilot screen enabled us to identify azelastine, a well-known histamine H1 antagonist, as a potent 5-HT3 antagonist. This interesting result was confirmed with electrophysiological experiments. The method described here is easy to implement and could be applicable for other LGICs, opening new ways for the screening of chemical libraries.