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The cannabinoid CB1 receptor is a G protein-coupled receptor (GPCR; receptor nomenclature follows Alexander et al., 2011) that is stimulated by full agonists, functionally similar to Δ9-tetrahydrocannabinol (Δ9-THC) (e.g. CP55940, WIN55212-2), and the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) (Howlett et al., 2002). CB1 receptors are expressed predominantly in the nervous system and are responsible for many of the neuronal effects produced by endocannabinoids and cannabinoid drugs. The CB1 receptor associates with Pertussis toxin-sensitive Gi/o proteins to regulate a variety of signal transduction pathways including inhibition of adenylyl cyclase, inhibition of L-, N- and P/Q-type Ca2+ channels, activation of focal adhesion kinase, induction of immediate early gene expression, and stimulation of nitric oxide production (see Howlett, 2005 and Pertwee, 2006). CB1 receptors also activate members of the mitogen-activated protein kinase (MAPK) family including extracellular signal-regulated kinases 1 and 2 (ERK1/2) (Bouaboula et al., 1995). These in vitro observations were confirmed by in vivo studies that showed acute Δ9-THC administration increased ERK1/2 activation in dorsal striatum, nucleus accumbens and hippocampus (Valjent et al. 2001; Derkinderen et al., 2003), whereas chronic Δ9-THC treatment increased phosphorylated ERK1/2 levels in prefrontal cortex and hippocampus (Rubino et al., 2004). Studies suggest alteration in ERK1/2 signalling in specific CB1 receptor-enriched brain regions is a key molecular adaptation that underlies the expression of cannabinoid tolerance and dependence (Rubino et al., 2005; Tonini et al. 2006).
The ERKs 1 and 2 are serine/threonine kinases that constitute the final component of the MAPK cascade [Raf/MAPK-ERK kinase (MEK)/ERK], which is considered a key junction point that mediates the integration and processing of information between signal transduction cascades in cells (Kyosseva, 2004). Dual phosphorylation of threonine and tyrosine residues that reside in the ERK1/2 activation loop is required for full activity (Chen et al., 2001). CB1 receptors regulate ERK1/2 phosphorylation/activation via several mechanisms that include Gi/o protein activation (Galve-Roperh et al., 2002; Davis et al. 2003), adenylate cyclase/protein kinase A (PKA) inhibition (Davis et al., 2003), receptor tyrosine kinase (RTK) transactivation (Bouaboula et al., 1997; Rubovitch et al. 2004; Korzh et al., 2008), phosphatidylinositol 3-kinase (PI-3K) activation (Galve-Roperh et al., 2002) and activation of the Src family kinase, Fyn (Derkinderen et al., 2003). Recent studies have demonstrated that CB1 receptor-mediated ERK activation is time-dependent (peak activation at 5 min followed by rapid inactivation) in HEK293 cells, and is regulated by CB1 receptor phosphorylation and desensitization, but not CB1 receptor internalization (Daigle et al., 2008).
The aim of the present study was to investigate the mechanisms that regulate the time-course of CB1 receptor-mediated ERK tyrosine phosphorylation in neuronal N18TG2 cells that express endogenous CB1 receptors. We found three phases of ERK phosphorylation: Phase I maximal ERK activation (0–5 min), Phase II decline in ERK activation (5–10 min) and Phase III plateau in ERK activation (>10 min). Cellular mechanisms responsible for each phase of CB1 receptor-mediated ERK activation differ, and include ligand-independent transactivation of multiple RTKs (Phase I and III), protein tyrosine phosphatase (PTP) activation (Phase I and III) and serine/threonine phosphatase activation/PKA inhibition (Phase II).
