Elsevier

Neuropharmacology

Volume 54, Issue 1, January 2008, Pages 36-44
Neuropharmacology

Rapid CB1 cannabinoid receptor desensitization defines the time course of ERK1/2 MAP kinase signaling

https://doi.org/10.1016/j.neuropharm.2007.06.005Get rights and content

Abstract

Molecular mechanisms regulating the development of physiological and behavioral tolerance to cannabinoids are not well understood. Two cellular correlates implicated in the development and maintenance of tolerance are CB1 cannabinoid receptor internalization and uncoupling of receptor signal transduction. Both processes have been proposed as mediators of tolerance because of observations that chronic Δ9-tetrahydrocannabinol (THC) treatment causes both region-specific decreases in CB1 receptors and G-protein coupling in the brain. To determine the balance of these two processes in regulating CB1 receptor signaling during sustained receptor stimulation, we evaluated the parameters affecting ERK1/2 MAP kinase activity in HEK293 cells stably expressing CB1 receptors. CB1 receptor stimulation by the potent CB1 receptor agonist, CP 55,940 transiently activated ERK1/2. To determine if CB1 receptor desensitization or internalization was responsible for the transient nature of ERK1/2 activation, we evaluated ERK1/2 phosphorylation in HEK293 cells expressing a desensitization-deficient CB1 receptor (S426A/S430A CB1). Here, the duration of S426A/S430A CB1 receptor-mediated activation of ERK1/2 was markedly prolonged relative to wild-type receptors, and was dynamically reversed by SR141716A. Interestingly, the S426A/S430A CB1 receptor was still able to recruit βarrestin-2, a key mediator of receptor desensitization, to the cell surface following agonist activation. In contrast to a central role for desensitization, pharmacological and genetic approaches suggested CB1 receptor internalization is dispensable in the transient activation of ERK1/2. This study indicates that the duration of ERK1/2 activation by CB1 receptors is regulated by receptor desensitization and underscores the importance of G-protein uncoupling in the regulation of CB1 receptor signaling.

Introduction

Most CNS effects of cannabinoids, the principal psychoactive ingredients of marijuana and their endogenous counterparts, are mediated by the CB1 cannabinoid receptor. It is well established that the CB1 receptor couples in an inhibitory manner to voltage-dependent Ca2+ channels and adenylyl cyclase and activates G-protein-regulated inwardly rectifying K+ (GIRK) channels (Howlett et al., 2002, Howlett and Fleming, 1984, Mackie and Hille, 1992, Mackie et al., 1995). In vitro studies have demonstrated that cannabinoids can also induce the activation of mitogen-activated protein (MAP) kinases of the extracellular signal-regulated kinase (ERK) superfamily (Bouaboula et al., 1995). Consistent with these findings, acute THC administration has been reported to increase CB1 receptor-mediated ERK1/2 activation in the dorsal striatum, nucleus accumbens, and hippocampus (Derkinderen et al., 2003, Valjent et al., 2001), while chronic THC treatment has been shown to increase ERK1/2 protein levels in the prefrontal cortex and hippocampus in vivo (Rubino et al., 2004).

Chronic administration of cannabinoids leads to the rapid development of tolerance. This tolerance is accompanied by changes in CB1 receptor number and/or function (Bass and Martin, 2000, Breivogel et al., 2003, Gonzalez et al., 2005, Sim-Selley, 2003) and the inhibition of cannabinoid-mediated plasticity in different brain regions (Hoffman et al., 2003, Mato et al., 2004). The uncoupling of G-proteins from their cognate receptor, or desensitization, terminates receptor signaling and has been implicated as one of the cellular adaptations underlying tolerance (Martin et al., 2004). G-protein-coupled receptors (GPCRs) can be desensitized following agonist activation through phosphorylation by GPCR kinases (GRKs) and subsequent βarrestin binding (Gainetdinov et al., 2004). The physical interaction of βarrestin with the phosphorylated receptor reduces the affinity of the GPCR for G-proteins, likely via a mechanism involving steric hindrance, thereby attenuating signaling (Dewire et al., 2007). Previous studies have found that the βarrestin-2 isoform of βarrestin and GRK3 are capable of desensitizing CB1 receptor-mediated activation of GIRK channels and inhibition of neurotransmission (Jin et al., 1999, Kouznetsova et al., 2002). Furthermore, serines 426 and 430 in the proximal carboxy-terminus of CB1 are the likely GRK3 phosphorylation sites underlying βarrestin-2 mediated CB1 receptor desensitization of GIRK activation (Jin et al., 1999).

