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Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling

BACKGROUND: The physiological regulation of G protein-coupled receptors, through desensitization and internalization, modulates the length of the receptor signal and may influence the development of tolerance and dependence in response to chronic drug treatment. To explore the importance of receptor...

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Autores principales: Scearce-Levie, Kimberly, Lieberman, Michael D, Elliott, Heather H, Conklin, Bruce R
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2005
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC551613/
https://www.ncbi.nlm.nih.gov/pubmed/15707483
http://dx.doi.org/10.1186/1741-7007-3-3
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author Scearce-Levie, Kimberly
Lieberman, Michael D
Elliott, Heather H
Conklin, Bruce R
author_facet Scearce-Levie, Kimberly
Lieberman, Michael D
Elliott, Heather H
Conklin, Bruce R
author_sort Scearce-Levie, Kimberly
collection PubMed
description BACKGROUND: The physiological regulation of G protein-coupled receptors, through desensitization and internalization, modulates the length of the receptor signal and may influence the development of tolerance and dependence in response to chronic drug treatment. To explore the importance of receptor regulation, we engineered a series of G(i)-coupled receptors that differ in signal length, degree of agonist-induced internalization, and ability to induce adenylyl cyclase superactivation. All of these receptors, based on the kappa opioid receptor, were modified to be receptors activated solely by synthetic ligands (RASSLs). This modification allows us to compare receptors that have the same ligands and effectors, but differ only in desensitization and internalization. RESULTS: Removal of phosphorylation sites in the C-terminus of the RASSL resulted in a mutant that was resistant to internalization and less prone to desensitization. Replacement of the C-terminus of the RASSL with the corresponding portion of the mu opioid receptor eliminated the induction of AC superactivation, without disrupting agonist-induced desensitization or internalization. Surprisingly, removal of phosphorylation sites from this chimera resulted in a receptor that is constitutively internalized, even in the absence of agonist. However, the receptor still signals and desensitizes in response to agonist, indicating normal G-protein coupling and partial membrane expression. CONCLUSIONS: These studies reveal that internalization, desensitization and adenylyl cyclase superactivation, all processes that decrease chronic G(i)-receptor signals, are independently regulated. Furthermore, specific mutations can radically alter superactivation or internalization without affecting the efficacy of acute G(i )signaling. These mutant RASSLs will be useful for further elucidating the temporal dynamics of the signaling of G protein-coupled receptors in vitro and in vivo.
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spelling pubmed-5516132005-03-04 Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling Scearce-Levie, Kimberly Lieberman, Michael D Elliott, Heather H Conklin, Bruce R BMC Biol Research Article BACKGROUND: The physiological regulation of G protein-coupled receptors, through desensitization and internalization, modulates the length of the receptor signal and may influence the development of tolerance and dependence in response to chronic drug treatment. To explore the importance of receptor regulation, we engineered a series of G(i)-coupled receptors that differ in signal length, degree of agonist-induced internalization, and ability to induce adenylyl cyclase superactivation. All of these receptors, based on the kappa opioid receptor, were modified to be receptors activated solely by synthetic ligands (RASSLs). This modification allows us to compare receptors that have the same ligands and effectors, but differ only in desensitization and internalization. RESULTS: Removal of phosphorylation sites in the C-terminus of the RASSL resulted in a mutant that was resistant to internalization and less prone to desensitization. Replacement of the C-terminus of the RASSL with the corresponding portion of the mu opioid receptor eliminated the induction of AC superactivation, without disrupting agonist-induced desensitization or internalization. Surprisingly, removal of phosphorylation sites from this chimera resulted in a receptor that is constitutively internalized, even in the absence of agonist. However, the receptor still signals and desensitizes in response to agonist, indicating normal G-protein coupling and partial membrane expression. CONCLUSIONS: These studies reveal that internalization, desensitization and adenylyl cyclase superactivation, all processes that decrease chronic G(i)-receptor signals, are independently regulated. Furthermore, specific mutations can radically alter superactivation or internalization without affecting the efficacy of acute G(i )signaling. These mutant RASSLs will be useful for further elucidating the temporal dynamics of the signaling of G protein-coupled receptors in vitro and in vivo. BioMed Central 2005-02-11 /pmc/articles/PMC551613/ /pubmed/15707483 http://dx.doi.org/10.1186/1741-7007-3-3 Text en Copyright © 2005 Scearce-Levie et al; licensee BioMed Central Ltd.
spellingShingle Research Article
Scearce-Levie, Kimberly
Lieberman, Michael D
Elliott, Heather H
Conklin, Bruce R
Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling
title Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling
title_full Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling
title_fullStr Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling
title_full_unstemmed Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling
title_short Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling
title_sort engineered g protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC551613/
https://www.ncbi.nlm.nih.gov/pubmed/15707483
http://dx.doi.org/10.1186/1741-7007-3-3
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