Physical and functional interactions between the serotonin transporter and the neutral amino acid transporter ASCT2

International audienceThe activity of serotonergic systems depends on the reuptake of extracellular serotonin via its plasma membrane serotonin [5-HT (5-hydroxytryptamine)] transporter (SERT), a member of the Na(+)/Cl(-)-dependent solute carrier 6 family. SERT is finely regulated by multiple molecular mechanisms including its physical interaction with intracellular proteins. The majority of previously identified SERT partners that control its functional activity are soluble proteins, which bind to its intracellular domains. SERT also interacts with transmembrane proteins, but its association with other plasma membrane transporters remains to be established. Using a proteomics strategy, we show that SERT associates with ASCT2 (alanine-serine-cysteine-threonine 2), a member of the solute carrier 1 family co-expressed with SERT in serotonergic neurons and involved in the transport of small neutral amino acids across the plasma membrane. Co-expression of ASCT2 with SERT in HEK (human embryonic kidney)-293 cells affects glycosylation and cell-surface localization of SERT with a concomitant reduction in its 5-HT uptake activity. Conversely, depletion of cellular ASCT2 by RNAi enhances 5-HT uptake in both HEK-293 cells and primary cultured mesencephalon neurons. Mimicking the effect of ASCT2 down-regulation, treatment of HEK-293 cells and neurons with the ASCT2 inhibitor D-threonine also increases 5-HT uptake. Moreover, D-threonine does not enhance further the maximal velocity of 5-HT uptake in cells depleted of ASCT2. Collectively, these findings provide evidence for a complex assembly involving SERT and a member of another solute carrier family, which strongly influences the subcellular distribution of SERT and the reuptake of 5-HT

5-HT 4 Receptors Constitutively Promote the Non-Amyloidogenic Pathway of APP Cleavage and Interact with ADAM10

International audienceIn addition to the amyloidogenic pathway, amyloid precursor protein (APP) can be cleaved by α-secretases, producing soluble and neuroprotective APP alpha (sAPPα) (nonamyloidogenic pathway) and thus preventing the generation of pathogenic amyloid-β. However, the mechanisms regulating APP cleavage by α-secretases remain poorly understood. Here, we showed that expression of serotonin type 4 receptors (5-HT(4)Rs) constitutively (without agonist stimulation) induced APP cleavage by the α-secretase ADAM10 and the release of neuroprotective sAPPα in HEK-293 cells and cortical neurons. This effect was independent of cAMP production. Interestingly, we demonstrated that 5-HT(4) receptors physically interacted with the mature form of ADAM10. Stimulation of 5-HT(4) receptors by an agonist further increased sAPPα secretion, and this effect was mediated by cAMP/Epac signaling. These findings describe a new mechanism whereby a GPCR constitutively stimulates the cleavage of APP by α-secretase and promotes the nonamyloidogenic pathway of APP processing

Beta-arrestin 2 barcode of phosphorylation: impact on beta-arrestin-dependent signaling

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Deciphering beta-arrestin-dependent signaling by RPPA

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Relative contributions of G protein and beta-arrestin-dependent transduction to FSH-R mediated signaling

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p.N680S single nucleotide polymorphism of the FSHR affects FSH-induced β-arrestin recruitment, β-arrestin-dependent translocation of PKA catalytic subunit to the nucleus and CRE-driven transcription

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5‑HT₄ Receptors Constitutively Promote the Non-Amyloidogenic Pathway of APP Cleavage and Interact with ADAM10

In addition to the amyloidogenic pathway, amyloid precursor protein (APP) can be cleaved by α-secretases, producing soluble and neuroprotective APP alpha (sAPPα) (nonamyloidogenic pathway) and thus preventing the generation of pathogenic amyloid-β. However, the mechanisms regulating APP cleavage by α-secretases remain poorly understood. Here, we showed that expression of serotonin type 4 receptors (5-HT₄Rs) constitutively (without agonist stimulation) induced APP cleavage by the α-secretase ADAM10 and the release of neuroprotective sAPPα in HEK-293 cells and cortical neurons. This effect was independent of cAMP production. Interestingly, we demonstrated that 5-HT₄ receptors physically interacted with the mature form of ADAM10. Stimulation of 5-HT₄ receptors by an agonist further increased sAPPα secretion, and this effect was mediated by cAMP/Epac signaling. These findings describe a new mechanism whereby a GPCR constitutively stimulates the cleavage of APP by α-secretase and promotes the nonamyloidogenic pathway of APP processing

