Antagonism of Dopamine Receptor 2 Long Affects Cannabinoid Receptor 1 Signaling in a Cell Culture Model of Striatal Medium Spiny Projection Neurons

摘要

Activation of dopamine receptor 2 long (D-2L) switches the signaling of type 1 cannabinoid receptor (CB1) from G alpha(i) to G alpha s, a process thought to be mediated through CB1-D-2L heteromerization. Given the clinical importance of D-2 antagonists, the goal of this study was to determine if D-2 antagonists could modulate CB1 signaling. Interactions between CB1 and D2L, Gai, Gas, and beta-arrestin1 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-G alpha(i) complexes were detected by BRET2. Arachidonyl-2'-chloroethylamide (ACEA), a selective CB1 agonist, caused a rapid and transient increase in BRET efficiency (BRETEff) between G alpha(i)-Rluc and CB1-green fluorescent protein 2 (GFP(2)), and a G alpha i-dependent increase in ERK phosphorylation. Physical interactions between CB1 and D2L were observed using BRET2. Cotreatment of STHdh(Q7/Q7) cells with ACEA and haloperidol, a D-2 antagonist, inhibited BRETEff signals between G alpha i-Rluc and CB1-GFP(2) and reduced the E-Max and pEC(50) of ACEA-mediated Gai- dependent ERK phosphorylation. ACEA and haloperidol cotreatments produced a delayed and sustained increase in BRETEff between G alpha s-Rluc and CB1-GFP(2) and increased the E-Max and pEC(50) of ACEA-induced G alpha s-dependent cAMP response element-binding protein phosphorylation. In cells expressing CB1 and D-2L treated with ACEA, binding of haloperidol to D-2 receptors switched CB1 coupling from Gai to Gas. In addition, haloperidol treatment reduced ACEA-induced b-arrestin1 recruitment to CB1 and CB1 internalization. D-2 antagonists allosterically modulate cannabinoid-induced CB1 coupling, signaling, and beta-arrestin1 recruitment through binding to CB1-D-2L heteromers. These findings indicate that D-2 antagonism, like D-2 agonists, change agonist-mediated CB1 coupling and signaling.