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Jean Mazella - One of the best experts on this subject based on the ideXlab platform.
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Neurotensin and the Neurotensin Receptor 3 in microglial cells
Journal of Neuroscience Research, 2005Co-Authors: Stephane Martin, Eleni Dicou, Jeanpierre Vincent, Jean MazellaAbstract:Microglia motility plays a crucial role in response to lesion or exocytotoxic damage of the cerebral tissue. The neuropeptide Neurotensin elicited the migration of the human microglial cell line C13NJ by a mechanism dependent on both phosphatidylinositol-3 kinase (PI3 kinase) and mitogen-activated protein (MAP) kinases pathways. The effect of Neurotensin on cell migration was blocked by the Neurotensin Receptor-3 propeptide, a selective ligand of this Receptor. The type I Neurotensin Receptor-3 was the only known Neurotensin Receptor expressed in these microglial cells, and its activation led to the phosphorylation of both extracellular signaling-regulated kinases Erk1/2 and Akt. Furthermore, the effect of Neurotensin on cell migration was preceded by a profound modification of the F-actin cytoskeleton, particularly by the rapid formation of numerous cell filopodia. Both the motility and the filopodia appearance induced by Neurotensin were totally blocked by selective inhibitors of MAP kinases or PI3 kinase pathways. In the murine microglial cell line N11, the Neurotensin Receptor-3 is also the only Neurotensin Receptor expressed, and its activation by Neurotensin leads to the phosphorylation of both Erk1/2 and Akt. In these cells, Neurotensin induces the gene expression of several cytokines/chemokines, including MIP-2, MCP-1, interleukin-1β and tumor necrosis factor-α. This induction is dependent on both protein kinases pathways. We observed that the effect of Neurotensin on the cytokine/chemokine expression is also inhibited by the Neurotensin Receptor-3 propeptide. This is the demonstration that the Neurotensin Receptor-3 is functional and mediates both the migratory action of Neurotensin and its induction of chemokines/cytokines expression. © 2005 Wiley-Liss, Inc.
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involvement of the Neurotensin Receptor 3 in the Neurotensin induced migration of human microglia
The Journal of Neuroscience, 2003Co-Authors: Stephane Martin, Jeanpierre Vincent, Jean MazellaAbstract:Microglia motility plays a crucial role in response to lesion or exocytotoxic damage of the cerebral tissue. We used two in vitro assays, a wound-healing model and a chemotaxis assay, to show that the neuropeptide Neurotensin elicited the migration of the human microglial cell line C13NJ by a mechanism dependent on both phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways. The effect of Neurotensin on cell migration was blocked by the Neurotensin Receptor-3 propeptide, a selective ligand of this Receptor. We demonstrate, by using RT-PCR, photoaffinity labeling, and Western blot analysis, that the type I Neurotensin Receptor-3 was the only known Neurotensin Receptor expressed in these microglial cells and that its activation led to the phosphorylation of both extracellular signal-regulating kinases 1/2 and Akt. Furthermore, the effect of Neurotensin on cell migration was preceded by a profound modification of the F-actin cytoskeleton, particularly by the rapid formation of numerous cell filopodia. Both the motility and the filopodia appearance induced by Neurotensin were totally blocked by selective inhibitors of MAP kinases or PI 3-kinase pathways. This demonstrates that the Neurotensin Receptor-3 is functional and mediates the migratory actions of Neurotensin.
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involvement of the Neurotensin Receptor subtype ntr3 in the growth effect of Neurotensin on cancer cell lines
International Journal of Cancer, 2001Co-Authors: Claude Dal Farra, Jean Mazella, Jeanmarie Botto, Philippe Sarret, Valerie Navarro, Jeanpierre VincentAbstract:The expression of the 3 currently known Neurotensin Receptors was studied in human cancer cells of prostatic, colonic or pancreatic origin by means of RT-PCR analysis and binding experiments. All the cells selected for this work have been shown to exhibit a growth response to Neurotensin. We found that the 7 transmembrane domain, levocabastine insensitive Receptor (NTR1) is expressed in most but not all of the cells studied whereas the 7 transmembrane domain, levocabastine sensitive Receptor (NTR2) is present in none of these cells. The 100 kDa-type I Neurotensin Receptor (NTR3) is expressed in all the cells assayed. Moreover, we demonstrated that Neurotensin can stimulate the growth of CHO cells stably transfected with the NTR3. Taken together, our results strongly suggest that the NTR3 subtype could be involved in the growth response of human cancer cells to Neurotensin. © 2001 Wiley-Liss, Inc.
