BRET Analysis of GPCR Dimers in Neurons and Non-Neuronal Cells: Evidence for Inactive, Agonist, and Constitutive Conformations

International audienceG-protein-coupled receptors (GPCRs) are dimeric proteins, but the functional consequences of the process are still debated. Active GPCR conformations are promoted either by agonists or constitutive activity. Inverse agonists decrease constitutive activity by promoting inactive conformations. The histamine H3 receptor (H3R) is the target of choice for the study of GPCRs because it displays high constitutive activity. Here, we study the dimerization of recombinant and brain H3R and explore the effects of H3R ligands of different intrinsic efficacy on dimerization. Co-immunoprecipitations and Western blots showed that H3R dimers co-exist with monomers in transfected HEK 293 cells and in rodent brains. Bioluminescence energy transfer (BRET) analysis confirmed the existence of spontaneous H3R dimers, not only in living HEK 293 cells but also in transfected cortical neurons. In both cells, agonists and constitutive activity of the H3R decreased BRET signals, whereas inverse agonists and GTPγS, which promote inactive conformations, increased BRET signals. These findings show the existence of spontaneous H3R dimers not only in heterologous systems but also in native tissues, which are able to adopt a number of allosteric conformations, from more inactive to more active states

A Large-Scale Epidemiological Study to Identify Bacteria Pathogenic to Pacific Oyster Crassostrea gigas and Correlation Between Virulence and Metalloprotease-like Activity

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LINGO-1, a protein involved in various neurodevelopmental processes, interacts with neurofibromin (Nf1), the protein responsible for neurofibromatosis type I: molecular studies and functional implications

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Expression of SAA1, SAA2 and SAA4 genes in human primary monocytes and monocyte-derived macrophages

International audienceCirculating serum amyloid A (SAA) is increased in various inflammatory conditions. The human SAA protein family comprises the acute phase SAA1/SAA2, known to activate a large set of innate and adaptive immune cells, and the constitutive SAA4. The liver synthesis of SAA1/SAA2 is well-established but there is still an open debate on extrahepatic SAA expression especially in macrophages. We aimed to investigate the ability of human primary monocytes and monocyte-derived macrophages to express SAA1, SAA2 and SAA4 at both the transcriptional and protein levels, as previous studies almost exclusively dealt with monocytic cell lines. Monocytes and derived macrophages from healthy donors were stimulated under various conditions. In parallel with SAA, pro-inflammatory IL1A, IL1B and IL6 cytokine expression was assessed. While LPS alone was non-effective, a combined LPS/dexamethasone treatment induced SAA1 and to a lesser extent SAA2 transcription in human monocytes and macrophages. In contrast, as expected, pro-inflammatory cytokine expression was strongly induced following stimulation with LPS, an effect which was dampened in the presence of dexamethasone. Furthermore, in monocytes polarized towards a pro-inflammatory M1 phenotype, SAA expression in response to LPS/dexamethasone was potentiated; a result mainly seen for SAA1. However, a major discrepancy was observed between SAA mRNA and intracellular protein levels under the experimental conditions used. Our results demonstrate that human monocytes and macrophages can express SAA genes, mainly SAA1 in response to an inflammatory environment. While SAA is considered as a member of a large cytokine network, its expression in the monocytes-macrophages in response to LPS-dexamethasone is strikingly different from that observed for classic pro-inflammatory cytokines. As monocytes-macrophages are major players in chronic inflammatory diseases, it may be hypothesized that SAA production from macrophages may contribute to the local inflammatory microenvironment, especially when macrophages are compactly organized in granulomas as in sarcoidosis

Physical interaction between neurofibromin and serotonin 5-HT6receptor promotes receptor constitutive activity

