The Extended N-Terminal Domain Confers Atypical Chemokine Receptor Properties to CXCR3-B.

peer reviewedThe chemokine receptor CXCR3 plays a critical role in immune cell recruitment and activation. CXCR3 exists as two main isoforms, CXCR3-A and CXCR3-B, resulting from alternative splicing. Although the two isoforms differ only by the presence of an N-terminal extension in CXCR3-B, they have been attributed divergent functional effects on cell migration and proliferation. CXCR3-B is the more enigmatic isoform and the mechanisms underlying its function and signaling remain elusive. We therefore undertook an in-depth cellular and molecular comparative study of CXCR3-A and CXCR3-B, investigating their activation at different levels of the signaling cascades, including G protein coupling, β-arrestin recruitment and modulation of secondary messengers as well as their downstream gene response elements. We also compared the subcellular localization of the two isoforms and their trafficking under resting and stimulated conditions along with their ability to internalize CXCR3-related chemokines. Here, we show that the N-terminal extension of CXCR3-B drastically affects receptor features, modifying its cellular localization and preventing G protein coupling, while preserving β-arrestin recruitment and chemokine uptake capacities. Moreover, we demonstrate that gradual truncation of the N terminus leads to progressive recovery of surface expression and G protein coupling. Our study clarifies the molecular basis underlying the divergent effects of CXCR3 isoforms, and emphasizes the β-arrestin-bias and the atypical nature of CXCR3-B

Genetische regulatie van het calcofluorbindend exopolysaccharidevan A. brasilense 7030

SIGLEKULeuven Campusbibliotheek Exacte Wetenschappen / UCL - Université Catholique de LouvainBEBelgiu

Development by Genetic Immunization of Monovalent Antibodies Against Human Vasoactive Intestinal Peptide Receptor 1 (VPAC1), New Innovative, and Versatile Tools to Study VPAC1 Receptor Function

Multi-membrane spanning proteins, such as G protein-coupled receptors (GPCRs) and ion channels, are extremely difficult to purify as native proteins. Consequently, the generation of antibodies that recognize the native conformation can be challenging. By combining genetic immunization, phage display, and biopanning, we identified a panel of monovalent antibodies (nanobodies) targeting the vasoactive intestinal peptide receptor 1 (VPAC1) receptor. The nine unique nanobodies that were classified into four different families based on their CDR3 amino acid sequence and length, were highly specific for the human receptor and bind VPAC1 with moderate affinity. They all recognize a similar epitope localized in the extracellular N-terminal domain of the receptor and distinct from the orthosteric binding site. In agreement with binding studies, which showed that the nanobodies did not interfere with VIP binding, all nanobodies were devoid of any functional properties. However, we observed that the binding of two nanobodies was slightly increased in the presence of VPAC1 agonists [vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27)], but decreased in the presence of VPAC1 antagonist. As no evidence of allosteric activity was seen in VIP binding studies nor in functional assays, it is, therefore, possible that the two nanobodies may behave as very weak allosteric modulators of VPAC1, detectable only in some sensitive settings, but not in others. We demonstrated that the fluorescently labeled nanobodies detect VPAC1 on the surface of human leukocytes as efficiently as a reference mouse monoclonal antibody. We also developed a protocol allowing efficient detection of VPAC1 by immunohistochemistry in paraffin-embedded human gastrointestinal tissue sections. Thus, these nanobodies constitute new original tools to further investigate the role of VPAC1 in physiological and pathological conditions

Propagation of conformational changes during μ-opioid receptor activation

International audienceµ-Opioid receptors (µORs) are G-protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive and agonist-induced active states (Huang et al., ref. 2) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use solution-state NMR to examine the process of μOR activation using a purified receptor (mouse sequence) preparation in an amphiphile membrane-like environment. We obtain spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-protein-coupling interface (TM5 and TM6), similar to that observed for the β2-adrenergic receptor. Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and intracellular loop 1 and/or helix 8 may be involved in G-protein coupling specificity, as has been suggested for other family A G-protein-coupled receptors

Development by genetic immunization of monovalent antibodies against human vasoactive intestinal peptide receptor 1 (VPAC1), new innovative, and versatile tools to study VPAC1 receptor function

