Elsevier

Brain Research

Volume 980, Issue 2, 8 August 2003, Pages 197-205
Brain Research

Research report
Multiple mechanisms of CB1 cannabinoid receptors regulation

https://doi.org/10.1016/S0006-8993(03)02970-6Get rights and content

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.

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).

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