Crystal structure of the neurotensin receptor 1 (NTSR1-H4bmx) in complex with NTS8-13
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Neurotensin (NT) is a tridecapeptide displaying interesting antinociceptive properties through its action on its receptors, NTS1 and NTS2. Neurotensin-like compounds have been shown to exert better antinociceptive properties than morphine at equimolar doses. In this article, we characterized the molecular effects of a novel neurotensin (8-13) (NT(8-13)) analog containing an unnatural amino acid. This compound, named JMV2009, displays a Silaproline in position 10 in replacement of a proline in the native NT(8-13). We first examined the binding affinities of this novel NT(8-13) derivative at both NTS1 and NTS2 receptor sites by performing competitive displacement of iodinated NT on purified cell membranes. Then, we evaluated the ability of JMV2009 to activate NTS1-related G proteins as well as to promote the recruitment of β-arrestins 1 and 2 by using BRET-based cellular assays in live cells. We next assessed its ability to induce p42/p44 MAPK phosphorylation and NT receptors internalization using western blot and cell-surface ELISA, respectively. Finally, we determined the in vitro plasma stability of this NT derivative. This article is associated with the original article "Pain relief devoid of opioid side effects following central action of a silylated neurotensin analog" published in European Journal of Pharmacology[1]. The reader is directed to the associated article for results interpretation, comments, and discussion.
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Neurotensin receptor
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Abstract 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 ( K i =2.8 n M ) 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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Abstract The neuromodulatory peptide neurotensin has been described to functionally interact with dopaminergic pathways of the human brain. We employed radioligand binding studies to investigate the physical interaction between co‐expressed dopamine D 2L or D 3 and neurotensin NTS 1 or NTS 2 receptors. Substantial cross‐inhibitory effects of both receptor subtypes NTS 1 and NTS 2 on the agonist binding of D 2L or D 3 were detected in the presence of neurotensin. To identify ligand‐specific modulation and subtype‐dependent differences, the novel dopamine receptor agonists 5 and 6 bearing the 7‐OH‐DPAT pharmacophore were synthesized. Exceptional ligand specificity was observed for D 3 –NTS 2 co‐expression, which gave a 20‐fold decrease in affinity for biphenylcarboxamide 5 in the presence of neurotensin. Comparing the binding properties of dopaminergic compounds in the presence of neurotensin, dopamine receptor subtype‐selective profiles of the cross‐inhibitory effect of neurotensin were observed.
Neurotensin receptor
Endogenous agonist
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Dopaminergic systems have been described to functionally interact with the neuromodulatory peptide neurotensin. Employing fluorescence detected coimmunoprecipitation and radioligand binding experiments, we herein demonstrate that coexpression of dopamine D(2L) receptor and the neurotensin receptor subtype NTS(1) leads to physical interaction and the formation of heteromers in transfected human embryonic kidney 293 cells. In this in vitro system, a trans-inhibitory effect on the agonist binding affinity of D(2) was observed in presence of neurotensin. To correlate between the functional properties of dopaminergic agents and the magnitude of neurotensin-induced modulation of D(2L) binding affinities in cells coexpressing D(2L) and NTS(1), a structurally diverse set of dopamine receptor agonists, partial agonists, and antagonists was tested. Ligand specific profiles indicating substantial bias between ligand efficacy and transmodulation were discovered, suggesting a heteromerization-based functional selectivity. In the presence of neurotensin, the novel D(2) agonist FAUC 326 displayed a 34-fold decrease of binding affinity in cells coexpressing D(2L) and NTS(1).
Neurotensin receptor
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Endogenous agonist
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Abstract Proceeding investigations of G protein-coupled receptor (GPCR) heterocomplexes have demonstrated that the dopamine D2 receptor (D 2 R), one of the hub receptors in the physiology of schizophrenia, interacts with both the neurotensin NTS1 (NTS1R) and the serotonin 5-HT 2A receptor (5-HT 2A R) in cell lines and rodent brain tissue. In situ proximity ligation assay and BRET-based saturation experiments confirmed interacting receptor assemblies in HEK293T and neuronal HT22 cells. The NTS1R agonist NT(8-13) reduces the Gα q -mediated calcium signal in the NTS1R-D 2 R complex compared to the NTS1R monomer which could be reversed by D 2 R antagonists. The bivalent ligand CS148 (NTS1R-agonistic, D 2 R-antagonistic) increased the calcium response addressing the dimer, consistent with the effect of the monovalent ligands suggesting an allosteric D 2 R-mediated modulation. In contrast, the 5-HT 2A R-D 2 R heteromer did not show a calcium-altering receptor-receptor interaction. Despite their common coupling-preference for Gα q , 5-HT 2A R and NTS1R supposedly interact with D 2 R each in a unique mode. This remarkably diverse ligand-mediated signalling in two different D 2 R heteroreceptor complexes illustrates the complexity of receptor-receptor interactions and their potential of modifying cell responses to external stimuli. Therefore, GPCR heteromers may provide a very promising novel target for the therapy of neuropsychiatric disorders.
Allosteric modulator
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Inverse agonist
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