Antagonism of dopamine receptor 2 long affects cannabinoid receptor 1 signaling in a cell culture model of striatal medium spiny projection neurons

Activation of dopamine receptor 2 long (D_2L) switches the signaling of type 1 cannabinoid receptor (CB₁) from Gα_i to Gα_s, a process thought to be mediated through CB₁-D_2L heteromerization. Given the clinical importance of D₂ antagonists, the goal of this study was to determine if D₂ antagonists could modulate CB₁ signaling. Interactions between CB₁ and D_2L, Gα_i, Gα_s, and β-arrestin 1 were studied using bioluminescence resonance energy transfer 2 (BRET²) in STHdh^Q7/Q7 cells. CB₁-dependent extracellular regulated kinase (ERK)1/2, CREB phosphorylation, and CB₁ internalization following cotreatment of CB₁ agonist and D₂ antagonist were quantified. Preassembled CB₁-Gα_i complexes were detected by BRET². Arachidonyl-2′-chloroethylamide (ACEA), a selective CB₁agonist, caused a rapid and transient increase in BRET efficiency (BRET_Eff) between Gα_i-Rluc and CB₁-green fluorescent protein 2 (GFP²), and a Gα_i-dependent increase in ERK phosphorylation. Physical interactions between CB₁ and D_2L were observed using BRET². Cotreatment of STHdh^Q7/Q7 cells with ACEA and haloperidol, a D₂ antagonist, inhibited BRET_Eff signals between Gα_i-Rluc and CB₁-GFP² and reduced the E_Max and pEC₅₀ of ACEA-mediated Gα_i-dependent ERK phosphorylation. ACEA and haloperidol cotreatments produced a delayed and sustained increase in BRET_Eff between Gα_s-Rluc and CB₁-GFP² and increased the E_Max and pEC₅₀ of ACEA-induced Gα_s-dependent cAMP response element-binding protein phosphorylation. In cells expressing CB₁and D_2L treated with ACEA, binding of haloperidol to D₂ receptors switched CB₁ coupling from Gα_i to Gα_s. In addition, haloperidol treatment reduced ACEA-induced β-arrestin1 recruitment to CB₁and CB₁ internalization. D₂ antagonists allosterically modulate cannabinoid-induced CB₁ coupling, signaling, and β-arrestin1 recruitment through binding to CB₁-D_2L heteromers. These findings indicate that D₂ antagonism, like D₂ agonists, change agonist-mediated CB₁ coupling and signaling.