Abstract

Tolerance to compounds that target G protein-coupled receptors (GPCR), such as the cannabinoid type-1 receptor (CB₁R), is in part facilitated by receptor desensitization. Processes that mediate CB₁R desensitization include phosphorylation of CB₁R residues S426 and S430 by a GPCR kinase (GRK), and subsequent recruitment of the b-arrestin2 scaffolding protein. Tolerance to cannabinoid drugs is reduced in S426A/S430A mutant mice and b-arrestin2 knock out (KO) mice according to previous work in vivo. However, the presence of additional phosphorylatable residues on the CB₁R C-terminus made it unclear as to whether recruitment to S426 and S430 accounted for all desensitization and tolerance by b-arrestin2. Therefore, we assessed acute response and tolerance to the cannabinoids, delta-9-tetrahydrocannabinol (Δ⁹-THC) and CP55,940, in S426A/S430A x b-arrestin2 KO double mutant mice. We observed both delayed tolerance and increased sensitivity to the antinociceptive and hypothermic effects of CP55,940 in male S426A/S430A single and double mutant mice compared to wild-type littermates, but not with Δ⁹-THC. Female S426A/S430A single and double mutant mice were more sensitive to acute antinociception (CP55,940 and Δ⁹-THC) and hypothermia (CP55,940 only) exclusively after chronic dosing, and did not differ in the development of tolerance. These results indicate that phosphorylation of S426 and S430 are likely responsible for b-arrestin2-mediated desensitization, as double mutant mice did not differ from the S426A/S430A single mutant model in respect to cannabinoid tolerance and sensitivity. We also found antinociceptive and hypothermic effects from cannabinoid treatment demonstrate sex-, agonist-, and duration-dependent features.

Significance Statement A better understanding of the molecular mechanisms involved in tolerance will improve the therapeutic potential of cannabinoid drugs. This study determined that deletion of beta-arrestin2 does not enhance the reduction in cannabinoid tolerance observed in CB1R S426A/S430A mutant mice. This finding shows that GRK-mediated phosphorylation of S426 and S430 is fully responsible for the effects of beta-arresin2 on cannabinoid tolerance.