Differential Activation of Intracellular versus Plasmalemmal CB₂ Cannabinoid Receptors

Abstract

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 Ca2+ responses that were Gq protein-mediated. When microinjected, all agonists 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 CB2 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 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 Ca2+ 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.

Cannabinoid Receptors in the Central Nervous System: Their Signaling and Roles in Disease

“…CB 1 -G protein mediated signaling occurs at the cell surface and at intracellular compartments (Rozenfeld and Devi, 2008; Brailoiu et al, 2014). At the cell surface, CB 1 receptor ligands modulate the interaction between receptors and β-arrestin as a mechanism to influence β-arrestin mediated signaling (Flores-Otero et al, 2014).…”

“…Interestingly, these location-specific signaling events appear to be widespread among several GPCRs. For example, the LH receptor, β2 adrenergic receptor and the CB 2 receptor can signal from intracellular compartments either by β-arrestins or G proteins via a “super-complex” ultimately resulting in three different spatio-temporal signaling waves (Brailoiu et al, 2014; Irannejad and von Zastrow, 2014; Lyga et al, 2016; Nogueras-Ortiz and Yudowski, 2016; Thomsen et al, 2016). Constitutive activation also plays a role in their trafficking (Leterrier et al, 2006; McDonald et al, 2007).…”

Differential Activation of Cultured Neonatal Cardiomyocytes by Plasmalemmal Versus Intracellular G Protein-coupled Receptor 55

“…Increases in intracellular Ca 2ϩ concentration as well as alterations in ionic currents and membrane properties have been correlated previously with GPR55 activation (16 -18). Considering the emerging paradigm of functional intracellular G protein-coupled receptors (GPCRs) (19,20), which has also been suggested for the receptors of other lipid mediators (21) and for cannabinoid receptors (22)(23)(24)(25), we postulated that a GPR55-dependent signaling pathway may be initiated either at the plasma membrane or * This work was supported, in whole or in part, by National Institutes of Health within the cell. Thus, we used Ca 2ϩ and voltage imaging and both extracellular and intracellular administration (microinjection) of GPR55 ligands to test the involvement of this receptor in the regulation of cardiomyocyte signaling.…”

Regulation of Cell Surface CB2 Receptor during Human B Cell Activation and Differentiation

“…The biologic basis underlying these different CB 2 expression patterns has not yet been fully delineated, but there is growing evidence that the presence of GPCRs at different cellular locations is an important feature of these receptors that promotes functional heterogeneity with respect to downstream signaling and biologic responses (Flordellis 2012; Gaudet et al 2015). Along these lines, there is growing evidence that intracellular forms for both CB 1 and CB 2 are common and exert distinct biologic effects (Brailoiu et al 2011; Brailoiu et al 2014; Bernard et al 2012). In this setting, understanding the distribution, regulation, and dynamic balance between cell surface and intracellular CB 2 receptors is likely to provide important insight regarding cannabinoid receptor biology.…”

“…It is not yet clear whether cannabinoids routinely bind and activate intracellular CB 2 , but there is at least one report providing direct experimental evidence for this (Brailoiu et al 2014). It is also not clear why B cells exhibit a receptor expression pattern that is distinct from other leukocytes or whether this is a unique feature in cells obtained from peripheral blood or related to the specific stage of cell activation or differentiation.…”