Research reportMultiple mechanisms of CB1 cannabinoid receptors regulation
Introduction
Agonist-induced endocytosis of G-protein-coupled receptors (GPCRs) is an important process, mediating diverse roles in receptors regulation and signaling. Following activation of many GPCRs by agonists, the receptors undergo desensitization and internalization into endosomes. Receptors can then either be resensitized and recycled to the plasma membrane, or targeted to degradation (‘downregulation’). In recent years it has become evident that the process of endocytosis of GPCRs can serve an additional role in receptor signaling, of switching the coupling of GPCRs to alternative, mitogenic kinase cascades (for reviews, see Refs. [6], [34], [35]).
Many GPCRs undergo endocytosis via clathrin-coated pits. This process, which was extensively studied for the β2-adrenergic receptor (for review, see Ref. [20]), involves agonist-induced phosphorylation of the receptor, which promotes binding of β-arrestin proteins, followed by uncoupling of the receptor from G-proteins. Beta-arrestins associate with the AP-2 complex, which targets the receptor to clathrin-coated pits. Subsequently, the coated pits bud off and form coated vesicles. Although endocytosis of numerous receptors proceeds through this pathway, it has become clear that there are alternative, clathrin-independent mechanisms, that mediate agonist-induced endocytosis and downregulation [13], [35]. For example, several receptors such as the β1- and β2-adrenergic receptors [28], M2 acetylcholine receptor [7], and cholecystokinin receptor [26], were shown to sequester and undergo endocytosis via caveolae, which are uncoated invaginations on the surface of cells, and are believed now to be specialized membrane compartments with a role in signal transduction and endocytosis (for review, see Ref. [22]). The route by which the receptor undergoes endocytosis may be specific for the receptor type as was shown for D1 and D2 dopamine receptors [37], or to the cell type, as was shown for the β2-adrenergic receptor [21].
The cannabinoid CB1 receptor, which is predominantly expressed in the brain, belongs to the seven-transmembrane GPCRs family [15]. Previous studies have shown that CB1 receptors undergo agonist-induced internalization in transfected CHO cells [23], AtT20 cells [9], and HEK-293 cells [31], as well as in cultured hippocampal neurons and F-11 cells, that naturally express CB1 receptors [3]. Evidence for agonist-induced downregulation (a decrease in total cell receptors) of CB1 receptors was obtained, so far, only following chronic treatment with cannabinoids in vivo. Thus, prolonged administration of cannabinoid agonists reduced the density of cannabinoid receptors in distinct brain regions of rats [2], [19], [25], [27], as well as in the mouse cerebelum [5]. In addition to ligand-induced homologous endocytosis, receptors can also be internalized and downregulated by heterologous mechanisms, namely, through activation of another type of receptor in the same cell. Heterologous downregulation of CB1 receptors was recently found by us in HEK-293 cells which were cotransfected with CB1-cannabinoid and δ-opioid receptors, following prolonged exposure to an opioid agonist [31].
Compared to the large amount of information that has been gathered regarding the mechanisms of endocytosis and downregulation of other GPCRs, the knowledge on the routes and mechanisms of cannabinoid receptors endocytosis is still limited. The common method for studying GPCRs endocytosis and downregulation is by measuring the disappearance of binding sites from the cell surface following treatment with an agonist. However, as cannabinoids are hydrophobic ligands that can penetrate the cell, this approach did not prove useful when working with whole CHO cells, as there was no decrease in cannabinoid binding sites in cells that were treated with a cannabinoid agonist [23]. Therefore, the information available on CB1 endocytosis mechanisms has come, so far, from confocal microscopy studies, which used immunocytochemically labeled CB1 receptors. Using this approach, Hsieh et al. [9] demonstrated that, in AtT20 cells transfected with CB1 receptors, agonists caused rapid internalization of the receptors via clathrin coated pits. Rapid recycling of receptors which was dependent on phosphatase activity was also shown. CB1 receptors internalization did not require activated G-proteins in transfected AtT20 cells [9], or in hippocampal neurons in culture [3].
