Related Experiment Video
Updated: Jun 3, 2026
Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods
Published on: June 25, 2019
Temporal profiling of orexin receptor-arrestin-ubiquitin complexes reveals differences between receptor subtypes
Matthew B Dalrymple1, Werner C Jaeger, Karin A Eidne
1Laboratory for Molecular Endocrinology-G Protein-Coupled Receptors, Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Perth, Western Australia 6009, Australia.
Abstract:
Orexin G protein-coupled receptors (OxRs) and their cognate agonists have been implicated in a number of disorders since their recent discovery, ranging from narcolepsy to formation of addictive behavior. Bioluminescence resonance energy transfer assays of agonist-occupied OxRs provided evidence for a strong dose-dependent interaction with both trafficking proteins β-arrestin 1 and 2 that required unusually high agonist concentrations compared with inositol phosphate signaling. This appears to be reflected in functional differences in potency with respect to orexin A (OxA) and OxR2-dependent ERK1/2 phosphorylation after 90 min compared with 2 min, potentially consistent with β-arrestin-mediated versus G protein-mediated signaling, respectively. Furthermore, extended bioluminescence resonance energy transfer kinetic data monitoring OxA-dependent receptor-β-arrestin and β-arrestin-ubiquitin proximity suggested subtype-specific differences in receptor trafficking, with OxR2 activation resulting in more sustained receptor-β-arrestin-ubiquitin complex formation than elicited by OxR1 activation. Enzyme-linked immunosorbent assay (ELISA) data also revealed that OxR1 underwent significantly more rapid recycling compared with OxR2. Finally, we have observed sustained OxA-dependent ERK1/2 phosphorylation in the presence of OxR2 compared with OxR1. Although both OxR subtypes could be classified as class B receptors for β-arrestin usage based on the initial strength of interaction with both β-arrestins, our temporal profiling revealed tangible differences between OxR subtypes. Consequently, OxR1 appears to fit uneasily into the commonly used β-arrestin classification scheme. More importantly, it is hoped that this improved profiling capability, enabling the subtleties of protein complex formation, stability, and duration to be assessed in live cells, will help unlock the therapeutic potential of targeting these receptors.
Insights
Orexin receptors (OxRs) show distinct signaling dynamics. OxR2 activation leads to sustained ERK1/2 phosphorylation and receptor trafficking, unlike OxR1, offering new therapeutic targets.
Area of Science:
- Pharmacology
- Cell Biology
- Neuroscience
Background:
- Orexin receptors (OxRs) are G protein-coupled receptors implicated in narcolepsy and addiction.
- Understanding OxR signaling pathways is crucial for developing targeted therapeutics.
Purpose of the Study:
- To investigate the distinct signaling mechanisms and trafficking dynamics of OxR1 and OxR2 subtypes.
- To explore the temporal differences in beta-arrestin and ERK1/2 phosphorylation mediated by OxRs.
Main Methods:
- Bioluminescence resonance energy transfer (BRET) assays to study receptor-protein interactions and trafficking.
- Enzyme-linked immunosorbent assay (ELISA) to assess receptor recycling.
- Monitoring ERK1/2 phosphorylation at different time points.
Main Results:
- Both OxR1 and OxR2 interact with beta-arrestins, but require high agonist concentrations.
- OxR2 activation results in sustained beta-arrestin-ubiquitin complex formation and ERK1/2 phosphorylation compared to OxR1.
- OxR1 exhibits more rapid recycling than OxR2.
Conclusions:
- Temporal profiling reveals significant differences in OxR1 and OxR2 signaling and trafficking.
- OxR1 does not fit neatly into the standard beta-arrestin classification scheme.
- These findings provide a foundation for unlocking the therapeutic potential of OxR-targeting drugs.
Related Concept Videos
01:22Opioid Receptors: Overview
01:15Receptor Downregulation in MVBs
The EGFR can initiate signaling pathways that lead to cell proliferation, migration, and differentiation. Overexpression of EGFR stimulates cells to proliferate. Excessive EGFR activation may...