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The principal finding of this study is that CB1 receptor-stimulated ERK tyrosine phosphorylation/activation occurs in three phases that are regulated by distinct cellular mechanisms. Phase I (0–5 min) initiates maximal ERK activation, Phase II involves a rapid decline in ERK activation (5–10 min), and Phase III maintains a plateau in ERK activation (>10 min). Phase I is mediated by CB1 receptor-stimulated transactivation of the Flk-1 VEGFR, IGF-1R and EGFR in a ligand-independent fashion mediated by Gi/oβγ-mediated activation of PI-3K and tyrosine phosphatase (PTP1B and Shp1) – regulation of Src kinase (Figure 7). Phase I is under permissive regulation by inhibition of adenylyl cyclase/PKA, while Phase II requires adenylyl cyclase/PKA inhibition plus serine/threonine phosphatase activation. The plateau in ERK phosphorylation in Phase III involves CB1 regulation of RTKs and is regulated via signalling mechanisms, similar to those used in Phase I. Blocking the CB1 receptor stimulus during Phase I or Phase III by the competitive antagonist SR141716 was sufficient to terminate ERK phosphorylation, indicating the stimulus must be continuously applied, rather than initially applied as a one-time trigger for an ensuing process. These results are in agreement with the time-course observed for exogenous CB1 receptors expressed in HEK293 cells, in which the agonist CP55940 induced rapid, transient ERK activation peaking at 5 min and rapidly decaying by 15–30 min, but with no apparent establishment of a sustained Phase III in these cells (Daigle et al., 2008). Expression of an S426A/S430A CB1 receptor mutant in HEK293 cells established that phosphorylation at this domain is required for the development of Phase II ‘desensitization’ (Daigle et al., 2008), possibly by attenuating Gi/o stimulation.
Figure 7. Signalling pathways utilized by the CB1 receptor to stimulate Phase I maximal ERK tyrosine phosphorylation in N18TG2 cell nucleus and cytosol. CB1 receptor-stimulated maximal ERK activation occurs via ligand-independent transactivation of the Flk-1 VEGF, EGF and IGF-1 receptor tyrosine kinases (RTK). The components involved in the transactivation process include Gi/oβγ subunit-mediated activation of PI-3K and Src kinase activation. A key event in Src kinase activation may be the dephosphorylation of Tyr527 by the tyrosine phosphatases PTP1B and Shp1. CB1 receptor-stimulated maximal ERK activation involves the traditional Raf/MEK/ERK cascade and results in an increase in ERK activation in both nucleus and cytosol. However, Phase I CB1 receptor-stimulated ERK activation does not involve net nuclear translocation of ERK in N18TG2 cells.
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Our findings expand upon previous studies showing CB1 receptor transactivation of VEGFRs to regulate ERK activation in N18TG2 cells (Rubovitch et al., 2004). Those studies reported that the CB1 receptor agonist desacetyllevonantradol potentiated Ca2+ influx into N18TG2 cells via VEGFR transactivation and the subsequent activation of ERK (Rubovitch et al., 2004). Desacetyllevonantradol-mediated ERK phosphorylation was attenuated by inhibition of matrix metalloproteinases and protein kinase C (Rubovitch et al., 2004), both of which can play a role in ligand-dependent RTK transactivation (Belcheva and Coscia, 2002). In contrast, our studies indicate that both Phase I and Phase III CB1 receptor-mediated ERK activations occur via ligand-independent transactivation of multiple RTKs, a discrepancy that may stem from methodological differences. In those studies (Rubovitch et al., 2004), N18TG2 cells were treated with desacetyllevonantradol for 10 min, which our studies demonstrate coincides with Phase II Gi/o protein desensitization. It is possible that the response to matrix metalloproteinase-mediated release of RTK-stimulating ligands may become evident as Gi/o protein regulation is suppressed.
Although our studies identified an absolute requirement for Flk-1 VEGFR transactivation in CB1 receptor-mediated ERK phosphorylation in N18TG2 cells, the use of RTK inhibitors designed to inhibit EGFRs and IGF-1Rs inhibited CB1 receptor-mediated ERK phosphorylation. The combination of EGFR and IGF-1R inhibitors produced additive inhibition of CB1 receptor-stimulated ERK phosphorylation in N18TG2 cells. One explanation is that EGF and IGF-1 receptors are transactivated by Flk-1 VEGFRs, as there is a precedent for crosstalk between RTKs to regulate ERK. For example, Shc-EGFR complexes were responsible for IGF-1-stimulated, ligand-dependent EGFR-driven ERK phosphorylation in a COS-7 cell model (Roudabush et al., 2000). In addition, PDGF stimulation of PDGFR–EGFR heterodimers resulted in EGFR transactivation and EGFR-mediated ERK phosphorylation in rat aortic vascular smooth muscle cells (Saito et al., 2001).
