In vivo delta opioid receptor internalization controls behavioral effects of agonists.
BACKGROUND
GPCRs regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Stimulation of a GPCR by an extracellular ligand triggers receptor signaling via G proteins, and this process is highly regulated. Receptor activation is typically accompanied by desensitization of receptor signaling, a complex feedback regulatory process of which receptor internalization is postulated as a key event. The in vivo significance of GPCR internalization is poorly understood. In fact, the majority of studies have been performed in transfected cell systems, which do not adequately model physiological environments and the complexity of integrated responses observed in the whole animal.


RESULTS
In this study, we used knock-in mice expressing functional fluorescent delta opioid receptors (DOR-eGFP) in place of the native receptor to correlate receptor localization in neurons with behavioral responses. We analyzed the pain-relieving effects of two delta receptor agonists with similar signaling potencies and efficacies, but distinct internalizing properties. An initial treatment with the high (SNC80) or low (AR-M100390) internalizing agonist equally reduced CFA-induced inflammatory pain. However, subsequent drug treatment produced highly distinct responses. Animals initially treated with SNC80 showed no analgesic response to a second dose of either delta receptor agonist. Concomitant receptor internalization and G-protein uncoupling were observed throughout the nervous system. This loss of function was temporary, since full DOR-eGFP receptor responses were restored 24 hours after SNC80 administration. In contrast, treatment with AR-M100390 resulted in retained analgesic response to a subsequent agonist injection, and ex vivo analysis showed that DOR-eGFP receptor remained G protein-coupled on the cell surface. Finally SNC80 but not AR-M100390 produced DOR-eGFP phosphorylation, suggesting that the two agonists produce distinct active receptor conformations in vivo which likely lead to differential receptor trafficking.


CONCLUSIONS
Together our data show that delta agonists retain full analgesic efficacy when receptors remain on the cell surface. In contrast, delta agonist-induced analgesia is abolished following receptor internalization, and complete behavioral desensitization is observed. Overall these results establish that, in the context of pain control, receptor localization fully controls receptor function in vivo. This finding has both fundamental and therapeutic implications for slow-recycling GPCRs.
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, Illkirch, France.
Amynah A A
AA
Pradhan
Jérôme A J
JA
Becker
Grégory
G
Scherrer
Petra
P
Tryoen-Toth
Dominique
D
Filliol
Audrey
A
Matifas
Dominique
D
Massotte
Claire
C
Gavériaux-Ruff
Brigitte L
BL
Kieffer
D000818:Animals; D001522:Behavior, Animal / Q000187:drug effects* / Q000502:physiology*; D001549:Benzamides / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D010146:Pain / Q000188:drug therapy / Q000503:physiopathology; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D011487:Protein Conformation; D017465:Receptors, Opioid, delta / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001549:Benzamides; D008024:Ligands; C415566:N,N-diethyl-4-(phenylpiperidin-4-ylidenemethyl)benzamide; D010879:Piperazines; D010880:Piperidines; D017465:Receptors, Opioid, delta; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C088464:4-(alpha-(4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl)-N,N-diethylbenzamide