Mutation of putative GRK phosphorylation sites in the cannabinoid receptor 1 (CB1R) confers resistance to cannabinoid tolerance and hypersensitivity to cannabinoids in mice

Daniel J. Morgan, Brian J. Davis, Chris S. Kearn, David Marcus, Alex J. Cook, Jim Wager-Miller, Alex Straiker, Michael H. Myoga, Jeffrey Karduck, Emma Leishman, Laura J. Sim-Selley, Traci A. Czyzyk, Heather B. Bradshaw, Dana E. Selley, Ken Mackie

Research output: Contribution to journalArticle

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Abstract

For many G-protein-coupled receptors (GPCRs), including cannabinoid receptor 1 (CB1R), desensitization has been proposed as a principal mechanism driving initial tolerance to agonists. GPCR desensitization typically requires phosphorylation by a G-proteincoupled receptor kinase (GRK) and interaction of the phosphorylated receptor with an arrestin. In simple model systems, CB1R is desensitized by GRK phosphorylation at two serine residues (S426 and S430). However, the role of these serine residues in tolerance and dependence for cannabinoids in vivo was unclear. Therefore, we generated mice where S426 and S430 were mutated to nonphosphorylatable alanines (S426A/S430A). S426A/S430A mutant mice were more sensitive to acutely administered delta-9-tetrahydrocannabinol (Δ9-THC), have delayed tolerance to Δ9-THC, and showed increased dependence for Δ9-THC. S426A/S430A mutants also showed increased responses to elevated levels of endogenous cannabinoids. CB1R desensitization in the periaqueductal gray and spinal cord following7dof treatment with Δ9-THC was absent in S426A/S430A mutants. Δ9-THC-induced downregulation of CB1Rin the spinal cord was also absent in S426A/S430A mutants. Cultured autaptic hippocampal neurons from S426A/S430A mice showed enhanced endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and reduced agonist-mediated desensitization of DSE. These results indicate that S426 and S430 play major roles in the acute response to, tolerance to, and dependence on cannabinoids. Additionally, S426A/S430A mice are a novel model for studying pathophysiological processes thought to involve excessive endocannabinoid signaling such as drug addiction and metabolic disease. These mice also validate the approach of mutating GRK phosphorylation sites involved in desensitization as a general means to confer exaggerated signaling to GPCRs in vivo.

Original languageEnglish (US)
Pages (from-to)5152-5163
Number of pages12
JournalJournal of Neuroscience
Volume34
Issue number15
DOIs
StatePublished - Jan 1 2014

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Cannabinoid Receptors
Dronabinol
Cannabinoids
Hypersensitivity
Phosphotransferases
Phosphorylation
G-Protein-Coupled Receptors
Mutation
Endocannabinoids
Serine
Spinal Cord
Arrestin
Periaqueductal Gray
Metabolic Diseases
Alanine
Substance-Related Disorders
Down-Regulation
Psychologic Desensitization
Neurons

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)

Cite this

Morgan, Daniel J. ; Davis, Brian J. ; Kearn, Chris S. ; Marcus, David ; Cook, Alex J. ; Wager-Miller, Jim ; Straiker, Alex ; Myoga, Michael H. ; Karduck, Jeffrey ; Leishman, Emma ; Sim-Selley, Laura J. ; Czyzyk, Traci A. ; Bradshaw, Heather B. ; Selley, Dana E. ; Mackie, Ken. / Mutation of putative GRK phosphorylation sites in the cannabinoid receptor 1 (CB1R) confers resistance to cannabinoid tolerance and hypersensitivity to cannabinoids in mice. In: Journal of Neuroscience. 2014 ; Vol. 34, No. 15. pp. 5152-5163.
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abstract = "For many G-protein-coupled receptors (GPCRs), including cannabinoid receptor 1 (CB1R), desensitization has been proposed as a principal mechanism driving initial tolerance to agonists. GPCR desensitization typically requires phosphorylation by a G-proteincoupled receptor kinase (GRK) and interaction of the phosphorylated receptor with an arrestin. In simple model systems, CB1R is desensitized by GRK phosphorylation at two serine residues (S426 and S430). However, the role of these serine residues in tolerance and dependence for cannabinoids in vivo was unclear. Therefore, we generated mice where S426 and S430 were mutated to nonphosphorylatable alanines (S426A/S430A). S426A/S430A mutant mice were more sensitive to acutely administered delta-9-tetrahydrocannabinol (Δ9-THC), have delayed tolerance to Δ9-THC, and showed increased dependence for Δ9-THC. S426A/S430A mutants also showed increased responses to elevated levels of endogenous cannabinoids. CB1R desensitization in the periaqueductal gray and spinal cord following7dof treatment with Δ9-THC was absent in S426A/S430A mutants. Δ9-THC-induced downregulation of CB1Rin the spinal cord was also absent in S426A/S430A mutants. Cultured autaptic hippocampal neurons from S426A/S430A mice showed enhanced endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and reduced agonist-mediated desensitization of DSE. These results indicate that S426 and S430 play major roles in the acute response to, tolerance to, and dependence on cannabinoids. Additionally, S426A/S430A mice are a novel model for studying pathophysiological processes thought to involve excessive endocannabinoid signaling such as drug addiction and metabolic disease. These mice also validate the approach of mutating GRK phosphorylation sites involved in desensitization as a general means to confer exaggerated signaling to GPCRs in vivo.",
author = "Morgan, {Daniel J.} and Davis, {Brian J.} and Kearn, {Chris S.} and David Marcus and Cook, {Alex J.} and Jim Wager-Miller and Alex Straiker and Myoga, {Michael H.} and Jeffrey Karduck and Emma Leishman and Sim-Selley, {Laura J.} and Czyzyk, {Traci A.} and Bradshaw, {Heather B.} and Selley, {Dana E.} and Ken Mackie",
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Morgan, DJ, Davis, BJ, Kearn, CS, Marcus, D, Cook, AJ, Wager-Miller, J, Straiker, A, Myoga, MH, Karduck, J, Leishman, E, Sim-Selley, LJ, Czyzyk, TA, Bradshaw, HB, Selley, DE & Mackie, K 2014, 'Mutation of putative GRK phosphorylation sites in the cannabinoid receptor 1 (CB1R) confers resistance to cannabinoid tolerance and hypersensitivity to cannabinoids in mice', Journal of Neuroscience, vol. 34, no. 15, pp. 5152-5163. https://doi.org/10.1523/JNEUROSCI.3445-12.2014

