Mécanismes de l'internalisation du récepteur CCK2 : bases pharmacologiques et structurales

Le récepteur CCK2 est un récepteur couplé aux protéines G (RCPG) possédant deux ligands naturels, la cholécystokinine et la gastrine, retrouvées au niveau du système nerveux central et du système gastro-intestinal. Le récepteur CCK2 est impliqué dans de nombreux processus physiologiques et physiopathologiques dont les cancers. La présence membranaire des RCPG est hautement régulée otamment par le mécanisme d'internalisation après timulation par un agoniste. Les beta-arrestines initient l'internalisation de nombreux RCPG et sont également capables d'agir comme des protéines d'échafaudage permettant aux récepteurs d'émettre un signal indépendamment des protéines G. De plus, des ligands dits " biaisés " sont capables d'activer sélectivement les voies de transductions du signal dépendantes des protéines G ou dépendantes des beta-arrestines. Les mécanismes de l'internalisation du récepteur CCK2 n'étaient pas connus et nos objectifs ont été, dans un premier temps, de caractériser ces mécanismes, puis de montrer que des ligands synthétiques du RCCK2 possèdent une activité " biaisée " et enfin de caractériser la conformation du récepteur CCK2 recrutant les beta-arrestines. Dans ce travail, nous rapportons que l'internalisation du récepteur CCK2 est un phénomène faisant intervenir les beta-arrestines, la clathrine et la dynamine. Nous localisons la zone d'interaction des beta-arrestines sur le récepteur CCK2, en identifiant un groupe de sérinethréonine située dans la partie distale de l'extrémité C-terminale du récepteur. Cependant, l'utilisation de mutants du récepteur présentant un recrutement beta-arrestines défectueux ou de cellules invalidées pour les deux beta-arrestines montre que le récepteur CCK2 est toujours capable de s'internaliser, suggérant la présence d'un mécanisme alternatif. Nous identifions des ligands synthétiques du récepteur CCK2 avec une activité " biaisée " capables d'activer des voies de transduction dépendantes des protéines G mais ne recrutant pas les beta-arrestines. De plus, un ligand, le GV150,013X n'est pas capable d'inhiber le recrutement des beta-arrestines induit par la CCK alors qu'il inhibe la production d'inositol phosphate de façon compétitive. La mutation d'acides aminés suffit pour diminuer le recrutement des beta-arrestines au RCCK2 sans altérer sa capacité à activer la PLC. Ces résultats sont les premiers à montrer l'existence d'une conformation moléculaire d'un RCPG recrutant les beta-arrestines distincte de celle activant la voie dépendante de la protéine G. Les résultats de cette étude apportent des informations quant à la régulation pharmacologique de l'internalisation du récepteur CCK2 et supportent le concept de sélectivité fonctionnelle des RCPG.The CCK2 receptor (CCK2R) is a G-protein coupled receptor (GPCR) and binds to two different natural ligands, cholecystokinin and gastrin, which are mostly localized in the gastro-intestinal tract and the central nervous system. The CCK2 receptor is involved in various physiological and pathological processes including cancers. The localization of GPCR at the cell membrane is highly regulated notably by internalization of the receptors following agonist stimulation. The -arrestins initiate internalization of many GPCR and act as scaffolding proteins leading GPCR to trigger G-protein independent signal transduction. Recently, biased ligands have been discovered that selectively activates either G-protein or -arrestins mediated signaling pathways. The mechanisms of CCK2R internalization were not known and the purpose of our study was first to characterize these mechanisms and then to show that synthetic ligands of CCK2R may have a biased activity towards -arrestins recruitment. Finally, we aimed at characterizing a conformational state of the CCK2R recruiting -arrestins distinct from the state activating G-proteins. The CCK2 receptor (CCK2R) is a G-protein coupled receptor (GPCR) and binds to two different natural ligands, cholecystokinin and gastrin, which are mostly localized in the gastro-intestinal tract and the central nervous system. The CCK2 receptor is involved in various physiological and pathological processes including cancers. The localization of GPCR at the cell membrane is highly regulated notably by internalization of the receptors following agonist stimulation. The -arrestins initiate internalization of many GPCR and act as scaffolding proteins leading GPCR to trigger G-protein independent signal transduction. Recently, biased ligands have been discovered that selectively activates either G-protein or -arrestins mediated signaling pathways. The mechanisms of CCK2R internalization were not known and the purpose of our study was first to characterize these mechanisms and then to show that synthetic ligands of CCK2R may have a biased activity towards -arrestins recruitment. Finally, we aimed at characterizing a conformational state of the CCK2R recruiting -arrestins distinct from the state activating G-proteins. In this work, we report that internalization of CCK2R is a process involving both beta-arrestine1 and 2 as well as clathrin and dynamin. We characterized the binding site of -arrestin on CCK2R, by identifying a cluster of serine/threonine in the distal part of the receptor C-terminus. However, CCK2R mutants lacking this region or receptors expressed in cells deleted of both -arrestins still displayed strong internalization of receptors suggesting the existence of an alternative mechanism that does not require -arrestins. We also identified synthetic ligands of CCK2R that display biased activity, by activating G-protein mediated signaling pathways without recruiting -arrestins. Furthermore, a CCK2R ligand, GV150,013X was not able to inhibit CCK-induced -arrestins recruitment whereas it acts as a potent competitive antagonist on CCK-induced inositol phosphates production. Else, the mutation of specific single amino acid into the binding site of CCK2R was sufficient to significantly reduce -arrestins recruitment without altering CCK-induced G-protein mediated signaling pathways. To our knowledge, these results show for the first time, that a GPCR conformational state recruiting b-arrestins is different from a state activating G-proteins. This study brings new insights into the pharmacological regulation of CCK2R internalization and strongly supports the concept of "functional selectivity"