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sortilin Neurotensin Receptor 3 a new tool to investigate Neurotensin signaling and cellular trafficking
Cellular Signalling, 2001Co-Authors: Jean MazellaAbstract:The identification of gp95sortilin, a sorting protein, as being the 100 kDa Neurotensin (NT) Receptor, a non-G-protein coupled Receptor, constitutes a new and interesting but intriguing step in the neuropeptide signaling as well as in cellular trafficking. The isolation of the same protein by three different experimental approaches sum up the complexity for researchers involved in the functional significance of the so-called sortilin/Neurotensin Receptor 3 (NTR3). This review will concentrate on the putative physiological and cellular roles of sortilin/NTR3 as most results so far have proposed hypothetical conclusions rather than concrete evidence.
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Stable Expression of the Mouse Levocabastine-Sensitive Neurotensin Receptor in HEK 293 Cell Line: Binding Properties, Photoaffinity Labeling, and Internalization Mechanism☆
Biochemical and biophysical research communications, 1998Co-Authors: Jeanmarie Botto, Jeanpierre Vincent, Philippe Sarret, Joëlle Chabry, Jean MazellaAbstract:The recently cloned new subtype of G protein-coupled Neurotensin Receptor (NTRL) was stably expressed in the HEK 293 cell line in order to investigate its binding and internalization properties. The expressed Receptor exhibited the typical binding characteristics of the low affinity, levocabastine-sensitive binding site previously described in rat and mouse brain and was detected as a protein with an apparent MW of 45 kDa by photoaffinity labeling. Although intracellular modulation of adenylate cyclase, guanylate cyclase and phospholipase C was not detected after application of Neurotensin or levocabastine on NTRL-transfected cells, this Receptor was able to internalize iodinated Neurotensin. The internalization process was followed by recycling of Receptors to the cell membrane. By contrast, no recycling was observed with the high affinity Neurotensin Receptor (NTRH). The differential intracellular routing of NTRH and NTRL after internalization is most probably the consequence of their divergent carboxy-terminal sequences.
Reinhard Grisshammer - One of the best experts on this subject based on the ideXlab platform.
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towards monitoring conformational changes of the gpcr Neurotensin Receptor 1 by single molecule fret
Proceedings of SPIE--the International Society for Optical Engineering, 2018Co-Authors: Thomas Heitkamp, Reinhard Grisshammer, Michael BorschAbstract:Neurotensin Receptor 1 (NTSR1) is a G protein-coupled Receptor that is important for signaling in the brain and the gut. Its agonist ligand Neurotensin (NTS), a 13-amino-acid peptide, binds with nanomolar affinity from the extracellular side to NTSR1 and induces conformational changes that trigger intracellular signaling processes. Our goal is to monitor the conformational dynamics of single fluorescently labeled NTSR1. For this, we fused the fluorescent protein mNeonGreen to the C terminus of NTSR1, purified the Receptor fusion protein from E. coli membranes, and reconstituted NTSR1 into liposomes with E. coli polar lipids. Using single-molecule anisotropy measurements, NTSR1 was found to be monomeric in liposomes, with a small fraction being dimeric and oligomeric, showing homoFRET. Similar results were obtained for NTSR1 in detergent solution. Furthermore, we demonstrated agonist binding to NTSR1 by time-resolved single-molecule Forster resonance energy transfer (smFRET), using Neurotensin labeled with the fluorophore ATTO594.
