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