Role of gut microbiota and bacterial translocation in acute intestinal injury and mortality in patients admitted in ICU for septic shock

IntroductionSepsis is a life-threatening organ dysfunction with high mortality rate. The gut origin hypothesis of multiple organ dysfunction syndrome relates to loss of gut barrier function and the ensuing bacterial translocation. The aim of this study was to describe the evolution of gut microbiota in a cohort of septic shock patients over seven days and the potential link between gut microbiota and bacterial translocation.MethodsSixty consecutive adult patients hospitalized for septic shock in intensive care units (ICU) were prospectively enrolled. Non-inclusion criteria included patients with recent or scheduled digestive surgery, having taken laxatives, pre- or probiotic in the previous seven days, a progressive digestive neoplasia, digestive lymphoma, chronic inflammatory bowel disease, moribund patient, and pregnant and lactating patients. The primary objective was to evaluate the evolution of bacterial diversity and richness of gut microbiota during seven days in septic shock. Epidemiological, clinical and biological data were gathered over seven days. Gut microbiota was analyzed through a metagenomic approach. 100 healthy controls were selected among healthy blood donors for reference basal 16S rDNA values.ResultsSignificantly lower bacterial diversity and richness was observed in gut microbiota of patients at Day 7 compared with Day 0 (p<0.01). SOFA score at Day 0, Acute Gastrointestinal Injury (AGI) local grade, septic shock origin and bacterial translocation had an impact on alpha diversity. A large increase in Enterococcus genus was observed at Day 7 with a decrease in Enterobacterales, Clostridiales, Bifidobacterium and other butyrate-producing bacteria.DiscussionThis study shows the importance of bacterial translocation during AGI in septic shock patients. This bacterial translocation decreases during hospitalization in ICUs in parallel to the decrease of microbiota diversity. This work highlights the role of gut microbiota and bacterial translocation during septic shock

Mécanismes de l'internalisation du récepteur CCK2 (bases pharmacologiques et structurales)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Regulation of membrane cholecystokinin-2 receptor by agonists enables classification of partial agonists as biased agonists.

International audienceGiven the importance of G-protein-coupled receptors as pharmacological targets in medicine, efforts directed at understanding the molecular mechanism by which pharmacological compounds regulate their presence at the cell surface is of paramount importance. In this context, using confocal microscopy and bioluminescence resonance energy transfer, we have investigated internalization and intracellular trafficking of the cholecystokinin-2 receptor (CCK2R) in response to both natural and synthetic ligands with different pharmacological features. We found that CCK and gastrin, which are full agonists on CCK2R-induced inositol phosphate production, rapidly and abundantly stimulate internalization. Internalized CCK2R did not rapidly recycle to plasma membrane but instead was directed to late endosomes/lysosomes. CCK2R endocytosis involves clathrin-coated pits and dynamin and high affinity and prolonged binding of β-arrestin1 or -2. Partial agonists and antagonists on CCK2R-induced inositol phosphate formation and ERK1/2 phosphorylation did not stimulate CCK2R internalization or β-arrestin recruitment to the CCK2R but blocked full agonist-induced internalization and β-arrestin recruitment. The extreme C-terminal region of the CCK2R (and more precisely phosphorylatable residues Ser(437)-Xaa(438)-Thr(439)-Thr(440)-Xaa(441)-Ser(442)-Thr(443)) were critical for β-arrestin recruitment. However, this region and β-arrestins were dispensable for CCK2R internalization. In conclusion, this study allowed us to classify the human CCK2R as a member of class B G-protein-coupled receptors with regard to its endocytosis features and identified biased agonists of the CCK2R. These new important insights will allow us to investigate the role of internalized CCK2R*β-arrestin complexes in cancers expressing this receptor and to develop new diagnosis and therapeutic strategies targeting this receptor

Distinct CCK-2 Receptor Conformations Associated with β-Arrestin-2 Recruitment or Phospholipase-C Activation Revealed by a Biased Antagonist

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Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration.

