Gain of affinity for VEGF165 binding within the VEGFR2/NRP1 cellular complex detected by an HTRF-based binding assay

Auriau, Johanna; Roujeau, Clara; Choucair, Zakia Belaid; Oishi, Atsuro; Derviaux, Carine; Roux, Thomas; Trinquet, Eric; Hermine, Olivier; Jockers, Ralf; Dam, Julie

doi:10.1016/j.bcp.2018.09.014

Cited by 8 publications

(4 citation statements)

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“…To study the effect of ligand binding on the proximity between leptin and EGF and their cognate receptors within the LepR:EGFR complex, we next used Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) in living HEK293 cells ( Supplementary Figure 3F ). Using targeted fluorescently-labeled SNAP receptors (energy donor, Terbium ( Tb )) and ligands (energy acceptor, d2 ), TR-FRET signals the presence of a ligand at ≤10 nm from a specific and unique receptor within a heteromeric receptor complex 33 . Because leptin does not bind to EGFR, the incubation of fluorescent leptin-d2 even at high concentrations with fluorescent SNAP-EGFR did not yield any TR-FRET signal ( Figure 2I , Supplementary Figure 3F ).…”

Section: Resultsmentioning

confidence: 99%

Leptin brain entry via a tanycytic LepR–EGFR shuttle controls lipid metabolism and pancreas function

et al. 2021

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Summary Metabolic health depends on the brain’s ability to control food intake and nutrient use versus storage, processes that require peripheral signals such as the adipocyte-derived hormone, leptin, to cross brain barriers and mobilize regulatory circuits. We have previously shown that hypothalamic tanycytes shuttle leptin into the brain to reach target neurons. Here, using multiple complementary models, we show that tanycytes express functional leptin receptor (LepRb), respond to leptin by triggering Ca2+ waves and target-protein phosphorylation, and that their transcytotic transport of leptin requires the activation of a LepR:EGFR complex by leptin and EGF sequentially. Selectively deleting LepR in tanycytes blocks leptin entry into the brain, inducing not only increased food intake and lipogenesis but glucose intolerance through attenuated insulin secretion by pancreatic β-cells, possibly via altered sympathetic nervous tone. Tanycytic LepRb:EGFR-mediated transport of leptin could thus be crucial to the pathophysiology of diabetes in addition to obesity, with therapeutic implications.

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Section: Resultsmentioning

confidence: 99%

Leptin brain entry via a tanycytic LepR–EGFR shuttle controls lipid metabolism and pancreas function

et al. 2021

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“…The expression level of GPCR-YFP was monitored by measuring Fluorescence of YFP by Mithras. The expression level of Snap-AT1R and Snap-β 2 AR was labeled with Tag-lite and measured Fluorescence signal as previously described …”

Section: Methodsmentioning

confidence: 99%

β-Arrestin-2 BRET Biosensors Detect Different β-Arrestin-2 Conformations in Interaction with GPCRs

2019

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β-Arrestins are critical regulators of G proteincoupled receptors (GPCRs) that desensitize G protein signaling, promote receptor internalization, and initiate signaling on their own. Recent structural findings indicate that β-arrestins adopt different conformations upon interaction with agonist-activated GPCRs. Here, we established a β-arrestin-2 conformational bioluminescence resonance energy transfer (BRET) sensor composed of the bright Nanoluc BRET donor and the red-shifted CyOFP1 BRET acceptor. The sensor monitors early intramolecular conformational changes of β-arrestin-2 in complex with a wide panel of different class A and class B GPCRs upon agonist activation and with orphan GPCRs known to spontaneously recruit βarrestin-2. The introduction of the R170E mutant in the β-arrestin-2 sensor allowed the detection of a partially active β-arrestin-2 conformation, which is not dependent on receptor phosphorylation, while the deletion of the β-arrestin-2 finger-loop region detected the "tail-conformation" corresponding to the interaction of β-arrestin with the carboxyl-terminal domain of GPCRs. The new sensors combine the advantages of the BRET technique in terms of sensitivity, robustness, and suitability for real-time measurements with a high responsiveness toward early conformational changes to help to elucidate the different conformational states of β-arrestins associated with GPCR activation in living cells.

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“…In the cytosol, enzymes such as amino acid oxidases, cyclooxygenases, lipoxygenases, nitric oxide (NO) synthase, and xanthine oxidase also generate ROS such as superoxide anions, peroxides and others [32][33][34]. ROS modulate several specific signaling pathways that regulate several cellular functions as well as pathological mechanisms in several human diseases [35][36][37][38]. The cyclooxygenases and lipoxygenases generate superoxide anions that drive arachidonic acid metabolism and inflammation, which play an important role in several cancers, whereas, the xanthine oxidase is implicated in oxidative stress during ischemia/ reperfusion injury [39][40][41].…”

Section: Oxidative Stressmentioning

confidence: 99%

Role of mitochondrial quality control in the pathogenesis of nonalcoholic fatty liver disease

Toan

Zhou

2020

Aging

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Nutrient oversupply and mitochondrial dysfunction play central roles in nonalcoholic fatty liver disease (NAFLD). The mitochondria are the major sites of β-oxidation, a catabolic process by which fatty acids are broken down. The mitochondrial quality control (MQC) system includes mitochondrial fission, fusion, mitophagy and mitochondrial redox regulation, and is essential for the maintenance of the functionality and structural integrity of the mitochondria. Excessive and uncontrolled production of reactive oxygen species (ROS) in the mitochondria damages mitochondrial components, including membranes, proteins and mitochondrial DNA (mtDNA), and triggers the mitochondrial pathway of apoptosis. The functionality of some damaged mitochondria can be restored by fusion with normally functioning mitochondria, but when severely damaged, mitochondria are segregated from the remaining functional mitochondrial network through fission and are eventually degraded via mitochondrial autophagy, also called as mitophagy. In this review, we describe the functions and mechanisms of mitochondrial fission, fusion, oxidative stress and mitophagy in the development and progression of NAFLD.

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Gain of affinity for VEGF165 binding within the VEGFR2/NRP1 cellular complex detected by an HTRF-based binding assay

Cited by 8 publications

References 77 publications

Leptin brain entry via a tanycytic LepR–EGFR shuttle controls lipid metabolism and pancreas function

Leptin brain entry via a tanycytic LepR–EGFR shuttle controls lipid metabolism and pancreas function

β-Arrestin-2 BRET Biosensors Detect Different β-Arrestin-2 Conformations in Interaction with GPCRs

Role of mitochondrial quality control in the pathogenesis of nonalcoholic fatty liver disease

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