RESUMEN
G2A is a GPCR abundantly expressed in immune cells. G2A-/- mice showed higher lethality, higher plasma cytokines, and an impaired bacterial clearance in response to a murine model of sepsis (cecal ligation and puncture), which were blocked by GdCl3, an inhibitor of Kupffer cells. Anti-IL-10 Ab reversed the impaired bacterial clearance in G2A-/- mice. Indomethacin effectively blocked both the increased i.p. IL-10 levels and the impaired bacterial clearance, indicating that disturbed PG system is the proximal cause of these phenomena. Stimulation with LPS/C5a induced an increase in Escherichia coli phagocytosis and intracellular cAMP levels in G2A+/+ peritoneal macrophages but not G2A-/- cells, which showed more PGE2/nitrite release and intracellular reactive oxygen species levels. Heterologous coexpression of G2A and adenosine receptor type 2b (A2bAR) induced a synergistic increase in cAMP signaling in a ligand-independent manner, with the evidence of physical interaction of G2A with A2bAR. BAY 60-6583, a specific agonist for A2bAR, increased intracellular cAMP levels in Kupffer cells from G2A+/+ but not from G2A-/- mice. Both G2A and A2bAR were required for antiseptic action of lysophosphatidylcholine. These results show inappropriate activation of G2A-/- Kupffer cells to septic insults due to an impaired cAMP signaling possibly by lack of interaction with A2bAR.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Infecciones por Escherichia coli/inmunología , Escherichia coli/fisiología , Macrófagos del Hígado/inmunología , Macrófagos Peritoneales/fisiología , Receptor de Adenosina A2B/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sepsis/metabolismo , Animales , Anticuerpos Bloqueadores , Proteínas de Ciclo Celular/genética , Células Cultivadas , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Humanos , Interleucina-10/inmunología , Interleucina-10/metabolismo , Macrófagos Peritoneales/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Receptor Cross-Talk , Receptor de Adenosina A2B/genética , Receptores Acoplados a Proteínas G/genética , Sepsis/genética , Transducción de SeñalRESUMEN
Emotional responses, such as fear and anxiety, are fundamentally important behavioral phenomena with strong fitness components in most animal species. Anxiety-related disorders continue to represent a major unmet medical need in our society, mostly because we still do not fully understand the mechanisms of these diseases. Animal models may speed up discovery of these mechanisms. The zebrafish is a highly promising model organism in this field. Here, we report the identification of a chemokine-like gene family, samdori (sam), and present functional characterization of one of its members, sam2 We show exclusive mRNA expression of sam2 in the CNS, predominantly in the dorsal habenula, telencephalon, and hypothalamus. We found knockout (KO) zebrafish to exhibit altered anxiety-related responses in the tank, scototaxis and shoaling assays, and increased crh mRNA expression in their hypothalamus compared with wild-type fish. To investigate generalizability of our findings to mammals, we developed a Sam2 KO mouse and compared it to wild-type littermates. Consistent with zebrafish findings, homozygous KO mice exhibited signs of elevated anxiety. We also found bath application of purified SAM2 protein to increase inhibitory postsynaptic transmission onto CRH neurons of the paraventricular nucleus. Finally, we identified a human homolog of SAM2, and were able to refine a candidate gene region encompassing SAM2, among 21 annotated genes, which is associated with intellectual disability and autism spectrum disorder in the 12q14.1 deletion syndrome. Taken together, these results suggest a crucial and evolutionarily conserved role of sam2 in regulating mechanisms associated with anxiety.
Asunto(s)
Ansiedad/genética , Trastorno del Espectro Autista/genética , Quimiocinas/genética , Miedo , Mutación , Animales , Trastornos de Ansiedad , Conducta Animal , Condicionamiento Psicológico/fisiología , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Variación Genética , Proteínas Fluorescentes Verdes/metabolismo , Homocigoto , Humanos , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/metabolismo , Conducta Social , Pez CebraRESUMEN
G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and are involved in a variety of pathological conditions including cancer and cardiovascular, metabolic, neurological, and autoimmune diseases. GPCRs are being intensively investigated as targets for therapeutic intervention, and the ß-arrestin recruitment assay has become a popular tool for analyzing GPCR activation. Here, we report a high-throughput method for cloning GPCR cDNAs into adenoviral bimolecular fluorescence complementation (BiFC) vectors and performing the ß-arrestin BiFC assay in cells transduced with recombinant adenoviruses. An analysis of the activation of somatostatin receptor 2 (SSTR2) with the adenovirus-based ß-arrestin BiFC assay showed that the assay is suitable for quantifying SSTR2 activation in response to specific agonists or antagonists. Furthermore, the adenovirus-based ß-arrestin BiFC assay was able to detect the activation of a broad range of GPCRs. Collectively, our data indicate that the adenovirus-based ß-arrestin BiFC assay can serve as a simple and universal platform for studying GPCR activation and thus will be useful for high-throughput screening of drugs that target GPCRs.
