Temporal profiling of orexin receptor-arrestin-ubiquitin complexes reveals differences between receptor subtypes
| Autor: | Werner C. Jaeger, Kevin D. G. Pfleger, Karin A. Eidne, Matthew B. Dalrymple |
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| Rok vydání: | 2011 |
| Předmět: |
Receptor recycling
Receptors Neuropeptide Time Factors Arrestins Receptor Endocytosis Enzyme-Linked Immunosorbent Assay Receptor Regulation Biology Biochemistry Receptors G-Protein-Coupled 03 medical and health sciences Orexin-A Orexin Receptors Chlorocebus aethiops Arrestin Animals Humans Phosphorylation Receptor Molecular Biology beta-Arrestins 030304 developmental biology 0303 health sciences Beta-Arrestins Ubiquitin 030302 biochemistry & molecular biology Receptor Recycling Cell Biology Molecular biology Receptor Desensitization Orexin receptor Endocytosis Orexin Kinetics beta-Arrestin 1 COS Cells Signal transduction G Protein-coupled Receptors (GPCR) Protein Binding Signal Transduction |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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