In the present study we investigated the routes of CB1 cannabinoid receptors endocytosis in HEK-293 cells transfected with CB1 receptors (HEK-CB). Endocytosis was measured by radioligand binding to membranes prepared from cells that were pre-exposed to a cannabinoid agonist. Pre-exposure to the cannabinoid agonist desacetyllevonantradol (DALN) decreased the density of CB1 binding sites in membranes prepared from the cells. Internalization of the receptors was found to proceed via two distinct pathways, through clathrin-coated pits and through caveolae. When both pathways were blocked, endocytosis of the receptors continued, indicating that compensatory mechanisms may exist. In contrast to HEK-CB cells, in N18TG2 neuroblastoma cells, that naturally express CB1 receptors, DALN-induced internalization of CB1 receptors was not mediated by caveolae-like membrane domains. Furthermore, while in HEK-293 cells, that had been cotransfected with CB1-cannabinoid and δ-opioid receptors, prolonged exposure to the opioid agonist etorphine induced heterologous downregulation of CB1 receptors, in N18TG2 cells, that naturally coexpress these two receptors, a long treatment with etorphine did not induce heterologous downregulation of cannabinoid receptors.
The data indicate that CB1 receptors can internalize via several distinct routes in the same cell, and that the process of endocytosis of CB1 receptors varies between different cell types.
Section snippets
Cell culture
HEK-293 cells were transfected with CB1 receptor DNA as previously described [32]. Cells (5×10−4 cells/ml) were seeded in 30 mm Petri dishes 24 h before transfection. The cells were transfected with 2 μg/dish mouse CB1 receptor DNA in pcDNA3 vector (HEK-CB). Selection was done in the presence of 1 mg/ml G418, and the selected clones were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 40 U/ml penicillin, 40 U/ml streptomycin and 0.25 mg/ml G418 (to
Results
Exposure of HEK-CB cells to the cannabinoid agonist DALN induced a dose- and time-dependent reduction in the binding of the radiolabeled antagonist [3H]SR141716 (Fig. 1). Exposure to 10−6 M DALN for 1.5 h reduced CB1 binding from 2.0±0.14 pmol/mg protein in control cells to 1.27±0.13 pmol/mg protein in DALN-pretreated cells, a reduction of 38% (n=32; P<0.001). Exposure of the cells to 10−6 M DALN for longer periods, up to 24 h, did not induce further reduction in [3H]SR141716 binding (Fig. 1A).
Discussion
Agonist-induced endocytosis of GPCRs mediates short-term regulation of receptors following their activation, and may also take part in long-term regulation of receptors that have been exposed to an agonist repeatedly, or for long periods of time. Compared to the extensive studies on other GPCRs endocytic mechanisms, the information regarding cannabinoid CB1 receptors endocytosis is limited. The present study was designed to investigate CB1 receptors regulation following agonist-treatment, and
Acknowledgements
This study was supported by The Israel Science Foundation founded by The Israel Academy of Sciences and Humanities (grant # 184-99).
References (37)
- et al.
Isolation and expression of a mouse CB1 cannabinoid receptor gene. Comparison of binding properties with those of native CB1 receptors in mouse brain and N18TG2 neuroblastoma cells
Biochem. Pharmacol.
(1997) - et al.
Cannabinoid receptor down-regulation without alteration of the inhibitory effect of CP 55,940 on adenylyl cyclase in the cerebellum of CP 55,940-tolerant mice
Brain Res.
(1996) - et al.
Dynamic targeting of the agonist-stimulated m2 muscarinic acetylcholine receptor to caveolae in cardiac myocytes
J. Biol Chem.
(1997) - et al.
Beta(2)-adrenergic receptor down-regulation. Evidence for a pathway that does not require endocytosis
J. Biol. Chem.
(1999) - et al.
Ligand-induced cleavage of the V2 vasopressin receptor by a plasma membrane metalloproteinase
J. Biol. Chem.
(1995) - et al.
The emergence of clathrin-independent pinocytic pathways
Curr. Opin. Cell Biol.
(1995) - et al.
CB1 cannabinoid receptor: cellular regulation and distribution in N18TG2 neuroblastoma cells
Mol. Brain Res.
(1998) - et al.
Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells
J. Biol. Chem.
(2000) - et al.
Chronic cannabinoid administration alters cannabinoid receptor binding in rat brain: a quantitative autoradiographic study
Brain Res.
(1993) - et al.
Differential targeting of beta-adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway
J. Biol. Chem.
(2000)
Independence of, and interactions between, cannabinoid and opioid signal transduction pathways in N18TG2 cells
Brain Res.