The ramifications of CB1 receptor signalling that depends entirely upon RTKs are (i) selectivity in cellular response based upon specific RTKs that are expressed; and (ii) either additivity, synergism or competition with growth factors to which RTKs would otherwise respond. Crosstalk between CB1 receptors and RTKs was first reported in Chinese hamster ovary cells expressing recombinant human CB1 receptors (Bouaboula et al., 1997). In that model system, the CB1 antagonist SR141716 blocked MAPK activation in response to endogenously expressed insulin and IGF-1 receptors, suggesting the requirement for functional coupling of CB1 receptors to these RTKs (Bouaboula et al., 1997). In U373MG glioblastoma and NCI-H292 lung carcinoma cells, cannabinoid agonists induced activation of protein kinase B and ERK1/2 via ligand-dependent transactivation of EGFRs (Hart et al., 2004), suggesting the response to endocannabinoids might not be observed if the EGFRs were fully stimulated by matrix metalloproteinase-mediated release of EGF ligands catalysed by other stimuli. 2-AG and anandamide evoked TrkB–CB1 receptor complex formation and tyrosine phosphorylation of TrkB in PC12 pheochromocytoma cells co-expressing exogenous TrkB and CB1 receptors, suggesting that these receptors form a complex or are held in close proximity in a common membrane domain (such as a lipid raft structure) (Berghuis et al., 2005). Migration of developing, CB1-expressing interneurons in response to anandamide occurred through Src kinase-mediated TrkB transactivation and was additive with the effects of brain-derived neurotrophic factor (BDNF) (Berghuis et al., 2005). However, CB1 agonists inhibited BDNF-induced morphological differentiation, which also occurred via a Src kinase-dependent mechanism. In other transactivation studies, anandamide precluded nerve growth factor-stimulated TrkA-induced PC12 cell differentiation via CB1 receptor-mediated inhibition of Rap1/B-Raf/ERK (Rueda et al., 2002). These investigations of RTK downstream functions seem to suggest a competitive interaction exists such that CB1 agonists can stimulate RTKs in the absence of other signals. However, if the cognate growth factor is available, CB1 receptor-mediated RTK transactivation appears to be competitive or not observed at all.
We propose that during Phase I, Gi/oβγ subunits mediate the sequential activation of PI-3K and Src kinase to stimulate CB1 receptor-mediated RTK transactivation in N18TG2 cells. Evidence from other cellular systems supports a pathway by which Gi/oβγ subunits bind to and activate PI-3K (Lopez-Ilasaca et al., 1997; Stephens et al. 1997), while Src kinase functions as a mediator of Gi/oβγ subunit-stimulated RTK phosphorylation and ERK activation (Luttrell et al., 1996; 1997). Studies using U373MG human astrocytoma cells indicated that CB1 receptors can activate ERK via Gi/oβγ subunit-mediated PI-3K activation (Galve-Roperh et al., 2002). Although CB1 receptors were not coupled to RTKs or a Src kinase in U373MG cells (Galve-Roperh et al., 2002), CB1 receptor-stimulated TrkB transactivation in PC12 cells was mediated by a Src kinase (Berghuis et al., 2005), and CB1 receptor-regulated hippocampal ERK activity was mediated by the Src kinase Fyn (Derkinderen et al., 2003). Our studies suggest that Phases I and III of CB1 receptor-stimulated ERK activation are regulated by specific tyrosine phosphatases that activate Src kinase (Somani et al., 1997; Roskoski, 2005). These studies demonstrate the complex interplay that tyrosine phosphorylation/dephosphorylation can exert on the endpoint of ERK activation, allowing for multiple points of intervention by concurrent signal transduction pathways.
