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Internalization of G protein-coupled receptors in single olfactory receptor neurons
M L Rankin1, R S Alvania, E L Gleason
1Department of Biological Sciences, Louisiana State University, Baton Rouge 70803, USA.
Abstract:
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.
Insights
Olfactory neurons use a clathrin-dependent pathway to internalize receptors after odorant stimulation. This process is crucial for receptor desensitization in channel catfish (Ictalurus punctatus).
Area of Science:
- Neuroscience
- Cell Biology
- Sensory Biology
Background:
- G protein-coupled receptors (GPCRs) desensitize via phosphorylation and internalization.
- Olfactory receptor neurons (ORNs) utilize GPCRs for odor detection.
- Clathrin-mediated endocytosis is a common pathway for receptor internalization.
Purpose of the Study:
- To investigate the existence of a clathrin-dependent receptor internalization pathway in channel catfish ORNs.
- To determine if odorant stimulation triggers receptor internalization in these neurons.
Main Methods:
- Western blotting and immunocytochemistry to detect clathrin and dynamin in ORNs.
- FM1-43 fluorescence dye uptake assay to monitor receptor internalization.
- Pharmacological manipulation using okadaic acid and a metabotropic glutamate receptor antagonist.
Main Results:
- Clathrin and dynamin were localized in catfish ORN cell bodies, dendrites, and dendritic knobs.
- Stimulation with L-glutamate induced FM1-43 dye internalization in ORNs.
- Internalized dye was concentrated in cell bodies and dendritic knobs.
- Internalization was not affected by okadaic acid but was reduced by a metabotropic glutamate receptor antagonist.
Conclusions:
- Channel catfish ORNs possess a functional, clathrin-dependent, receptor-mediated internalization pathway.
- This pathway is likely involved in the desensitization of olfactory receptors following odorant stimulation.