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structure and dynamics of a constitutively active Neurotensin Receptor
Scientific Reports, 2016Co-Authors: Brian E. Krumm, Sangbae Lee, Supriyo Bhattacharya, Nagarajan Vaidehi, Istvan Botos, Courtney F White, Reinhard GrisshammerAbstract:Many G protein-coupled Receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in Receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 A crystal structure of a constitutively active, agonist-bound Neurotensin Receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free Receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular Receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered Receptor dynamics, possibly explains the reduced Neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.
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Construction of recombinant HEK293 cell lines for the expression of the Neurotensin Receptor NTSR1.
Methods of Molecular Biology, 2015Co-Authors: Su Xiao-bo, Joseph Shiloach, Reinhard GrisshammerAbstract:Abstract G protein-coupled Receptors (GPCRs) are associated with a wide array of diseases and are targets of most of the medicines sold worldwide. Despite their clinical importance, only 25 unique GPCR structures have been determined as of April 2014. The first step for structural studies is to establish the expression of correctly folded, functional Receptors in recombinant host cells at quantities to allow subsequent purification and crystallization trials. Here we describe the T-REx™-inducible expression system to construct and select a stable HEK293 cell line for high-level expression of functional Neurotensin Receptor type I (NTSR1). We also present the protocols used for the adaptation of the cells into suspension culture, as well as the optimization of the induction parameters for NTSR1 expression, which led to 1 mg of purified NTSR1 per liter suspension culture in bioreactors.
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Structural prerequisites for G-protein activation by the Neurotensin Receptor
Nature Communications, 2015Co-Authors: Brian E. Krumm, Priyanka Shah, Jim F. White, Reinhard GrisshammerAbstract:We previously determined the structure of Neurotensin Receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist Neurotensin. This Receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A^3.49, L310A^6.37, F358A^7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F358^7.42 causes the conserved W321^6.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na^+ ion pocket and linking the agonist with residues in the lower Receptor part implicated in GPCR activation. In the intracellular Receptor half, the bulkier L310^6.37 side chain dictates the position of R167^3.50 of the highly conserved D/ERY motif. These residues, together with the presence of E166^3.49 provide determinants for G-protein activation by NTSR1. The structural basis of how G-protein coupled Receptors respond to unique stimuli remains poorly understood. Here, Krumm et al. present new structures of the Neurotensin Receptor and reveal insights into how ligand binding is linked to structural rearrangements associated with Receptor activation.
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structural prerequisites for g protein activation by the Neurotensin Receptor
Nature Communications, 2015Co-Authors: Brian E. Krumm, Priyanka Shah, Jim F. White, Reinhard GrisshammerAbstract:We previously determined the structure of Neurotensin Receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist Neurotensin. This Receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist with residues in the lower Receptor part implicated in GPCR activation. In the intracellular Receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1.
Peter Gmeiner - One of the best experts on this subject based on the ideXlab platform.
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structure based evolution of subtype selective Neurotensin Receptor ligands
ChemistryOpen, 2014Co-Authors: Carolin Schaab, Harald Hübner, Jurgen Einsiedel, Ralf C Kling, Timothy Clark, Dieter Seebach, Peter GmeinerAbstract:Subtype-selective agonists of the Neurotensin Receptor NTS2 represent a promising option for the treatment of neuropathic pain, as NTS2 is involved in the mediation of μ-opioid-independent anti-nociceptive effects. Based on the crystal structure of the subtype NTS1 and previous structure–activity relationships (SARs) indicating a potential role for the sub-pocket around Tyr11 of NT(8–13) in subtype-specific ligand recognition, we have developed new NTS2-selective ligands. Starting from NT(8–13), we replaced the tyrosine unit by β2-amino acids (type 1), by heterocyclic tyrosine bioisosteres (type 2) and peptoid analogues (type 3). We were able to evolve an asymmetric synthesis of a 5-substituted azaindolylalanine and its application as a bioisostere of tyrosine capable of enhancing NTS2 selectivity. The S-configured test compound 2 a, [(S)-3-(pyrazolo[1,5-a]pyridine-5-yl)-propionyl11]NT(8–13), exhibits substantial NTS2 affinity (4.8 nm) and has a nearly 30-fold NTS2 selectivity over NTS1. The (R)-epimer 2 b showed lower NTS2 affinity but more than 600-fold selectivity over NTS1.