Following binding of agonist and activation, CB1 receptors internalize via clathrin-coated pits. However, the details and regulation of this process are now only emerging (Coutts et al., 2001, Hsieh et al., 1999, Leterrier et al., 2004, McDonald et al., 2007, Rinaldi-Carmona et al., 1998). Specifically, while βarrestin-2 is a multi-functional protein (Dewire et al., 2007), whose binding has also been proposed to regulate many forms of receptor endocytosis (Claing et al., 2002), its involvement in CB1 receptor internalization remains unknown.

In these studies we used HEK293 cells stably expressing CB1 to investigate the relative contributions of CB1 receptor desensitization and internalization to the time course of ERK1/2 phosphorylation following CB1 receptor activation. We found that CB1 receptor desensitization primarily determines the time course of ERK1/2 activation. In contrast, CB1 receptor internalization is required for neither the onset nor the decay of ERK1/2 phosphorylation.

Section snippets

Reagents and drugs

CP 55,940 ((−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol) and SR141716A (4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide) were provided by the National Institute of Drug Abuse Drug Supply Program (RTI, Research Triangle Park, NC). Concanavalin A, was purchased from Vector Laboratories (Burlingame, CA). Pertussis toxin and cholera toxin were purchased from List Biological Laboratories, Inc. (USA).

Generation of mutant CB1 receptor constructs

The CB1

Mutation of serines 426 and 430 of the CB1 receptor prolong the time course of ERK1/2 activation in a SR141716A reversible manner

To test the hypothesis that putative GRK phosphorylation sites (serines 426 and 430) (Jin et al., 1999) in the proximal carboxy-terminus of the CB1 receptor determine the rate of ERK1/2 inactivation, the kinetics of ERK1/2 phosphorylation were evaluated following agonist stimulation of wild-type or S426A/S430A CB1 receptors stably expressed in HEK293 cells. A maximally efficacious concentration (100 nM) of the CB1 agonist, CP 55,940, was used for subsequent experiments (EC50 = 0.97 ± 0.06 nM for

Discussion

The principal finding of this study is that CB1 receptor desensitization dictates the kinetics of agonist-mediated ERK1/2 phosphorylation (activity). Using HEK293 cells stably expressing modified CB1 receptors, we have shown that mutation of serines 426 and 430 of CB1 to alanines dramatically prolongs the duration of ERK1/2 phosphorylation during protracted receptor stimulation (Fig. 1A). Importantly, the sustained phosphorylation of ERK1/2 (at time points ≤35 min) is a direct result of

Acknowledgements

This work was supported by DA07278 (T.D.), DA11322 (K.M.), and DA00286 (K.M.). We would like to thank Giovanna Cacciola for generating the concentration–response curve for ERK1/2 activation by CP 55,940 and for help with the initial assay development.

References (40)

  • J.G. Li et al.

    U50,488H-induced internalization of the human kappa opioid receptor involves a beta-arrestin- and dynamin-dependent mechanism. Kappa receptor internalization is not required for mitogen-activated protein kinase activation

    J. Biol. Chem.

    (1999)
  • B.R. Martin et al.

    Signaling pathways involved in the development of cannabinoid tolerance

    Trends Pharmacol. Sci.

    (2004)
  • T. Rubino et al.

    Modulation of extracellular signal-regulated kinases cascade by chronic delta 9-tetrahydrocannabinol treatment

    Mol. Cell. Neurosci.

    (2004)
  • H. Andersson et al.

    Membrane assembly of the cannabinoid receptor 1: impact of a long N-terminal tail

    Mol. Pharmacol.

    (2003)
  • S. Arttamangkul et al.

    Separation of mu-opioid receptor desensitization and internalization: endogenous receptors in primary neuronal cultures

    J. Neurosci.

    (2006)
  • M. Bouaboula et al.

    Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1

    Biochem. J.

    (1995)
  • R.E. Campbell et al.

    A monomeric red fluorescent protein

    Proc. Natl. Acad. Sci. U.S.A.

    (2002)
  • A.A. Coutts et al.

    Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons

    J. Neurosci.

    (2001)
  • P. Derkinderen et al.

    Regulation of extracellular signal-regulated kinase by cannabinoids in hippocampus

    J. Neurosci.

    (2003)
  • S.M. Dewire et al.

    beta-Arrestins and cell signaling

    Annu. Rev. Physiol.

    (2007)
  • Cited by (129)

    • Mechanisms of cannabinoid tolerance

      2023, Biochemical Pharmacology
    • The endocannabinoid system in the visual process

      2023, Journal of Photochemistry and Photobiology
    View all citing articles on Scopus
    View full text