Differential Activation of Intracellular versus Plasmalemmal CB(2) Cannabinoid Receptors

[Image: see text] The therapeutic and psychoactive properties of cannabinoids have long been recognized. The type 2 receptor for cannabinoids (CB(2)) 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 CB(2)-based therapeutic agents have been related to its intricate pharmacology, including the species specificity and functional selectivity of the CB(2)-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 CB(2) ligands and concurrent calcium imaging in CB(2)-expressing U2OS cells. We found that extracellular administration of anandamide was ineffective, whereas 2-arachidonoyl glycerol (2-AG) and WIN55,212-2 triggered delayed, CB(2)-dependent Ca(2+) responses that were Gq protein-mediated. When microinjected, all agonists elicited fast, transient, and dose-dependent elevations in intracellular Ca(2+) concentration upon activation of Gq-coupled CB(2) receptors. The CB(2) dependency was confirmed by the sensitivity to AM630, a selective CB(2) antagonist, and by the unresponsiveness of untransfected U2OS cells to 2-AG, anandamide, or WIN55,212-2. Moreover, we provide functional and morphological evidence that CB(2) receptors are localized at the endolysosomes, while their activation releases Ca(2+) from inositol 1,4,5-trisphosphate-sensitive- and acidic-like Ca(2+) stores. Our results support the functionality of intracellular CB(2) receptors and their ability to couple to Gq and elicit Ca(2+) signaling. These findings add further complexity to CB(2) receptor pharmacology and argue for careful consideration of receptor localization in the development of CB(2)-based therapeutic agents.