International audienceActive G protein-coupled receptor (GPCR) conformations not only are promoted by agonists but also occur in their absence, leading to constitutive activity. Association of GPCRs with intracellular protein partners might be one of the mechanisms underlying GPCR constitutive activity. Here, we show that serotonin 5 hydroxytryptamine 6 (5-HT6) receptor constitutively activates the Gs/adenylyl cyclase pathway in various cell types, including neurons. Constitutive activity is strongly reduced by silencing expression of the Ras-GTPase activating protein (Ras-GAP) neurofibromin, a 5-HT6 receptor partner. Neurofibromin is a multidomain protein encoded by the NF1 gene, the mutation of which causes Neurofibromatosis type 1 (NF1), a genetic disorder characterized by multiple benign and malignant nervous system tumors and cognitive deficits. Disrupting association of 5-HT6 receptor with neurofibromin Pleckstrin Homology (PH) domain also inhibits receptor constitutive activity, and PH domain expression rescues 5-HT6 receptor-operated cAMP signaling in neurofibromin-deficient cells. Furthermore, PH domains carrying mutations identified in NF1 patients that prevent interaction with the 5-HT6 receptor fail to rescue receptor constitutive activity in neurofibromin-depleted cells. Further supporting a role of neurofibromin in agonist-independent Gs signaling elicited by native receptors, the phosphorylation of cAMP-responsive element-binding protein (CREB) is strongly decreased in prefrontal cortex of Nf1+/− mice compared with WT mice. Moreover, systemic administration of a 5-HT6 receptor inverse agonist reduces CREB phosphorylation in prefrontal cortex of WT mice but not Nf1+/− mice. Collectively, these findings suggest that disrupting 5-HT6 receptor–neurofibromin interaction prevents agonist-independent 5-HT6 receptor-operated cAMP signaling in prefrontal cortex, an effect that might underlie neuronal abnormalities in NF1 patients

Expression des gènes SAA par les monocytes et macrophages humains

National audienceLes taux des protéines serum amyloid A (SAA) sont augmentés par diverses conditions inflammatoires. Chez l’Homme, la famille de protéines SAA comporte les protéines de phase aigüe SAA1/SAA2, connues pour activer de nombreuses cellules immunitaires, et la protéine constitutive SAA4. La synthèse hépatique des protéines SAA1/SAA2 est bien établie mais une expression extra-hépatique est encore débattue, particulièrement dans les macrophages. De plus, des auteurs ont mis en évidence la présence d’ARNm SAA dans des cellules spumeuses de plaques d’athéromes. Nous avons étudié la capacité des monocytes et macrophages dérivés de monocytes humains à exprimer SAA1, SAA2 et SAA4, au niveau transcriptionnel et protéique, tandis que les études précédentes ont principalement investigué des lignées monocytaires. Des monocytes et macrophages dérivés de monocytes de donneurs sains ont été traités par différentes conditions. L’expression des cytokines pro-inflammatoires IL1A, IL1B et IL6 a été étudiée en parallèle de l’expression des gènes SAA. Nous avons mis en évidence l’expression du gène SAA1, et de SAA2 à une moindre mesure, lorsque les cellules sont simultanément traitées par un agent pro-inflammatoire et un agent anti-inflammatoire. L’agent pro-inflammatoire seul n’avait pas d’effet sur l’expression des gènes SAA, bien qu’il induise, comme attendu, l’expression des cytokines pro-inflammatoires, effet amoindri par l’ajout du stimulus anti-inflammatoire. De plus, la polarisation des monocytes en phénotype pro-inflammatoire M1 potentialisait l’expression des gènes SAA, principalement SAA1. Nous avons cependant observé un écart entre les taux d’ARNm SAA et les taux intracellulaires de protéines. Nos résultats montrent que les monocytes et macrophages humains peuvent exprimer les gènes SAA, principalement SAA1, dans un environnement inflammatoire. Les monocytes et macrophages étant des acteurs majeurs de maladies inflammatoires, il est possible d’émettre l’hypothèse que la production de SAA par les monocytes et macrophages peut contribuer à la persistance d’un microenvironnement local inflammatoire et à la formation des plaques d’athéromes