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Efficient Synthesis of Heterocyclic Neurotensin Receptor Ligands by Microwave-Assisted Aminocarbonylation
Synthesis, 2013Co-Authors: Christopher Lang, Peter GmeinerAbstract:Marking the heterocyclic Neurotensin Receptor antagonist SR142948A as a lead compound, the development of an efficient and a practical synthetic route to heterocyclic aryl carboxamides is reported. Thus, a highly efficient and flexible access to these carboxamides was elaborated by taking advantage of microwave-assisted aminocarbonylation reaction mediated by Mo(CO) 6 , Herrmann’s palladacycle, [( t -Bu) 3 PH]BF 4 , and DBU.
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Development of a Metabolically Stable Neurotensin Receptor 2 (NTS2) Ligand
ChemMedChem, 2012Co-Authors: Cornelia Held, Manuel Plomer, Jasmin Meltretter, Harald Hübner, Monika Pischetsrieder, Peter GmeinerAbstract:Subtype-selective Neurotensin Receptor 2 (NTS2) ligands can be used as molecular probes to investigate the physiological role of Neurotensinergic systems and serve as lead compounds to initiate the development of drugs for the treatment of tonic pain. Starting from our recently described NTS2 ligand 1, structural variants of type 2 were synthesized to further improve binding affinity and selectivity to gain metabolic stability. The peptide–peptoid hybrid 2 b showed excellent NTS2 binding affinity (Ki=2.8 nM) and 22 000-fold selectivity over NTS1, as well as metabolic stability over 32 h in a serum degradation assay. Employing a MAPK-driven luciferase reporter gene assay and an IP accumulation assay, the Neurotensin mimetic 2 b displayed respective inhibitions of constitutive activity exceeding 4.3- and 3.9-fold that of the inverse agonist activity of the endogenous ligand Neurotensin.
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discovery of highly potent and Neurotensin Receptor 2 selective Neurotensin mimetics
Journal of Medicinal Chemistry, 2011Co-Authors: Jurgen Einsiedel, Cornelia Held, Manuel Plomer, Harald Hübner, Maud Hervet, Nuska Tschammer, Peter GmeinerAbstract:The Neurotensin Receptor subtype 2 (NTS2) is involved in the modulation of tonic pain sensitivity and psychiatric diseases and is, therefore, regarded as a highly attractive pharmacological target protein. Aiming to discover NTS2 selective ligands, we herein describe the identification of screening hits and the chemical synthesis of structural variants leading to the highly potent and NTS2 selective peptide−peptoid hybrids of type 3. The Neurotensin mimetics 3a and 3e−g incorporating an N-(4-hydroxyphenethyl)glycine substructure exhibit single digit nanomolar affinity (Ki = 4.3−8.8 nM) and 1900−12000 fold selectivity over the Neurotensin Receptor subtype 1 (NTS1). According to functional experiments, the test compounds 3a and 3e−g displayed an inhibition of constitutive mitogen-activated protein kinase (MAPK) activity exceeding 2.6−4.6 times the inverse agonist activity of the endogenous ligand Neurotensin.
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novel insights into gpcr peptide interactions mutations in extracellular loop 1 ligand backbone methylations and molecular modeling of Neurotensin Receptor 1
Bioorganic & Medicinal Chemistry, 2008Co-Authors: Steffen Harterich, Jurgen Einsiedel, Susanne Koschatzky, Peter GmeinerAbstract:Abstract Investigating prototypical interactions between NT(8–13) and the human Neurotensin Receptor 1 (hNTR1), we created a Receptor–ligand model that was validated by site-directed mutagenesis and structure–activity relationship studies. Stabilization of the extracellular loop 1 (EL1) by π-stacking clusters proved to be important for agonist binding when substitution of six conserved amino acids by alanine resulted in an agonist specific loss of maximal binding capacity. In agreement with our modeling studies, EL1 seems to adopt a clamp-type border area controlling the shape of the binding site crevice. Employing chemically manipulated peptide analogs as molecular probes, the impact of backbone modifications on Receptor–ligand interaction, especially the influence on ligand conformation, was examined in binding studies and explained by in silico analysis.
