Cannabinoid agonists increase the interaction between β-Arrestin 2 and ERK1/2 and upregulate β-Arrestin 2 and 5-HT2A receptors

Abstract

We have recently reported that selective cannabinoid 2 (CB2) receptor agonists upregulate 5-HT2A receptors by enhancing ERK1/2 signaling in prefrontal cortex (PFCx). Increased activity of cortical 5-HT2A receptors has been associated with several neuropsychiatric disorders such as anxiety and schizophrenia. Here we examine the mechanisms involved in this enhanced ERK1/2 activation in rat PFCx and in a neuronal cell model. Sprague-Dawley rats treated with a non-selective cannabinoid agonist (CP55940, 50 μg/kg, 7 days, i.p.) showed enhanced co-immunoprecipitation of β-Arrestin 2 and ERK1/2, enhanced pERK protein levels, and enhanced expression of β-Arrestin 2 mRNA and protein levels in PFCx. In a neuronal cell line, we found that selective CB2 receptor agonists upregulate β-Arrestin 2, an effect that was prevented by selective CB2 receptor antagonist JTE-907 and CB2 shRNA lentiviral particles. Additionally, inhibition of clathrin-mediated endocytosis, ERK1/2, and the AP-1 transcription factor also prevented the cannabinoid receptor-induced upregulation of β-Arrestin 2. Our results suggest that sustained activation of CB2 receptors would enhance β-Arrestin 2 expression possibly contributing to its increased interaction with ERK1/2 thereby driving the upregulation of 5-HT2A receptors. The CB2 receptor-mediated upregulation of β-Arrestin 2 would be mediated, at least in part, by an ERK1/2-dependent activation of AP-1. These data could provide the rationale for some of the adverse effects associated with repeated cannabinoid exposure and shed light on some CB2 receptor agonists that could represent an alternative therapeutic because of their minimal effect on serotonergic neurotransmission.

G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

“…Here we treated cells with CP55940 for 72 h in order to assess the effect of repeated cannabinoid agonist exposure on the expression of GRKs because our previous findings show that repeated, but not single, exposure to cannabinoid agonists up-regulates 5-HT 2A receptor protein expression (1)(2)(3)(4). CP55940 treatment in these cells significantly (p Ͻ 0.01) increased GRK5 protein levels (67 Ϯ 3% increase compared with vehicle-treated controls) without significant (p Ͼ 0.05) changes in the protein levels of GRK6 (Fig.…”

“…Effect of Selective CB 1 or CB 2 Receptor Agonists on GRK5 mRNA-CLU213 cells were incubated with either vehicle (ethanol, 0.01% final concentration), GP1a 1 nM (selective CB 2 agonist, K i ϭ 0.037 and 353 nM for CB 2 and CB 1 receptors, respectively) (30), or 15 nM ACEA (selective CB 1 agonist, K i ϭ 1.4 nM and 3.1 M for CB 1 and CB 2 receptors, respectively) (31) for 72 h. Cells were washed (three times) with PBS every 24 h, and fresh vehicle, GP1a, or ACEA was added. qRT-PCR for GRK5 was performed.…”

“…Cells were analyzed for GRK5, ␤-arrestin 2, CB 1 Calcium Assay to measure 5-HT 2A Receptor Activity-Optimal conditions were elucidated using different buffers, plating densities, agonists, time courses, etc., and through reference to previously published protocols (34,35). Cells were plated at 30,000 cells/well in complete medium and grown to 90% confluence on black-sided 96-well plates.…”

“…Co-immunoprecipitation-These experiments were conducted with the Thermo Scientific Pierce co-immunoprecipitation kit following the manufacturer's protocol and as described in detail previously (1). The ␤-arrestin 2 and ERK1/2 antibody was purchased from Santa Cruz Biotechnology, Inc.…”

“…We have recently reported that repeated exposure to cannabinoid agonists induces a strong up-regulation and increases the activity of 5-HT 2A 2 receptors in rat prefrontal cortex (PFCx) and in two neuronal cell models (1)(2)(3)(4). This cannabinoid-mediated up-regulation of 5-HT 2A receptors was 1) induced by nonselective CB 1 /CB 2 and selective CB 2 receptor agonists (2, 4) and 2) inhibited by selective CB 2 , but not CB 1 , shRNA lentiviral particles, suggesting that CB 2 receptors mediate this phenomenon (3).…”