Multiple mechanisms of CB1 cannabinoid receptors regulation
ABSTRACT Agonist-induced regulation of cannabinoid CB1 receptors was examined in HEK-293 cells transfected with CB1 receptors and in neuroblastoma N18TG2 cells that naturally express CB1 receptors. In HEK-293 cells, CB1 receptors internalization proceeded, in parallel, via clathrin-coated pits and caveolae. Simultaneous disruption of both pathways induced compensatory endocytic mechanism(s). In N18TG2 cells, endocytosis was not mediated by caveolae-like membrane domains. Heterologous, opioid-induced, downregulation of CB1 receptors was evident in HEK-293 but not N18TG2 cells. The data demonstrate the existence of multiple pathways of CB1 receptors regulation.
- SourceAvailable from: Ana Juknat
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- "Lipid raft/caveolae have been proposed to compartmentalize the endocannabinoid signalling machinery in several cellular systems (Keren and Sarne, 2003; McFarland et al., 2004; 2008 Bari et al., 2005a,b; 2006; 2008; McFarland and Barker, 2005; Sarnataro et al., 2005, 2006; Oddi et al., 2007; Placzek et al., 2008; Rimmerman et al., 2008; Maccarrone et al., 2009). Specifically, several lines of evidence support an association of the cannabinoid CB 1 receptor (nomenclature follows Alexander et al., 2011) with lipid raft/caveolae including: (i) CB1 receptor C-terminal acylation domain is required for proper interactions with lipid raft-associated G proteins (Mukhopadhyay et al., 1999; Barnett-Norris et al., 2005; Fay et al., 2005; Xie and Chen, 2005); (ii) CB1 receptor internalization in human embryonic kidney 293 cells-CB1 transfected cells that occurs via both caveolae and clathrin-coated pits (Keren and Sarne, 2003); (iii) increased CB1 receptor binding and signalling following cholesterol depletion in C6 glioma cells (Bari et al., 2005a,b; 2006); (iv) CB1 receptor association with lipid raft fractions/ non-lipid raft fractions in MDA-MB- 231 breast cancer cells which depends on receptor activation/ antagonism (Sarnataro et al., 2005; 2006); and (v) CB1 receptor localization within lipid rafts in human endothelial cells, and CB1 receptor co-localization with caveolin-1 in C6 glioma cells (Bari et al., 2006; 2008). "
ABSTRACT: N-acyl ethanolamines (NAEs) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids and along with related lipids are synthesized on demand from membrane phospholipids. Here, we have studied the compartmentalization of NAEs and 2-AG into lipid raft fractions isolated from the caveolin-1-lacking microglial cell line BV-2, following vehicle or cannabidiol (CBD) treatment. Results were compared with those from the caveolin-1-positive F-11 cell line. BV-2 cells were incubated with CBD or vehicle. Cells were fractionated using a detergent-free continuous OptiPrep density gradient. Lipids in fractions were quantified using HPLC/MS/MS. Proteins were measured using Western blot. BV-2 cells were devoid of caveolin-1. Lipid rafts were isolated from BV-2 cells as confirmed by co-localization with flotillin-1 and sphingomyelin. Small amounts of cannabinoid CB(1) receptors were found in lipid raft fractions. After incubation with CBD, levels and distribution in lipid rafts of 2-AG, N-arachidonoyl ethanolamine (AEA), and N-oleoyl ethanolamine (OEA) were not changed. Conversely, the levels of the saturated N-stearoyl ethanolamine (SEA) and N-palmitoyl ethanolamine (PEA) were elevated in lipid raft fractions. In whole cells with growth medium, CBD treatment increased AEA and OEA time-dependently, while levels of 2-AG, PEA and SEA did not change. Whereas levels of 2-AG were not affected by CBD treatment, the distribution and levels of NAEs showed significant changes. Among the NAEs, the degree of acyl chain saturation predicted the compartmentalization after CBD treatment suggesting a shift in cell signalling activity. LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.British Journal of Pharmacology 03/2011; 165(8):2436-49. DOI:10.1111/j.1476-5381.2011.01380.x · 4.84 Impact Factor
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- "CB1 receptor endocytosis is important in resensitization, as indicated by the finding that blockade of endocytic recycling enhances WIN55,212-2-induced desensitization of CB1 receptormediated cAMP inhibition (Wu et al., 2008). Longer agonist exposure (1.5 h) promotes down-regulation of CB1 receptors (Hsieh et al., 1999), as can briefer exposure to very high concentrations of agonists (Keren and Sarne, 2003; Martini et al., 2007). CB1 receptor down-regulation is associated with co-localization of the receptor with lysosomal markers, lysosome-associated membrane protein (LAMP)1 and 2 (Martini et al., 2007). "
ABSTRACT: The main pharmacological effects of marijuana, as well as synthetic and endogenous cannabinoids, are mediated through G-protein-coupled receptors (GPCRs), including CB(1) and CB(2) receptors. The CB(1) receptor is the major cannabinoid receptor in the central nervous system and has gained increasing interest as a target for drug discovery for treatment of nausea, cachexia, obesity, pain, spasticity, neurodegenerative diseases and mood and substance abuse disorders. Evidence has accumulated to suggest that CB(1) receptors, like other GPCRs, interact with and are regulated by several other proteins beyond the established role of heterotrimeric G-proteins. These proteins, which include the GPCR kinases, beta-arrestins, GPCR-associated sorting proteins, factor associated with neutral sphingomyelinase, other GPCRs (heterodimerization) and the novel cannabinoid receptor-interacting proteins: CRIP(1a/b), are thought to play important roles in the regulation of intracellular trafficking, desensitization, down-regulation, signal transduction and constitutive activity of CB(1) receptors. This review examines CB(1) receptor-interacting proteins, including heterotrimeric G-proteins, but with particular emphasis on non-G-protein entities, that might comprise the CB(1) receptosomal complex. The evidence for direct interaction with CB(1) receptors and potential functional roles of these interacting proteins is discussed, as are future directions and challenges in this field with an emphasis on the possibility of eventually targeting these proteins for drug discovery.British Journal of Pharmacology 06/2010; 160(3):454-66. DOI:10.1111/j.1476-5381.2010.00777.x · 4.84 Impact Factor
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- "somes (Hsieh et al. 1999; Leterrier et al. 2004; Martini et al. 2007). However, the endocytotic mechanism of CB1 receptors is not completely understood although recent findings suggest that several pathways including clathrincoated pits and caveolae might be involved in this process (Keren and Sarne 2003). Following CB1R activation and desensitization, the receptors are internalized, and then are either degraded in lysosomes or recycled back to the membrane for reactivation. "
ABSTRACT: Agonist-induced internalization of G protein-coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid-rafts- and clathrin-coated-pits-mediated pathways were involved in agonist-induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Delta9-THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212-2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist-induced CB1 receptor desensitization. Furthermore, co-expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co-expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212-2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Delta9-THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212-2, CP 55940 or 2-arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid-mediated effects.Journal of Neurochemistry 03/2008; 104(4):1132-43. DOI:10.1111/j.1471-4159.2007.05063.x · 4.28 Impact Factor