The cannabinoid CB1 receptor (CB1R) is a G protein-coupled receptor, which couples to the G i/o family of heterotrimeric G proteins. The receptor displays both basal and agonist-induced signaling and internalization. Although basal activity of CB1Rs is attributed to constitutive (agonist-independent) receptor activity, studies in neurons suggested a role of postsynaptic endocannabinoid (eCB) release in the persistent activity of presynaptic CB1Rs. To elucidate the role of eCBs in basal CB1R activity, we have investigated the role of diacylglycerol lipase (DAGL) in this process in Chinese hamster ovary (CHO) cells, which are not targeted specifically with eCBs. Agonist-induced G protein activation was determined by detecting dissociation G protein subunits expressed in CHO cells with bioluminescence resonance energy transfer (BRET), after labeling the ␣ and ␤ subunits with Renilla luciferase and enhanced yellow fluorescent protein (EYFP), respectively. Preincubation of the cells with tetrahydrolipstatin (THL), a known inhibitor of DAGLs, caused inhibition of the basal activity of CB1R. Moreover, preincubation of CHO and cultured hippocampal neurons with THL increased the number of CB1Rs on the cell membrane, which reflects its inhibitory action on CB1R internalization in non-simulated cells. In CHO cells co-expressing CB1R and angiotensin AT 1 receptors, angiotensin II-induced G o protein activation that was blocked by both a CB1R antagonist and THL. These data indicate that cell-derived eCB mediators have a general role in the basal activity of CB1Rs in non-neural cells and neurons, and that this mechanism can be stimulated by AT 1 receptor activation.

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In the past years, the relationship between the endocannabinoid system (ECS) and other hormonal and neuromodulatory systems has been intensively studied. G protein-coupled receptors (GPCRs) can stimulate endocannabinoid (eCB) production via activation of Gq/11 proteins and, in some cases, Gs proteins. In this review, we summarize the pathways through which GPCR activation can trigger eCB release, as well as the best known examples of this process throughout the body tissues. Angiotensin II-induced activation of AT1 receptors, similar to other Gq/11-coupled receptors, can lead to the formation of 2-arachido-noylglycerol (2-AG), an important eCB. The importance of eCB formation in angiotensin II action is supported by the finding that the hypertensive effect of angiotensin II, injected directly into the hypothalamic paraventricular nucleus of anaesthetized rats, can be abolished by AM251, an inverse agonist of CB1 cannabinoid receptors (CB1Rs). We conclude that activation of the ECS should be considered as a general consequence of the stimulation of Gq/11-coupled receptors, and may mediate some of the physiological effects of GPCRs.

CB 1 cannabinoid receptor (CB 1 R) undergoes both constitutive and agonist-induced internalization, but the underlying mechanisms of these processes and the role of β-arrestins in the regulation of CB 1 R function are not completely understood. In this study, we followed CB 1 R internalization using confocal microscopy and bioluminescence resonance energy transfer measurements in HeLa and Neuro-2a cells. We found that upon activation CB 1 R binds β-arrestin2 (β-arr2), but not β-arrestin1. Furthermore, both the expression of dominant-negative β-arr2 (β-arr2-V54D) and siRNA-mediated knock-down of β-arr2 impaired the agonist-induced internalization of CB 1 R. In contrast, neither β-arr2-V54D nor β-arr2-specific siRNA had a significant effect on the constitutive internalization of CB 1 R. However, both constitutive and agonist-induced internalization of CB 1 R were impaired by siRNA-mediated depletion of clathrin heavy chain. We conclude that although clathrin is required for both constitutive and agonist-stimulated internalization of CB 1 R, β-arr2 binding is only required for agonist-induced internalization of the receptor suggesting that the molecular mechanisms underlying constitutive and agonist-induced internalization of CB 1 R are different.