Mutation of putative GRK phosphorylation sites in the cannabinoid receptor 1 (CB1R) confers resistance to cannabinoid tolerance and hypersensitivity to cannabinoids in mice.
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-protein-coupled 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 (Δ(9)-THC), have delayed tolerance to Δ(9)-THC, and showed increased dependence for Δ(9)-THC. S426A/S430A mutants also showed increased responses to elevated levels of endogenous cannabinoids. CB1R desensitization in the periaqueductal gray and spinal cord following 7 d of treatment with Δ(9)-THC was absent in S426A/S430A mutants. Δ(9)-THC-induced downregulation of CB1R in 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.
Gill Center for Biomolecular Science, Indiana University, Bloomington, Indiana 47405, Department of Psychological and Brain Sciences, Indiana University, Bloomington, Indiana 47405, Department of Anesthesiology, University of Washington School of Medicine, Seattle, Washington 98195, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, and Department of Physiology, Mayo Clinic Arizona, Scottsdale, Arizona 85259.
Daniel J
DJ
Morgan
Brian J
BJ
Davis
Chris S
CS
Kearn
David
D
Marcus
Alex J
AJ
Cook
Jim
J
Wager-Miller
Alex
A
Straiker
Michael H
MH
Myoga
Jeffrey
J
Karduck
Emma
E
Leishman
Laura J
LJ
Sim-Selley
Traci A
TA
Czyzyk
Heather B
HB
Bradshaw
Dana E
DE
Selley
Ken
K
Mackie
D020816:Amino Acid Motifs; D000818:Animals; D063386:Cannabinoid Receptor Agonists / Q000494:pharmacology*; D059333:Central Nervous System Sensitization; D013759:Dronabinol / Q000494:pharmacology*; D004361:Drug Tolerance; D054768:G-Protein-Coupled Receptor Kinases / Q000378:metabolism; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D008564:Membrane Potentials; D051379:Mice; D020125:Mutation, Missense; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D010487:Periaqueductal Gray / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism / Q000502:physiology
D063386:Cannabinoid Receptor Agonists; D043884:Receptor, Cannabinoid, CB1; D013759:Dronabinol; D054768:G-Protein-Coupled Receptor Kinases
CB1; GPCR; THC; cannabinoid; desensitization; tolerance