Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes*

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...

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Main Authors:	Dalrymple, Matthew B., Jaeger, Werner C., Eidne, Karin A., Pfleger, Kevin D. G.
Format:	Online
Language:	English
Published:	American Society for Biochemistry and Molecular Biology 2011
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089514/

id	pubmed-3089514
recordtype	oai_dc
spelling	pubmed-30895142011-05-16 Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes* Dalrymple, Matthew B. Jaeger, Werner C. Eidne, Karin A. Pfleger, Kevin D. G. Signal Transduction 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. American Society for Biochemistry and Molecular Biology 2011-05-13 2011-03-04 /pmc/articles/PMC3089514/ /pubmed/21378163 http://dx.doi.org/10.1074/jbc.M111.223537 Text en © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version full access. Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) applies to Author Choice Articles
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Dalrymple, Matthew B. Jaeger, Werner C. Eidne, Karin A. Pfleger, Kevin D. G.
spellingShingle	Dalrymple, Matthew B. Jaeger, Werner C. Eidne, Karin A. Pfleger, Kevin D. G. Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes*
author_facet	Dalrymple, Matthew B. Jaeger, Werner C. Eidne, Karin A. Pfleger, Kevin D. G.
author_sort	Dalrymple, Matthew B.
title	Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes*
title_short	Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes*
title_full	Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes*
title_fullStr	Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes*
title_full_unstemmed	Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes*
title_sort	temporal profiling of orexin receptor-arrestin-ubiquitin complexes reveals differences between receptor subtypes*
description	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.
publisher	American Society for Biochemistry and Molecular Biology
publishDate	2011
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089514/
_version_	1611452473881395200

Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes*

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