CB1 receptor-mediated inhibition of the adenylyl cyclase/PKA pathway is a critical modulator in the regulation of Phase I and II ERK activation. This regulatory mechanism is predicted to exert a dominant effect under circumstances of concurrent neuromodulator-mediated stimulation of types 5/6 adenylyl cyclase or Ca2+ influx-mediated regulation of types 1/3/8 adenylyl cyclase (Rhee et al., 1998). PKA-mediated phosphorylation/inhibition of Raf is a well-defined mechanism for regulation of ERK activity (Kikuchi and Williams, 1996; Mischak et al. 1996). This mechanism played the predominant role in anandamide-stimulated ERK phosphorylation in N1E-115 neuroblastoma cells, in which net Raf dephosphorylation resulted in activation of the Raf/MEK/ERK cascade (Davis et al., 2003). Hippocampal ERK phosphorylation was also significantly influenced by modification of cAMP levels (Derkinderen et al., 2003). Other reports have attributed the sustained phase of the Raf/MEK/ERK cascade in neuronal cells to PKA-mediated activation of Rap-1, a Ras superfamily member that mediates inhibition of Raf-1 and activation of B-Raf (Vossler et al., 1997; Bouschet et al. 2003). Thus, CB1 receptor-mediated inhibition of adenylyl cyclase/PKA may be an important regulatory mechanism in Phase II by precluding PKA-Rap-1-mediated ERK activation.
Studies of recombinant WT and desensitization-deficient CB1 receptors expressed in HEK293 cells implicated phosphorylation of Ser426 and Ser430 in the Phase II off-rate (t1/2 = 3 min) of CB1-stimulated ERK phosphorylation (Daigle et al., 2008). When phosphorylated by GPCR kinase 3, Ser426 and Ser430 were responsible for the uncoupling of the CB1 receptor from G protein-mediated ion channel regulation in an oocyte model (Jin et al., 1999), suggesting a similar desensitization mechanism for CB1 receptor uncoupling to the ERK phosphorylation process (Daigle et al., 2008). Pretreatment of N18TG2 cells with the serine/threonine phosphatase inhibitor okadaic acid at concentrations that inhibit both PP1 and PP2A activity prevented the decline in Phase II ERK activation and resulted in an increase in net ERK1 and ERK2 tyrosine phosphorylation compared to control values following WIN55212-2 treatment for 10 min. PP1 and PP2A typically inhibit MAPK signalling by catalysing the dephosphorylation/inactivation of Raf, MEK or ERK (Zhou et al., 2002; Junttila et al. 2008).
Phase III CB1 receptor-mediated ERK activation may occur as signal components relocate from the plasma membrane to sub-cellular loci. Moreover, studies in neurons have suggested that sustained ERK activation is necessary for ERK nuclear translocation and regulation of gene expression by transcription factors such as Elk-1 (Traverse et al., 1992; Roux and Blenis, 2004). In the present study, the kinetics of CB1 agonist-mediated ERK phosphorylation were identical in N18TG2 cytosol and nucleus. However, CB1 receptor-mediated ERK phosphorylation appears to be dissociated from the process of ERK nuclear translocation in N18TG2 cells, inasmuch as ERK is present in the nucleus in the absence of exogenously applied CB1 receptor agonists and sustained CB1 receptor-mediated ERK activation did not evoke ERK nuclear accumulation. Nevertheless, Phase III sustained ERK activation during chronic cannabinoid exposure may underlie the cellular modifications necessary for expression of cannabinoid tolerance (Rubino et al., 2004; 2005; Tonini et al., 2006).
In conclusion, the information gained regarding the cellular regulation of CB1 receptor-stimulated ERK activation reveals how protein kinases, protein phosphatases and CB1 receptor-mediated RTK transactivation play a role in the complex signalling networks that regulate cellular function. A thorough analysis of how each of these signalling processes participate in CB1 receptor regulation of the MAPK cascade can provide targets for modification of cellular behaviour in either specific cell types or states of disease. At present, there is a growing body of evidence that CB1 receptor agonists and antagonists have therapeutic benefits in modulating cellular processes that involve synaptic plasticity and neuronal remodelling in pathologies such as substance abuse and neurodegenerative diseases. Targeting the cellular signalling mechanisms utilized by CB1 receptors may provide new intervention strategies that can maximize benefits and reduce risks associated with the therapeutic use of cannabinoid ligands.