Mutation of putative GRK phosphorylation sites in the cannabinoid receptor 1 (CB1R) confers resistance to cannabinoid tolerance and hypersensitivity to cannabinoids in mice. / Morgan, Daniel J.; Davis, Brian J.; Kearn, Chris S.; Marcus, David; Cook, Alex J.; Wager-Miller, Jim; Straiker, Alex; Myoga, Michael H.; Karduck, Jeffrey; Leishman, Emma; Sim-Selley, Laura J.; Czyzyk, Traci A.; Bradshaw, Heather B.; Selley, Dana E.; Mackie, Ken.

In: Journal of Neuroscience, Vol. 34, No. 15, 01.01.2014, p. 5152-5163.

Research output: Contribution to journalArticle

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T1 - Mutation of putative GRK phosphorylation sites in the cannabinoid receptor 1 (CB1R) confers resistance to cannabinoid tolerance and hypersensitivity to cannabinoids in mice

AU - Morgan, Daniel J.

AU - Davis, Brian J.

AU - Kearn, Chris S.

AU - Marcus, David

AU - Cook, Alex J.

AU - Wager-Miller, Jim

AU - Straiker, Alex

AU - Myoga, Michael H.

AU - Karduck, Jeffrey

AU - Leishman, Emma

AU - Sim-Selley, Laura J.

AU - Czyzyk, Traci A.

AU - Bradshaw, Heather B.

AU - Selley, Dana E.

AU - Mackie, Ken

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N2 - For many G-protein-coupled receptors (GPCRs), including cannabinoid receptor 1 (CB1R), desensitization has been proposed as a principal mechanism driving initial tolerance to agonists. GPCR desensitization typically requires phosphorylation by a G-proteincoupled receptor kinase (GRK) and interaction of the phosphorylated receptor with an arrestin. In simple model systems, CB1R is desensitized by GRK phosphorylation at two serine residues (S426 and S430). However, the role of these serine residues in tolerance and dependence for cannabinoids in vivo was unclear. Therefore, we generated mice where S426 and S430 were mutated to nonphosphorylatable alanines (S426A/S430A). S426A/S430A mutant mice were more sensitive to acutely administered delta-9-tetrahydrocannabinol (Δ9-THC), have delayed tolerance to Δ9-THC, and showed increased dependence for Δ9-THC. S426A/S430A mutants also showed increased responses to elevated levels of endogenous cannabinoids. CB1R desensitization in the periaqueductal gray and spinal cord following7dof treatment with Δ9-THC was absent in S426A/S430A mutants. Δ9-THC-induced downregulation of CB1Rin the spinal cord was also absent in S426A/S430A mutants. Cultured autaptic hippocampal neurons from S426A/S430A mice showed enhanced endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and reduced agonist-mediated desensitization of DSE. These results indicate that S426 and S430 play major roles in the acute response to, tolerance to, and dependence on cannabinoids. Additionally, S426A/S430A mice are a novel model for studying pathophysiological processes thought to involve excessive endocannabinoid signaling such as drug addiction and metabolic disease. These mice also validate the approach of mutating GRK phosphorylation sites involved in desensitization as a general means to confer exaggerated signaling to GPCRs in vivo.

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