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