Long-term interactions between opioid and cannabinoid agonist at the cellular level: cross-desensitization and down-regulation
Brain Res.
Divers pathways mediate delta-opioid receptor down regulation within the same cell
Mol. Brain Res.
Role of endocytosis in mediating downregulation of G-protein-coupled receptors
Trends Pharmacol. Sci.
Cholesterol depletion inhibits epidermal growth factor receptor transactivation by angiotensin II in vascular smooth muscle cells: role of cholesterol-rich microdomains and focal adhesions in angiotensin II signaling
J. Biol. Chem.
Chronic delta9-tetrahydrocannabinol treatment produces a time-dependent loss of cannabinoid receptors and cannabinoid receptor-activated G proteins in rat brain
J. Neurochem.
Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons
J. Neurosci.
Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin
J. Cell Biol.
Cited by (34)
Mechanistic insights into the protective effect of paracetamol against rotenone-induced Parkinson's disease in rats: Possible role of endocannabinoid system modulation
2021, International ImmunopharmacologyCitation Excerpt :Increased levels of endogenous cannabinoids were previously reported in animal models of PD [139–142] and in PD’s human patients [143]. Prolonged CB1 receptor activation seems to be leading subsequently to its downregulation [144,145], an effect that may be reversed by PCM. It was demonstrated that CB1 receptor activation reduced pro-inflammatory mediators’ levels including NF‐κB, COX-2, and iNOS and reduced lipid peroxidation in an animal model of neuro-inflammation [21].
Long-term application of cannabinoids leads to dissociation between changes in cAMP and modulation of GABA <inf>A</inf> receptors of mouse trigeminal sensory neurons
2019, Neurochemistry InternationalCitation Excerpt :While most studies focused on CB1 turn-over and adaptation in the central nervous system, fewer examined the longer term effects of the spinal cord and the peripheral nervous system (Falenski et al., 2010; Fan et al., 1996; Rubino et al., 2000a; Sim-Selley, 2003). In vitro studies used a variety of different cell lines and cultures, leading to controversial results (Coutts et al., 2001; Grimsey et al., 2010; Hsieh et al., 1999; Keren and Sarne, 2003; Laprairie et al., 2014; Rinaldi-Carmona et al., 1998). We employed a model system of TG cultures to find out if 24 h application of CB1 agonists can change CB1 expression and function, and alter their cellular targets.
Early alteration of distribution and activity of hippocampal type-1 cannabinoid receptor in Alzheimer's disease-like mice overexpressing the human mutant amyloid precursor protein
2018, Pharmacological ResearchCitation Excerpt :This could explain the observed decrease in the amount of caveolin-1 co-immunoprecipitated with CB1 in transgenic mice compared to wild type ones (Fig. 3E). Furthermore, considering the functional role of caveolin-1 in sequestering CB1 receptor within caveolae [15,19–21], the interference in the interaction between CB1 and caveolin-1 due to the aberrant expression of APP in Tg2576 mice could also explain the mislocalization of CB1 receptor in membrane subdomains observed in subfractionation experiments (Fig. 2). It should be noted that we could not assess if the observed changes in CB1 activity in our model are also present in mice overexpressing the wild-type form of human APP, not being available this murine model.
Palmitoylation of cysteine 415 of CB<inf>1</inf> receptor affects ligand-stimulated internalization and selective interaction with membrane cholesterol and caveolin 1
2017, Biochimica et Biophysica Acta - Molecular and Cell Biology of LipidsDifferential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB<inf>1</inf> cannabinoid receptor
2013, Molecular and Cellular EndocrinologyCitation Excerpt :Similar to most GPCRs, CB1R internalizes upon agonist stimulation. This has been demonstrated in many cell lines, including CHO (Rinaldi-Carmona et al., 1998), AtT20 (Hsieh et al., 1999; Jin et al., 1999; Roche et al., 1999), F11 (Coutts et al., 2001), neuroblastoma N18TG2 (Keren and Sarne, 2003) and HEK293 (Keren and Sarne, 2003; Leterrier et al., 2004) cells, as well as in hippocampal neurons, which naturally express CB1R (Coutts et al., 2001; Leterrier et al., 2006). According to different studies, this agonist-induced CB1R endocytosis occurs via clathrin- and/or caveolin-mediated pathways in different cell types (Bari et al., 2008; Hsieh et al., 1999; Keren and Sarne, 2003; Wu et al., 2008).