Andreas Plückthun - One of the best experts on this subject based on the ideXlab platform.
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probing the conformation states of Neurotensin Receptor 1 variants by nmr site directed methyl labeling
ChemBioChem, 2021Co-Authors: Inguna Goba, Andreas Plückthun, Matthias Hillenbrand, David Goricanec, Dominik Schum, Franz HagnAbstract:G protein-coupled Receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein-stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope-labeled methyl probe, we show that the activity and the conformation state of stabilized Neurotensin Receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist-induced activity of the Receptor. Furthermore, this assay can be used as a readout when re-introducing agonist-dependent signaling into a highly stabilized, and thus rigidified, Receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively 19 F-labeled Receptors are not sufficient to resolve signal overlap for a more detailed analysis.
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spr based fragment screening with Neurotensin Receptor 1 generates novel small molecule ligands
PLOS ONE, 2017Co-Authors: Sylwia Huber, Philipp Heine, Andreas Plückthun, Fabio Casagrande, Melanie N Hug, Lisha Wang, Lutz Kummer, Michael HennigAbstract:The Neurotensin Receptor 1 represents an important drug target involved in various diseases of the central nervous system. So far, the full exploitation of potential therapeutic activities has been compromised by the lack of compounds with favorable physicochemical and pharmacokinetic properties which efficiently penetrate the blood-brain barrier. Recent progress in the generation of stabilized variants of solubilized Neurotensin Receptor 1 and its subsequent purification and successful structure determination presents a solid starting point to apply the approach of fragment-based screening to extend the chemical space of known Neurotensin Receptor 1 ligands. In this report, surface plasmon resonance was used as primary method to screen 6369 compounds. Thereby 44 hits were identified and confirmed in competition as well as dose-response experiments. Furthermore, 4 out of 8 selected hits were validated using nuclear magnetic resonance spectroscopy as orthogonal biophysical method. Computational analysis of the compound structures, taking the known crystal structure of the endogenous peptide agonist into consideration, gave insight into the potential fragment-binding location and interactions and inspires chemistry efforts for further exploration of the fragments.
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A cleavable ligand column for the rapid isolation of large quantities of homogeneous and functional Neurotensin Receptor 1 variants from E. coli
Protein Expression and Purification, 2015Co-Authors: Pascal Egloff, Mattia Deluigi, Philipp Heine, Stefanie Balada, Andreas PlückthunAbstract:G protein-coupled Receptors (GPCRs) are key players of cell signaling, thus representing important drug targets for the treatment of human diseases. Since inherent difficulties in Receptor production and handling have precluded the application of many in vitro experiments, major questions about GPCR mechanisms and dynamics remain elusive to date. We recently used directed evolution in Escherichia coli on Neurotensin Receptor 1 (NTR1) for the generation of GPCR variants with greatly elevated functional expression levels and with excellent stability in detergent micelles. In this work we outline a highly efficient purification method for our evolved Receptor variants, which is based on the application of an inexpensive, disposable high-affinity ligand column as the initial purification step. The ligand resin allows isolation of correctly folded GPCR variants directly from whole E. coli cell lysates at the scale of 10 mg and it permits preparations of agonist- and antagonist-bound Receptor samples. The purification principle presented here was key to the first structures of signaling-active NTR1 variants (Egloff et al., 2014). Since E. coli is uniquely suitable for the production of fully deuterated proteins, our method provides the basis for an array of NMR experiments that were not feasible for GPCRs to date, but which will shed light on novel aspects of Receptor function and dynamics.
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structure of signaling competent Neurotensin Receptor 1 obtained by directed evolution in escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Pascal Egloff, Philipp Heine, Stefanie Balada, Matthias Hillenbrand, Christoph Klenk, A Batyuk, Karola M Schlinkmann, Daniel Scott, Marco Schutz, Andreas PlückthunAbstract:Crystallography has advanced our understanding of G protein–coupled Receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using Neurotensin Receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three Neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 A resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type–like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein–coupled Receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many Receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein–coupled Receptor signaling, Receptor maturation, and desensitization.
