Cannabinoid 2 receptors regulate dopamine 2 receptor expression by a beta-arrestin 2 and GRK5-dependent mechanism in neuronal cells

We have previously reported that the repeated exposure to cannabinoids upregulates and enhances the activity of serotonin 2A (5-HT_2A) and dopamine 2 (D₂) receptors and facilitates the formation of D₂-5-HT_2A receptor heterodimers in the rat prefrontal cortex and two neuronal cell lines. Because the repeated exposure to cannabinoids has been associated with adverse neuropsychiatric disorders, this study investigated the mechanisms that underly the cannabinoid-mediated regulation of D₂ receptor expression in a neuronal cell model, CLU213 cells. We initially tested the effects of repeated exposure (72 h) to a non-selective cannabinoid agonist (1 nM CP55940), a selective CB₁ receptor agonist (15 nM ACEA), or a selective CB₂ receptor drug (1 nM GP1a) on the expression of postsynaptic D₂ (D₂L) receptors in CLU213 cells. Repeated CP55940, GP1a, or ACEA treatments significantly increased D₂L receptor protein levels (99 % ± 7%, 30 % ± 7%, and 39 % ± 5% increases compared with control levels, respectively). Repeated exposure to both GP1a and ACEA increased D₂L receptor protein levels by 73 % ± 8%. Interestingly, CP55940 and GP1a, but not ACEA, upregulated D₂ mRNA. Using cells that were stably transfected with short-hairpin RNA (shRNA) lentiviral particles targeting CB₂ receptors, G protein-coupled receptor kinase 5 (GRK5), and β-arrestin 2, we found that CB₂ receptors regulated D₂ expression through a mechanism that is dependent on GRK5, β-arrestin 2, and extracellular signal-related kinase (ERK)1/2. We also found that repeated exposure to either ACEA or GP1a selectively stimulated the protein and mRNA expression of GRK proteins. ACEA significantly upregulated GRK2 proteins, whereas GP1a upregulated GRK5 protein expression. Our results identified mechanisms associated with the upregulation of D₂ receptors in neuronal cells after the repeated exposure to cannabinoids. These data can shed light on the mechanisms that can be targeted to prevent potential adverse effects, while simultaneously determining the therapeutic benefits of cannabinoids.