Multiple mechanistically distinct modes of endocannabinoid mobilization at central amygdala glutamatergic synapses.

The central amygdala (CeA) is a key structure at the limbic-motor interface regulating stress responses and emotional learning. Endocannabinoid (eCB) signaling is heavily implicated in the regulation of stress-response physiology and emotional learning processes; however, the role of eCBs in the modulation of synaptic efficacy in the CeA is not well understood. Here we describe the subcellular localization of CB1 cannabinoid receptors and eCB synthetic machinery at glutamatergic synapses in the CeA and find that CeA neurons exhibit multiple mechanistically and temporally distinct modes of postsynaptic eCB mobilization. These data identify a prominent role for eCBs in the modulation of excitatory drive to CeA neurons and provide insight into the mechanisms by which eCB signaling and exogenous cannabinoids could regulate stress responses and emotional learning

Transactivation of epidermal growth factor receptor EGFR by CB1 cannabinoid receptor agonists

Cannabinoids have long been used as therapeutic agents. The recent identification of the endogenous cannabinoid system in the brain, namely the abundance of the CB1 receptor (CB1R) in the CNS and the discovery of the endogenous ligands, has suggested that the cannabinoid neuromodulatory system plays important roles in many physiological processes, such as in memory, as well as holds therapeutic promise in a wide range of diseases ranging from mood and anxiety disorders to movement disorders and spinal cord injury. The CB1 receptor exerts its effects through several signalling events, which include the activation of the extracellular signal-regulated kinase, or ERKs. The mechanism by which this G-protein coupled receptor triggers ERK activation is not, however, well understood. To investigate the molecular events coupling CB1 activation to PKC and ERK activation, we generated clonal SHSY-5Y neuroblastoma cell lines which stably overexpress EGFP-CB1R and assessed whether this coupling could be mediated via transactivation of growth factor receptors with intrinsic tyrosine kinase activity. We first established using both immunoprecipitation and immunocytochemistry and GFP fluorescent imaging that the EGFP-CB1R was properly glycosylated and targeted to plasma membranes. We now report that the CB1R agonist methanandamide specifically stimulated, in a time-dependent manner, tyrosine phosphorylation of several cellular proteins, including phosphorylation of the EGF receptor and Src kinase on tyrosine residues 1148 and 416, respectively, and of ERK2. These effects were abolished by the specific CB1 antagonist AM251 and by the EGFR antagonist AG1478. These data provide first evidence that in a neuronal cellular background, the CB1R intracellular signalling engages the specific transactivation of the tyrosine kinase EGF receptor to regulate ERK activation and downstream transcriptional events. ©ΦApmakon-Túttoς

CB1R-dependent activation of Fyn tyrosine kinase and protein kinase C Delta, PKCδ, in lipid rafts

Cannabinoid 1 receptors (CB1Rs) are heptahelical transmembrane receptors which may exert their effects through the activation of the extracellular signal-regulated kinases (ERKs). We have previously shown that stably overexpressed CB1R in neuroblastoma cells (SH-SY5Y-CB1R cell line) is coupled to ERK activation via a mechanism that involves cannabinoid-induced transactivation of the EGF receptor and PKC activation. In a new line of experiments, EGFR transactivation by cannabinoid agonists was further supported by assessments of Ras activity. Ras assays revealed elevated Ras activity after Methanandamide treatment, which was abolished by the EGFR inhibitor AG1478. In analyzing this mechanism, we investigated the subcellular trafficking of the CB1R in basal conditions and in response to agonist stimulation in SH-SY5Y-CB1R cells. We found that under basal conditions, CB1R was mainly distributed in subcellular fractions which contain plasma-membrane, mitochondria or ER membranes, whereas after treatment with the CB1 agonist Methanandamide we observed redistribution of the receptor into the lipid rafts fractions. Moreover, we found that the activated (phosphorylated) species of EGFR also appeared in the lipid rafts after Methanandamide and importantly this effect was completely abolished by AG1478. To address what molecular events couple CB1R activation to ERK activation, we investigated whether members of Src family tyrosine kinases mediate this coupling. We found that PP1 and PP2 inhibitors of the Src family of tyrosine kinases in particular of Fyn kinase, abolish the methanandamide-dependent ERK activation. Furthermore, Methanandamide treatment induced tyrosine phosphorylation, an event that was inhibited by PP1, as well as by inhibitors of novel PKCs. In addition, Methanandamide-induced phosphorylation- activation of PKCs was also partially inhibited by Fyn and PKC inhibitors. Next, using immunoprecipitations we found that the novel PKC isoform delta, PKCδ, was tyrosine-phosphorylated in response to Methanandamide treatment and that this tyrosine phosphorylation was abolished by PP1 and Ro31-8220 (an inhibitor of classic and novel PKCs) but not by Go6976 (an inhibitor of classic PKC isoforms). These results suggest that in CB1R signaling a) Fyn activation may lie upstream of PKCδ, and b) a novel PKC isoform other than PKCδ activates Fyn which in turn activates PKCδ. ©PHARMAKON-Press