Abstract
Activation of dopamine receptor 2 long (D2L) switches the signaling of type 1 cannabinoid receptor (CB1) from Gαi to Gαs, a process thought to be mediated through CB1-D2L 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 D2L, Gαi, Gαs, and β-arrestin1 were studied using bioluminescence resonance energy transfer 2 (BRET2) in STHdhQ7/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-Gαi complexes were detected by BRET2. Arachidonyl-2′-chloroethylamide (ACEA), a selective CB1 agonist, caused a rapid and transient increase in BRET efficiency (BRETEff) between Gαi-Rluc and CB1–green fluorescent protein 2 (GFP2), and a Gαi-dependent increase in ERK phosphorylation. Physical interactions between CB1 and D2L were observed using BRET2. Cotreatment of STHdhQ7/Q7 cells with ACEA and haloperidol, a D2 antagonist, inhibited BRETEff signals between Gαi-Rluc and CB1-GFP2 and reduced the EMax and pEC50 of ACEA-mediated Gαi-dependent ERK phosphorylation. ACEA and haloperidol cotreatments produced a delayed and sustained increase in BRETEff between Gαs-Rluc and CB1-GFP2 and increased the EMax and pEC50 of ACEA-induced Gαs-dependent cAMP response element-binding protein phosphorylation. In cells expressing CB1 and D2L treated with ACEA, binding of haloperidol to D2 receptors switched CB1 coupling from Gαi to Gαs. In addition, haloperidol treatment reduced ACEA-induced β-arrestin1 recruitment to CB1 and CB1 internalization. D2 antagonists allosterically modulate cannabinoid-induced CB1 coupling, signaling, and β-arrestin1 recruitment through binding to CB1-D2L heteromers. These findings indicate that D2 antagonism, like D2 agonists, change agonist-mediated CB1 coupling and signaling.
Footnotes
- Received January 20, 2016.
- Accepted April 4, 2016.
This work was supported by a partnership grant from the Canadian Institutes of Health Research, Nova Scotia Health Research Foundation, and the Huntington Society of Canada [ROP-97185] to E.M.D.-W., and a Canadian Institutes of Health Research operating grant [MOP-97768] to M.E.M.K. A.M.B. is supported by scholarships from Dalhousie University and King Abdul Aziz University, Jeddah, Saudi Arabia. R.B.L. is supported by studentships from the Canadian Institutes of Health Research, the Huntington Society of Canada, Killam Trusts, and Nova Scotia Health Research Foundation. Primary laboratory of origin: E.M.D.-W.
This work was previously presented in abstract as: Bagher AM, Laprairie RB, Kelly ME, and Denovan-Wright EM (2014) Influence of the dopamine receptor Type 2 (D2) antagonist on the cannabinoid receptor Type 1 (CB1) function. The 24th Annual International Cannabinoid Research Society Symposium on the Cannabinoids; 2014 June 29th; Baveno, Italy. Abstr. International Cannabinoid Research Society, Research Triangle Park, NC.
↵This article has supplemental material available at molpharm.aspetjournals.org.
- Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|