Marielle Saclier and Houda Yacoub-Youssef : Both should be considered as first authorInternational audienceMacrophages (MPs) exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for adult skeletal muscle repair, notably by acting on myogenic precursor cells. However, these interactions have not been fully characterized. Here, we explored both in vitro and in vivo, in human, the interactions of differentially activated MPs with myogenic precursor cells (MPCs) during adult myogenesis and skeletal muscle regeneration. We showed in vitro that through the differential secretion of cytokines and growth factors, proinflammatory MPs inhibited MPC fusion while anti-inflammatory MPs strongly promoted MPC differentiation by increasing their commitment into differentiated myocytes and the formation of mature myotubes. Furthermore, the in vivo time course of expression of myogenic and MP markers was studied in regenerating human healthy muscle after damage. We observed that regenerating areas containing proliferating MPCs were preferentially associated with MPs expressing proinflammatory markers. In the same muscle, regenerating areas containing differentiating myogenin-positive MPCs were preferentially coupled to MPs harboring anti-inflammatory markers. These data demonstrate for the first time in human that MPs sequentially orchestrate adult myogenesis during regeneration of damaged skeletal muscle. These results support the emerging concept that inflammation, through MP activation, controls stem cell fate and coordinates tissue repair

AMPK Activation Regulates LTBP4-Dependent TGF-β1 Secretion by Pro-inflammatory Macrophages and Controls Fibrosis in Duchenne Muscular Dystrophy

International audienceChronic inflammation and fibrosis characterize Duchenne muscular dystrophy (DMD). We show that pro-inflammatory macrophages are associated with fibrosis in mouse and human DMD muscle. DMD-derived Ly6Cpos macrophages exhibit a profibrotic activity by sustaining fibroblast production of collagen I. This is mediated by the high production of latent-TGF-β1 due to the higher expression of LTBP4, for which polymorphisms are associated with the progression of fibrosis in DMD patients. Skewing macrophage phenotype via AMPK activation decreases ltbp4 expression by Ly6Cpos macrophages, blunts the production of latent-TGF-β1, and eventually reduces fibrosis and improves DMD muscle force. Moreover, fibro-adipogenic progenitors are the main providers of TGF-β-activating enzymes in mouse and human DMD, leading to collagen production by fibroblasts. In vivo pharmacological inhibition of TGF-β-activating enzymes improves the dystrophic phenotype. Thus, an AMPK-LTBP4 axis in inflammatory macrophages controls the production of TGF-β1, which is further activated by and acts on fibroblastic cells, leading to fibrosis in DMD

Coupling between Myogenesis and Angiogenesis during Skeletal Muscle Regeneration Is Stimulated by Restorative Macrophages

Summary: In skeletal muscle, new functions for vessels have recently emerged beyond oxygen and nutrient supply, through the interactions that vascular cells establish with muscle stem cells. Here, we demonstrate in human and mouse that endothelial cells (ECs) and myogenic progenitor cells (MPCs) interacted together to couple myogenesis and angiogenesis in vitro and in vivo during skeletal muscle regeneration. Kinetics of gene expression of ECs and MPCs sorted at different time points of regeneration identified three effectors secreted by both ECs and MPCs. Apelin, Oncostatin M, and Periostin were shown to control myogenesis/angiogenesis coupling in vitro and to be required for myogenesis and vessel formation during muscle regeneration in vivo. Furthermore, restorative macrophages, which have been previously shown to support myogenesis in vivo, were shown in a 3D triculture model to stimulate myogenesis/angiogenesis coupling, notably through Oncostatin M production. Our data demonstrate that restorative macrophages orchestrate muscle regeneration by controlling myogenesis/angiogenesis coupling. : In this study, Chazaud et al. demonstrate that endothelial cells (ECs) and myogenic progenitor cells (MPCs) interacted to couple myogenesis and angiogenesis during skeletal muscle regeneration. EC- and MPC-derived Apelin, Oncostatin M, and Periostin controlled myogenesis/angiogenesis coupling and were required for myogenesis and vessel formation. They show that, via the production of Oncostatin M, restorative macrophages promoted myogenesis/angiogenesis coupling. Keywords: muscle stem cells, myogenesis, angiogenesis, skeletal muscle regeneration, macrophage