Expression des gènes SAA par les monocytes et macrophages humains

National audienceLes taux des protéines serum amyloid A (SAA) sont augmentés par diverses conditions inflammatoires. Chez l’Homme, la famille de protéines SAA comporte les protéines de phase aigüe SAA1/SAA2, connues pour activer de nombreuses cellules immunitaires, et la protéine constitutive SAA4. La synthèse hépatique des protéines SAA1/SAA2 est bien établie mais une expression extra-hépatique est encore débattue, particulièrement dans les macrophages. De plus, des auteurs ont mis en évidence la présence d’ARNm SAA dans des cellules spumeuses de plaques d’athéromes. Nous avons étudié la capacité des monocytes et macrophages dérivés de monocytes humains à exprimer SAA1, SAA2 et SAA4, au niveau transcriptionnel et protéique, tandis que les études précédentes ont principalement investigué des lignées monocytaires. Des monocytes et macrophages dérivés de monocytes de donneurs sains ont été traités par différentes conditions. L’expression des cytokines pro-inflammatoires IL1A, IL1B et IL6 a été étudiée en parallèle de l’expression des gènes SAA. Nous avons mis en évidence l’expression du gène SAA1, et de SAA2 à une moindre mesure, lorsque les cellules sont simultanément traitées par un agent pro-inflammatoire et un agent anti-inflammatoire. L’agent pro-inflammatoire seul n’avait pas d’effet sur l’expression des gènes SAA, bien qu’il induise, comme attendu, l’expression des cytokines pro-inflammatoires, effet amoindri par l’ajout du stimulus anti-inflammatoire. De plus, la polarisation des monocytes en phénotype pro-inflammatoire M1 potentialisait l’expression des gènes SAA, principalement SAA1. Nous avons cependant observé un écart entre les taux d’ARNm SAA et les taux intracellulaires de protéines. Nos résultats montrent que les monocytes et macrophages humains peuvent exprimer les gènes SAA, principalement SAA1, dans un environnement inflammatoire. Les monocytes et macrophages étant des acteurs majeurs de maladies inflammatoires, il est possible d’émettre l’hypothèse que la production de SAA par les monocytes et macrophages peut contribuer à la persistance d’un microenvironnement local inflammatoire et à la formation des plaques d’athéromes

Physical interaction between neurofibromin and serotonin 5-HT 6

International audienceActive G protein-coupled receptor (GPCR) conformations not only are promoted by agonists but also occur in their absence, leading to constitutive activity. Association of GPCRs with intracellular protein partners might be one of the mechanisms underlying GPCR constitutive activity. Here, we show that serotonin 5 hydroxytryptamine 6 (5-HT6) receptor constitutively activates the Gs/adenylyl cyclase pathway in various cell types, including neurons. Constitutive activity is strongly reduced by silencing expression of the Ras-GTPase activating protein (Ras-GAP) neurofibromin, a 5-HT6 receptor partner. Neurofibromin is a multidomain protein encoded by the NF1 gene, the mutation of which causes Neurofibromatosis type 1 (NF1), a genetic disorder characterized by multiple benign and malignant nervous system tumors and cognitive deficits. Disrupting association of 5-HT6 receptor with neurofibromin Pleckstrin Homology (PH) domain also inhibits receptor constitutive activity, and PH domain expression rescues 5-HT6 receptor-operated cAMP signaling in neurofibromin-deficient cells. Furthermore, PH domains carrying mutations identified in NF1 patients that prevent interaction with the 5-HT6 receptor fail to rescue receptor constitutive activity in neurofibromin-depleted cells. Further supporting a role of neurofibromin in agonist-independent Gs signaling elicited by native receptors, the phosphorylation of cAMP-responsive element-binding protein (CREB) is strongly decreased in prefrontal cortex of Nf1+/− mice compared with WT mice. Moreover, systemic administration of a 5-HT6 receptor inverse agonist reduces CREB phosphorylation in prefrontal cortex of WT mice but not Nf1+/− mice. Collectively, these findings suggest that disrupting 5-HT6 receptor–neurofibromin interaction prevents agonist-independent 5-HT6 receptor-operated cAMP signaling in prefrontal cortex, an effect that might underlie neuronal abnormalities in NF1 patients

LINGO family receptors are differentially expressed in the mouse brain and form native multimeric complexes

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