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selection and characterization of darpins specific for the Neurotensin Receptor 1
Protein Engineering Design & Selection, 2009Co-Authors: Peter Milovnik, Davide Ferrari, Casim A. Sarkar, Andreas PlückthunAbstract:We describe here the selection and characterization of designed ankyrin repeat proteins (DARPins) that bind specifically to the rat Neurotensin Receptor 1 (NTR1), a G-protein coupled Receptor (GPCR). The selection procedure using ribosome display and the initial clone analysis required <10 microg of detergent-solubilized, purified NTR1. Complex formation with solubilized GPCR was demonstrated by ELISA and size-exclusion chromatography; additionally, the GPCR could be detected in native membranes of mammalian cells using fluorescence microscopy. The main binding epitope in the GPCR lies within the 33 amino acids following the seventh transmembrane segment, which comprise the putative helix 8, and additional binding interactions are possibly contributed by the cytoplasmic loop 3, thus constituting a discontinuous epitope. Since the selected binders recognize the GPCR both in detergent-solubilized and in membrane-embedded forms, they will be potentially useful both in co-crystallization trials and for signal transduction experiments.
Jeanpierre Vincent - One of the best experts on this subject based on the ideXlab platform.
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Neurotensin and the Neurotensin Receptor 3 in microglial cells
Journal of Neuroscience Research, 2005Co-Authors: Stephane Martin, Eleni Dicou, Jeanpierre Vincent, Jean MazellaAbstract:Microglia motility plays a crucial role in response to lesion or exocytotoxic damage of the cerebral tissue. The neuropeptide Neurotensin elicited the migration of the human microglial cell line C13NJ by a mechanism dependent on both phosphatidylinositol-3 kinase (PI3 kinase) and mitogen-activated protein (MAP) kinases pathways. The effect of Neurotensin on cell migration was blocked by the Neurotensin Receptor-3 propeptide, a selective ligand of this Receptor. The type I Neurotensin Receptor-3 was the only known Neurotensin Receptor expressed in these microglial cells, and its activation led to the phosphorylation of both extracellular signaling-regulated kinases Erk1/2 and Akt. Furthermore, the effect of Neurotensin on cell migration was preceded by a profound modification of the F-actin cytoskeleton, particularly by the rapid formation of numerous cell filopodia. Both the motility and the filopodia appearance induced by Neurotensin were totally blocked by selective inhibitors of MAP kinases or PI3 kinase pathways. In the murine microglial cell line N11, the Neurotensin Receptor-3 is also the only Neurotensin Receptor expressed, and its activation by Neurotensin leads to the phosphorylation of both Erk1/2 and Akt. In these cells, Neurotensin induces the gene expression of several cytokines/chemokines, including MIP-2, MCP-1, interleukin-1β and tumor necrosis factor-α. This induction is dependent on both protein kinases pathways. We observed that the effect of Neurotensin on the cytokine/chemokine expression is also inhibited by the Neurotensin Receptor-3 propeptide. This is the demonstration that the Neurotensin Receptor-3 is functional and mediates both the migratory action of Neurotensin and its induction of chemokines/cytokines expression. © 2005 Wiley-Liss, Inc.
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involvement of the Neurotensin Receptor 3 in the Neurotensin induced migration of human microglia
The Journal of Neuroscience, 2003Co-Authors: Stephane Martin, Jeanpierre Vincent, Jean MazellaAbstract:Microglia motility plays a crucial role in response to lesion or exocytotoxic damage of the cerebral tissue. We used two in vitro assays, a wound-healing model and a chemotaxis assay, to show that the neuropeptide Neurotensin elicited the migration of the human microglial cell line C13NJ by a mechanism dependent on both phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways. The effect of Neurotensin on cell migration was blocked by the Neurotensin Receptor-3 propeptide, a selective ligand of this Receptor. We demonstrate, by using RT-PCR, photoaffinity labeling, and Western blot analysis, that the type I Neurotensin Receptor-3 was the only known Neurotensin Receptor expressed in these microglial cells and that its activation led to the phosphorylation of both extracellular signal-regulating kinases 1/2 and Akt. Furthermore, the effect of Neurotensin on cell migration was preceded by a profound modification of the F-actin cytoskeleton, particularly by the rapid formation of numerous cell filopodia. Both the motility and the filopodia appearance induced by Neurotensin were totally blocked by selective inhibitors of MAP kinases or PI 3-kinase pathways. This demonstrates that the Neurotensin Receptor-3 is functional and mediates the migratory actions of Neurotensin.