β-arrestin1 phosphorylation by GRK5 regulates G protein-independent 5-HT 4 receptor signalling

International audienceG protein‐coupled receptors (GPCRs) have been found to trigger G protein‐independent signalling. However, the regulation of G protein‐independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein‐independent 5‐HT4 receptor (5‐HT4R)‐operated Src/ERK (extracellular signal‐regulated kinase) pathway, but not the Gs pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor’ C‐terminus in both human embryonic kidney (HEK)‐293 cells and colliculi neurons. This inhibition required two sequences of events: the association of β–arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C‐t domain and the phosphorylation, by GRK5, of β–arrestin1 (at Ser412) bound to the receptor. Phosphorylated β‐arrestin1 in turn prevented activation of Src constitutively bound to 5‐HT4Rs, a necessary step in receptor‐stimulated ERK signalling. This is the first demonstration that β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent signalling

Radioresistance and genomic alterations in head and neck squamous cell cancer: Sub‐analysis of the ProfiLER protocol

International audienceBackgroundGenome analysis could provide tools to assess predictive molecular biomarkers of radioresistance.MethodsHead and neck squamous cell carcinoma patients included in ProfiLER study and who underwent a curative radiotherapy were screened. Univariate and Cox multivariate analyses were performed to explore the relationships between molecular abnormalities, infield relapse and complete tumor response after radiation.ResultsOne hundred and forty-three patients were analyzed. PIK3CA mutation and genomic instability of MAP kinases pathway were found to be prognostic factors of loco-regional relapse in multivariate analysis with respectively HR 0.33, 95% CI 0.13–0.83, p = 0.005 and HR 0.61, 95% CI 0.38–0.96, p = 0.025. Instability of apoptosis pathway was found to be a prognostic factor of complete response after radiotherapy with HR 0.24, 95% CI 0.07–0.88, p = 0.04.ConclusionThis sub analysis suggests that PIK3CA mutation, variation of copy number of MAP kinases and apoptosis pathways play a significant role in the radioresistance phenomeno

Structure-Based Design of PDZ Ligands as Inhibitors of 5‑HT_2A Receptor/PSD-95 PDZ1 Domain Interaction Possessing Anti-hyperalgesic Activity

Disrupting the interaction between the PDZ protein PSD-95 and the C-terminal domain of the 5-HT_2A serotonin receptor has been shown to reduce hyperalgesia in a rodent model of neuropathic pain. Here, we designed and synthesized PDZ ligands capable of binding to the first PDZ domain (PDZ1) of the PSD-95 protein and evaluated their biological activity <i>in vitro</i> and <i>in vivo</i>. A series of substituted indoles was identified by docking simulations, and six novel analogues were synthesized. Three analogues displayed strong interactions with the first PDZ domain (PDZ1) of PDZ-95 in ¹H–¹⁵N heteronuclear single-quantum coherence (HSQC) experiments and two of them were able to inhibit the interaction between PSD-95 and the 5-HT_2A receptor <i>in vitro</i>. We identified compound <b>8b</b> as the analogue able to significantly suppress mechanical hyperalgesia in an experimental model of traumatic neuropathic pain in the rat. This effect was suppressed by the coadministration of the 5-HT_2A receptor antagonist M100907, consistent with an inhibitory effect upon 5-HT_2A receptor/PSD-95 interaction. Finally, we determined an NMR-restraint driven model structure for the PSD95 PDZ1/<b>8b</b> complex, which confirms that indole <b>8b</b> binds to the putative PDZ-ligand binding site