A Predominant Role for Inhibition of the Adenylate Cyclase/Protein Kinase A Pathway in ERK Activation by Cannabinoid Receptor 1 in N1E-115 Neuroblastoma Cells

Abstract

Cannabinoids activate several members of the mitogen-activated protein kinase superfamily including p44 and p42 extracellular signal-regulated kinase (ERK). We used N1E-115 neuroblastoma cells and the cannabinoid receptor agonist WIN 55,212-2 (WIN) to examine the signal transduction pathways leading to the activation of ERK. ERK phosphorylation (activation) was measured by Western blot. The EC 50 for stimulation of ERK phosphorylation was 10 nM, and this effect was blocked by pertussis toxin and the CB1 (cannabinoid) receptor antagonist SR141716A. The MEK inhibitors PD 98059 and U0126 blocked ERK phosphorylation, as did the adenylate cyclase activator forskolin. The phosphatidylinositol (PI) 3-kinase inhibitor LY 294002 and the Src kinase inhibitor PP2 partially occluded the response but also decreased basal levels of phospho-ERK. The PI 3-kinase and Src pathways are known to promote cell survival in many systems; therefore, MTT (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan) conversion was used to examine the effects of these inhibitors on cellular viability. LY 294002 decreased the number of viable cells after 18 h of treatment; therefore, the inhibition of ERK by this inhibitor is probably because of cytotoxicity. Forskolin blocked ERK phosphorylation with an EC 50 of <3 M, and the protein kinase A (PKA) inhibitor H-89 enhanced ERK phosphorylation. c-Raf phosphorylation at an inhibitory PKA-regulated site (Ser 259 ) was also reduced by WIN. This is probably due to constitutive phosphatase activity because WIN did not directly stimulate PP1 or PP2A activity when measured using 6,8-difluoro-4-methylumbelliferyl phosphate as a fluorogenic substrate. These data implicate the inhibition of PKA as the predominant pathway for ERK activation by CB1 receptors in N1E-115 cells. PI 3-kinase and Src appear to contribute to ERK activation by maintaining activation of kinases, which prime the pathway and maintain cellular viability.

Chronic cannabinoid administration in vivo compromises extinction of fear memory

“…Activation of CB1 receptors stimulates a pertussis toxinsensitive G protein and inhibits adenylyl cyclase and PKA, leading to a decrease in constitutive phosphorylation of c-Raf at the inhibitory site (Ser259) and an increase in ERK phosphorylation (Davis et al 2003). ERK is known to be required for several forms of synaptic plasticity and learning and memory (English and Sweatt 1997;Impey et al 1998;Atkins et al 1998;Coogan et al 1999;Schafe and LeDoux 2000).…”

Mutations in the ‘DRY’ motif of the CB1 cannabinoid receptor result in biased receptor variants

“…8A). Recent data have suggested that CB 1 R-induced activation of p42/44 MAPK (ERK1/2), which was formerly suggested to occur via G-protein-dependent pathways (Galve-Roperh et al 2002, Davis et al 2003, Dalton & Howlett 2012, is also mediated by b-arrs (Ahn et al 2013a, Mahavadi et al 2014. Therefore, we aimed to study how the ERK1/2 responses correlate with the G-protein activation and/or b-arr recruitment of the biased CB 1 R mutants.…”

Endocannabinoids via CB1receptors act as neurogenic niche cues during cortical development

“…The CB 1 receptor, via canonical Gi-mediated inhibition of adenylyl cyclase, decreases cAMP concentration, and this in turn plays a prominent role by de-inhibiting the ERK pathway by protein kinase A [49,50]. In addition, G protein bg subunits liberated upon CB 1 receptor activation stimulate the ERK pathway in a PI3K-dependent manner [51].…”