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involvement of the Neurotensin Receptor subtype ntr3 in the growth effect of Neurotensin on cancer cell lines
International Journal of Cancer, 2001Co-Authors: Claude Dal Farra, Jean Mazella, Jeanmarie Botto, Philippe Sarret, Valerie Navarro, Jeanpierre VincentAbstract:The expression of the 3 currently known Neurotensin Receptors was studied in human cancer cells of prostatic, colonic or pancreatic origin by means of RT-PCR analysis and binding experiments. All the cells selected for this work have been shown to exhibit a growth response to Neurotensin. We found that the 7 transmembrane domain, levocabastine insensitive Receptor (NTR1) is expressed in most but not all of the cells studied whereas the 7 transmembrane domain, levocabastine sensitive Receptor (NTR2) is present in none of these cells. The 100 kDa-type I Neurotensin Receptor (NTR3) is expressed in all the cells assayed. Moreover, we demonstrated that Neurotensin can stimulate the growth of CHO cells stably transfected with the NTR3. Taken together, our results strongly suggest that the NTR3 subtype could be involved in the growth response of human cancer cells to Neurotensin. © 2001 Wiley-Liss, Inc.
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Stable Expression of the Mouse Levocabastine-Sensitive Neurotensin Receptor in HEK 293 Cell Line: Binding Properties, Photoaffinity Labeling, and Internalization Mechanism☆
Biochemical and biophysical research communications, 1998Co-Authors: Jeanmarie Botto, Jeanpierre Vincent, Philippe Sarret, Joëlle Chabry, Jean MazellaAbstract:The recently cloned new subtype of G protein-coupled Neurotensin Receptor (NTRL) was stably expressed in the HEK 293 cell line in order to investigate its binding and internalization properties. The expressed Receptor exhibited the typical binding characteristics of the low affinity, levocabastine-sensitive binding site previously described in rat and mouse brain and was detected as a protein with an apparent MW of 45 kDa by photoaffinity labeling. Although intracellular modulation of adenylate cyclase, guanylate cyclase and phospholipase C was not detected after application of Neurotensin or levocabastine on NTRL-transfected cells, this Receptor was able to internalize iodinated Neurotensin. The internalization process was followed by recycling of Receptors to the cell membrane. By contrast, no recycling was observed with the high affinity Neurotensin Receptor (NTRH). The differential intracellular routing of NTRH and NTRL after internalization is most probably the consequence of their divergent carboxy-terminal sequences.
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effects of sr 48692 on Neurotensin induced calcium activated chloride currents in the xenopus oocyte expression system agonist like activity on the levocabastine sensitive Neurotensin Receptor and absence of antagonist effect on the levocabastine ins
Neuroscience Letters, 1997Co-Authors: Jeanmarie Botto, Jeanpierre Vincent, Eric Guillemare, Jean MazellaAbstract:Abstract The effect of the drug SR 48692 on the Ca 2+ -activated Cl − current induced by Neurotensin on Xenopus oocytes injected with cRNAs encoding rodent high and low affinity Neurotensin Receptors, was examined. In this Receptor expression system, SR 48692 failed to antagonize electrophysiological measurement of Neurotensin-evoked current via the rat high affinity Neurotensin Receptor, whereas its application onto oocytes expressing the mouse low affinity Neurotensin Receptor triggered an inward current, as well as Neurotensin itself. However, no current activation was observed after application of the drug on oocytes expressing the rat high affinity Neurotensin Receptor. These observations in the oocyte expression system did not reflect typical antagonist properties of SR 48692 drug.