Understanding Biased CB1R Signaling Through Phosphoproteomics

Of the 50 million+ adults in the United States suffering from chronic pain, 19.6 million suffer from high-impact chronic pain that interferes with life or work activities. Though cannabinoid-based therapies offer a needed alternative to opioids for the treatment of chronic pain, tolerance to their therapeutic effects can rapidly develop, limiting their efficacy while also facilitating escalating consumption and dependence. Consequently, identification of the mechanism(s) of tolerance to the analgesic effects of cannabinoids may serve to prolong their clinical utility. Desensitization (uncoupling of a receptor from its G proteins) and internalization (loss of receptor from the cell surface) are cell-based correlates of tolerance that involve G protein-coupled receptor kinase phosphorylation. For cannabinoid type 1 receptors (CB1R), desensitization and internalization appear to be mediated by distinct receptor domains. Previous work in vitro suggests that desensitization of CB1R is mediated by residues S426 and S430 while six different carboxy terminal serines and threonines (T461, S463, S465, T466, T468, and S469) are critical for CB1R internalization. We subsequently evaluated the contribution of these 8 different phosphorylation sites in vivo through the creation first of a desensitization-resistant (S426A/S430A) and then an internalization-resistant six-point mutant (6PM) mouse by mutating each of the phosphorylation sites to a non-phosphorylated alanine. The central hypothesis is that internalization of CB1R counteracts tolerance to strongly internalizing cannabinoids, such as CP55,940, and that tolerance to CP55,940 would be more profoundly impacted in 6PM mice relative to Δ9-THC, a weakly internalizing, partial agonist. In contrast, desensitization of CB1R would have a greater effect on tolerance development to Δ9-THC compared to CP55,940. Behaviorally, we have repeatedly shown that desensitization-resistant S426A/S430A mice display enhanced sensitivity and delayed tolerance to Δ9-THC-mediated antinociception. Preliminary data collected from our recently generated six-point mutants (T461A/S463A/S465A/T466A/T468A/S469A; 6PM) has revealed decreased sensitivity and faster tolerance to CP55,940-induced antinociception. This proposal seeks to identify molecular neuroadaptations associated with cannabinoid tolerance that will complement our behavioral data, to be utilized in a larger proposal for NIDA funding on cannabinoid tolerance. Specifically, this pilot project will utilize the cutting-edge use of phosphoproteomics to identify agonist-specific downstream targets in commercially available HEK293 cells stably expressing HA-CB1R that are treated with either vehicle, Δ9-THC or CP55,940 at time points (5 and 60 minutes) sufficient to induce desensitization and/or internalization. Our hypothesis is that treatment with CP55,940, a strongly internalizing cannabinoid agonist, will result in robust phosphorylation of the six internalization residues while treatment with Δ9-THC, a weakly internalizing agonist, will result in phosphorylation at the residues involved in desensitization, S426 and S430, with limited phosphorylation at the six internalization residues. The results of this project will be utilized in a larger grant to devise the precise nature of diverse downstream signaling pathways dictated through CB1R agonist-specific binding with the eventual aim of devising appropriate therapeutic strategies to combat cannabis tolerance and dependence in managing chronic pain.