CB1 cannabinoid receptor-mediated increases in cyclic AMP accumulation are correlated with reduced Gi/o function

Area of Science:

• Molecular Pharmacology
• Neuroscience
• Cell Signaling

Background:

• Cannabinoid receptor 1 (CB1R) is known to activate Gi/o-dependent signaling pathways.
• Previous hypotheses suggested CB1R-mediated cAMP increases result from Gs activation.
• Direct investigation of CB1R-stimulated GTPγS binding to Gs was lacking.

Purpose of the Study:

• To investigate the coupling of CB1R to Gs proteins.
• To elucidate the mechanisms underlying CB1R-mediated cAMP production.
• To examine the influence of receptor mutations and co-expressed receptors on CB1R signaling.

Main Methods:

• Utilized three cellular models: pertussis toxin-treated N18TG2 cells, Chinese hamster ovary (CHO) cells expressing a mutated CB1R (L341A/A342L), and MN9D cells co-expressing CB1 and D2 dopamine receptors.
• Assayed cAMP levels using [3H]cAMP binding competition.
• Measured G protein activation via antibody-targeted scintillation proximity assay for [35S]GTPγS binding.

Main Results:

• In mutated CB1R-CHO cells, cannabinoid agonists stimulated cAMP, but Gs binding was unaffected, while Gi/o binding was reversed.
• In MN9D cells, CB1 and D2 dopamine receptor co-agonism increased cAMP, reversing the Gi/o activation typically seen with CB1 agonism alone.
• CB1R demonstrated low-efficacy coupling to Gs, distinct from its potent Gi/o coupling.

Conclusions:

• CB1R couples to both Gi/o and Gs proteins, with significantly higher efficacy for Gi/o.
• CB1R-mediated cAMP accumulation is primarily due to the attenuation of Gi/o's tonic inhibition of adenylyl cyclase.
• Receptor mutations and co-receptor activation can alter CB1R signaling bias, converting agonist responses at Gi/o to inverse agonism.