RESUMO
Desensitization of many G protein-coupled receptors after ligand binding generally involves phosphorylation of the receptors and internalization of the ligand-bound, phosphorylated receptors by a clathrin-mediated endocytic pathway. Olfactory receptor neurons from the channel catfish (Ictalurus punctatus) express the G protein-coupled odorant receptors and metabotropic glutamate receptors. To determine whether a clathrin-dependent receptor internalization pathway exists in olfactory receptor neurons, western blotting and immunocytochemistry were used to identify and localize clathrin and dynamin in isolated olfactory neurons. Clathrin and dynamin immunoreactivity was found in the cell bodies, dendrites, and dendritic knobs of the neurons. Using the activity-dependent fluorescent dye FM1-43 to monitor receptor internalization, we show that single olfactory neurons stimulated with the odorant amino acid L-glutamate internalized the dye. Odorant-stimulated neurons showed a consistent pattern of internalized FM1-43 fluorescence localized in the cell bodies and dendritic knobs. Odorant-stimulated internalization was unaffected by the caveolae activator okadaic acid and was significantly decreased by a metabotropic glutamate receptor antagonist, suggesting that a functional, clathrin-dependent, receptor-mediated internalization pathway exists in olfactory receptor neurons.
Assuntos
Caveolinas , Regulação para Baixo/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Neurônios Receptores Olfatórios/química , Neurônios Receptores Olfatórios/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Caveolina 1 , Clatrina/análise , Clatrina/genética , Dinaminas , Corantes Fluorescentes , GTP Fosfo-Hidrolases/análise , GTP Fosfo-Hidrolases/genética , Expressão Gênica/fisiologia , Ictaluridae , Proteínas de Membrana/análise , Dados de Sequência Molecular , Neurônios Receptores Olfatórios/citologia , Compostos de Piridínio , Compostos de Amônio Quaternário , Ratos , Homologia de Sequência de AminoácidosRESUMO
Epitope-tagged Xenopus nucleolin was expressed in Escherichia coli cells and in Xenopus oocytes either as a full-length wild-type protein or as a truncation that lacked the distinctive carboxy glycine/arginine-rich (GAR) domain. Both full-length and truncated versions of nucleolin were tagged at their amino termini with five tandem human c-myc epitopes. Whether produced in E. coli or in Xenopus, epitope-tagged full-length nucleolin bound nucleic acid probes in in vitro filter binding assays. Conversely, the E. coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation maintained slight binding activity. Indirect immunofluorescence staining showed that myc-tagged full-length nucleolin properly localized to the dense fibrillar regions within the multiple nucleoli of Xenopus oocyte nuclei. The epitope-tagged GAR truncation also translocated to the oocyte nuclei, but it failed to efficiently localize to the nucleoli. Our results show that the carboxy GAR domain must be present for nucleolin to efficiently bind nucleic acids in vitro and to associate with nucleoli in vivo.