Differential activation of intracellular versus plasmalemmal CB₂ Cannabinoid receptors

The therapeutic and psychoactive properties of cannabinoids have long been recognized. The type 2 receptor for cannabinoids (CB₂) has emerged as an important therapeutic target in several pathologies, as it mediates beneficial effects of cannabinoids while having little if any psychotropic activity. Difficulties associated with the development of CB₂-based therapeutic agents have been related to its intricate pharmacology, including the species specificity and functional selectivity of the CB₂- initiated responses. We postulated that a plasmalemmal or subcellular location of the receptor may contribute to the differential signaling pathways initiated by its activation. To differentiate between these two, we used extracellular and intracellular administration of CB₂ ligands and concurrent calcium imaging in CB₂-expressing U2OS cells. We found that extracellular administration of anandamide was ineffective, whereas 2-arachidonoyl glycerol (2-AG) and WIN55,212-2 triggered delayed, CB₂-dependent Ca ²⁺ responses that were Gq protein-mediated. When microinjected, allagonists elicited fast, transient, and dose-dependent elevations in intracellular Ca²⁺ concentration upon activation of Gq-coupled CB₂ receptors. The CB₂ dependency was confirmed by the sensitivity to AM630, a selective CB₂antagonist, and by the unresponsiveness of untransfected U2OS cells to 2-AG, anandamide,or WIN55,212-2. Moreover, we provide functional and morphological evidence that CB₂ receptors are localized at the endolysosomes, while their activation releases Ca²⁺ from inositol 1,4,5-trisphosphate-sensitive- and acidic-like Ca²⁺ stores. Our results support the functionality of intracellular CB₂ receptors and their ability to couple to Gq and elicit Ca ²⁺ signaling. These findings add further complexity to CB₂ receptor pharmacology and argue for careful consideration of receptor localization in the development of CB₂-based therapeutic agents.