Multi-membrane spanning proteins, such as G protein-coupled receptors (GPCRs) and ion channels, are extremely difficult to purify as native proteins. Consequently, the generation of antibodies that recognize the native conformation can be challenging. By combining genetic immunization, phage display, and biopanning, we identified a panel of monovalent antibodies (nanobodies) targeting the vasoactive intestinal peptide receptor 1 (VPAC1) receptor. The nine unique nanobodies that were classified into four different families based on their CDR3 amino acid sequence and length, were highly specific for the human receptor and bind VPAC1 with moderate affinity. They all recognize a similar epitope localized in the extracellular N-terminal domain of the receptor and distinct from the orthosteric binding site. In agreement with binding studies, which showed that the nanobodies did not interfere with VIP binding, all nanobodies were devoid of any functional properties. However, we observed that the binding of two nanobodies was slightly increased in the presence of VPAC1 agonists [vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27)], but decreased in the presence of VPAC1 antagonist. As no evidence of allosteric activity was seen in VIP binding studies nor in functional assays, it is, therefore, possible that the two nanobodies may behave as very weak allosteric modulators of VPAC1, detectable only in some sensitive settings, but not in others. We demonstrated that the fluorescently labeled nanobodies detect VPAC1 on the surface of human leukocytes as efficiently as a reference mouse monoclonal antibody. We also developed a protocol allowing efficient detection of VPAC1 by immunohistochemistry in paraffin-embedded human gastrointestinal tissue sections. Thus, these nanobodies constitute new original tools to further investigate the role of VPAC1 in physiological and pathological conditions.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Modular integrated secretory system engineering in Pichia pastoris to enhance G-protein coupled receptor expression

Membrane protein research is still hampered by the generally very low levels at which these proteins are naturally expressed, necessitating heterologous expression. Protein degradation, folding problems, and undesired post-translational modifications often occur, together resulting in low expression levels of heterogeneous protein products that are unsuitable for structural studies. We here demonstrate how the integration of multiple engineering modules in Pichia pastoris can be used to increase both the quality and the quantity of overexpressed integral membrane proteins, with the human CXCR4 G-protein coupled receptor as an example. The combination of reduced proteolysis, enhanced ER folding capacity, GlycoDelete-based N-Glycan trimming, and nanobody-based fold stabilization improved the expression of this GPCR in P. pastoris from a low expression level of a heterogeneously glycosylated, proteolyzed product to substantial quantities (2-3 mg/L shake flask culture) of a nonproteolyzed, homogeneously glycosylated proteoform. We expect that this set of tools will contribute to successful expression of more membrane proteins in a quantity and quality suitable for functional and structural studies

Development by genetic immunization of monovalent antibodies (nanobodies) behaving as antagonists of the human ChemR23 receptor

The generation of Abs that recognize the native conformation of G protein-coupled receptors can be a challenging task because, like most multimembrane-spanning proteins, they are extremely difficult to purify as native protein. By combining genetic immunization, phage display, and biopanning, we identified two functional monovalent Abs (nanobodies) targeting ChemR23. The two nanobodies (CA4910 and CA5183) were highly specific for the human receptor and bind ChemR23 with moderate affinity. Binding studies also showed that they share a common binding site that overlaps with that of chemerin, the natural ligand of ChemR23. Consistent with these results, we found that the nanobodies were able to antagonize chemerin-induced intracellular calcium increase. The inhibition was partial when chemerin was used as agonist and complete when the chemerin(149-157) nonapeptide was used as agonist. Engineering of a bivalent CA4910 nanobody resulted in a relatively modest increase in affinity but a marked enhancement of efficacy as an antagonist of chemerin induced intracellular calcium mobilization and a much higher potency against the chemerin(149-157) nonapeptide-induced response. We also demonstrated that the fluorescently labeled nanobodies detect ChemR23 on the surface of human primary cell populations as efficiently as a reference mouse mAb and that the bivalent CA4910 nanobody behaves as an efficient antagonist of chemerin-induced chemotaxis of human primary cells. Thus, these nanobodies constitute new tools to study the role of the chemerin/ChemR23 system in physiological and pathological conditions.status: publishe

Development by Genetic Immunization of Monovalent Antibodies (Nanobodies) Behaving as Antagonists of the Human ChemR23 Receptor.

The generation of Abs that recognize the native conformation of G protein-coupled receptors can be a challenging task because, like most multimembrane-spanning proteins, they are extremely difficult to purify as native protein. By combining genetic immunization, phage display, and biopanning, we identified two functional monovalent Abs (nanobodies) targeting ChemR23. The two nanobodies (CA4910 and CA5183) were highly specific for the human receptor and bind ChemR23 with moderate affinity. Binding studies also showed that they share a common binding site that overlaps with that of chemerin, the natural ligand of ChemR23. Consistent with these results, we found that the nanobodies were able to antagonize chemerin-induced intracellular calcium increase. The inhibition was partial when chemerin was used as agonist and complete when the chemerin(149-157) nonapeptide was used as agonist. Engineering of a bivalent CA4910 nanobody resulted in a relatively modest increase in affinity but a marked enhancement of efficacy as an antagonist of chemerin induced intracellular calcium mobilization and a much higher potency against the chemerin(149-157) nonapeptide-induced response. We also demonstrated that the fluorescently labeled nanobodies detect ChemR23 on the surface of human primary cell populations as efficiently as a reference mouse mAb and that the bivalent CA4910 nanobody behaves as an efficient antagonist of chemerin-induced chemotaxis of human primary cells. Thus, these nanobodies constitute new tools to study the role of the chemerin/ChemR23 system in physiological and pathological conditions.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

A genetically encoded biosensor reveals location bias of opioid drug action

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation