Cannabinoid Receptor 1 trafficking and the role of the intracellular pool: Implications for therapeutics

doi:10.1016/j.bcp.2010.06.007

Biochemical Pharmacology

Volume 80, Issue 7, 1 October 2010, Pages 1050-1062

https://doi.org/10.1016/j.bcp.2010.06.007 Get rights and content

Abstract

Cannabinoid Receptor 1 (CB₁), an abundant G-protein coupled receptor in the CNS, is currently of significant interest as a therapeutic target. Although the cellular control of receptor trafficking is intimately linked with drug effects, CB₁ trafficking is poorly defined in the current literature and conflicting evidence exists as to whether CB₁ should be classified as a recycling, degrading or “dual-fate” receptor. Of particular interest is the widely noted intracellular pool which has been speculated to form part of a constitutive internalization and recycling pathway. This study performs a detailed quantification of CB₁ trafficking in four cell lines, one of which expresses CB₁ endogenously. We demonstrate that, contrary to previous reports, CB₁ does not recycle following constitutive or agonist-induced internalization but instead exhibits a primarily degradative phenotype. Further, our data suggest that the intracellular pool does not contribute to cell surface re-population. These findings have significant implications for the interpretation of CB₁ biochemical studies and the design and application of cannabinoid therapeutics.

Graphical abstract

“Intracellular pool” CB₁ receptors do not contribute to cell surface re-population, which requires new protein synthesis as CB₁ does not recycle following internalization.

Introduction

Cannabinoid Receptor 1 (CB₁) is one of the most abundant G-protein coupled receptors (GPCRs) in the mammalian CNS and is also expressed at various sites in the periphery [1]. As the mediator of endocannabinoid (eCB) effects on a variety of brain and systemic functions, with postulated roles in a number of neurological and immune disorders (reviewed in [2]), CB₁ is the target of a number of therapeutics recently approved or currently in clinical trials (e.g. Marinol, Cesamet, Sativex).

For the majority of receptor-mediated drug treatments, the quantity of receptors present at the cell surface is a fundamental determinant of the potential cellular response. Considerable research in recent times has therefore focused on understanding GPCR intracellular trafficking. Following internalization, receptors can enter divergent post-endocytic pathways that confer distinct effects on cell signaling. Trafficking of receptors to degradative pathways leads to a prolonged loss of responsiveness (e.g. [3]) whereas the recycling of receptors back to the surface allows the cell to remain responsive to further activation (reviewed in [4]). Recent studies have revealed that the delivery of receptors to particular pathways is highly organized (e.g. [5], [6], [7]) and associated interacting proteins may present drug targets through which receptor localization and activity, and consequently cellular responses, could be controlled [8].

In the brain, CB₁ is expressed at the pre-synaptic cell membrane of axon terminals (e.g. [9], [10]). Plasma membrane expression in primary neuronal cultures is associated with axonal processes [11], [12], [13], and is also present in endogenously-expressing [14], [15] and transfected [16], [17], [18], [19], [20] immortalized cell lines. However, in the brain (e.g. [9], [21], [22]), as well as in each of these cell systems, a significant proportion of CB₁ is located in an “intracellular pool” in the cytoplasm. In both transfected cells and those that endogenously express CB₁, this intracellular pool displays only minimal co-localization with protein synthesis-associated organelles [18], [23]. This observation, combined with the ability of the receptor to constitutively endocytose [18] and correlation of results with other receptors that exhibit similar phenotypes (e.g. [24], [25], [26]) have led to the inference that this intracellular pool serves as a reservoir of endocytic origin. This reservoir may function as a source from which surface CB₁ is replenished to replace internalized receptor [18], suggesting that CB₁ exhibits a recycling phenotype.

In contrast to this recycling hypothesis, downregulation of CB₁ following chronic agonist stimulation has also been widely reported (reviewed in [27]), and recently GASP-1 [19], [28] and AP3 [23], adaptor proteins associated with sorting and delivery of receptors to lysosomes, were demonstrated to interact with CB₁. Furthermore, interruption of the GASP-1 interaction with a dominant-negative mutant prevented degradation [19]. Thus, there is evidence for both recycling and degradation of CB₁.

In this study, we have utilized a high-throughput receptor quantification technique [29] to perform a detailed characterization of CB₁ intracellular trafficking and to investigate the role of the intracellular pool in four cell lines, one of which expresses CB₁ endogenously. We demonstrate that CB₁ exhibits a primarily degradative phenotype and that the widely noted intracellular pool does not contribute to cellular re-sensitization.

Section snippets

Cell culture

Cell culture and molecular biology reagents were from Invitrogen (Carlsbad, CA) unless otherwise stated. Human embryonic kidney-293 (HEK) cells (ATCC #CRL-1573) were transfected with rat CB₁ chimerized with a single hemagglutinin (HA) tag [30], rat CB₁ chimerized with a single FLAG tag, or human Dopamine Receptor 1 (D₁) with three HA tags. AtT-20 and Chinese hamster ovary-K1 (CHO; ATCC #CRL-9618) cells were transfected with human CB₁ chimerized with three HA tags. CB₁ and D₁ constructs

CB₁ is present both at the cell surface and in a large intracellular pool in transfected and endogenously-expressing cell lines

The majority of the experiments presented in this study were performed with HEK cells stably expressing HA-tagged rat CB₁. To ensure that the results were not cell type or species specific, experiments central to the findings of this report were also performed on CHO and AtT-20 cells stably expressing HA-tagged human CB₁ and on Neuro-2a cells, a mouse neuroblastoma line that expresses CB₁ endogenously [15], [32].

We first characterized the subcellular localization of CB₁ in each of these models.

Discussion

As a highly prevalent receptor that modulates a range of brain and systemic functions, CB₁ is currently of significant interest as a pharmaceutical target. The number of functional receptors present at the plasma membrane is generally considered to be a major determinant of potential cell responsiveness to endogenous or exogenous ligands, yet we and others have noted that many cells that endogenously express CB₁, including neurons, exhibit large intracellular pools of receptors in addition to

Acknowledgements

We thank Prof Ken Mackie (Dept. of Anaesthesiology, University of Washington, Seattle, WA) for donating the rCB₁ HEK-293 cell line, wild-type AtT-20 cells and CB₁ antibodies, and Drs Emma Scotter and Leslie Schwarcz (Dept. of Pharmacology and Clinical Pharmacology, University of Auckland, NZ) for insightful suggestions and discussion. This research was funded by the Royal Society of New Zealand Marsden Fund. The Discovery-1™ and MetaMorph^® analysis facility (//www.fmhs.auckland.ac.nz/sms/pharmacology/discovery1

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Cannabinoid Receptor 1 trafficking and the role of the intracellular pool: Implications for therapeutics

Abstract

Graphical abstract

Introduction

Section snippets

Cell culture

CB1 is present both at the cell surface and in a large intracellular pool in transfected and endogenously-expressing cell lines

Discussion

Acknowledgements

Prog Neurobiol

Curr Opin Neurobiol

J Biol Chem

J Biol Chem

Mol Brain Res

J Biol Chem

J Biol Chem

Neuroscience

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Life Sci

Biochim Biophys Acta – Rev Biomembr

Trends Endocrinol Metab

J Biol Chem

J Biol Chem

Anal Biochem

Curr Opin Neurobiol

Pharmacol Biochem Behav

J Biol Chem

Neuroscience

J Biol Chem

Endocannabinoids and their receptors: physiology, pathology and pharmacology

Immunol Endocr Metab Agents Med Chem

Functional effects of long-term activation on human beta 2- and beta 3-adrenoceptor signalling

Br J Pharmacol

A kinase-regulated PDZ-domain interaction controls endocytic sorting of the β2-adrenergic receptor

Nature

Regulation of GPCRs by endocytic membrane trafficking and its potential implications

Annu Rev Pharmacol Toxicol

Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons

J Neurosci

The CB1 cannabinoid receptor is the major cannabinoid receptor at excitatory presynaptic sites in the hippocampus and cerebellum

J Neurosci

CB₁ is present both at the cell surface and in a large intracellular pool in transfected and endogenously-expressing cell lines