Bidirectional allosteric interactions between cannabinoid receptor 1 (CB₁) and dopamine receptor 2 long (D_2L) heterotetramers

Type 1 cannabinoid (CB₁) and dopamine 2 long form (D_2L) receptors can physically interact to form heteromers that display unique pharmacology in vitro compared to homomeric complexes. Co-expression of CB₁ and D_2L and co-application of CB₁ and D₂ agonists increases cAMP levels while administration of either agonist alone decreases cAMP levels. To understand the observed co-agonist response, our first goal of the current study was to define the stoichiometry of CB₁/D_2L/Gα protein complexes. Using bioluminescence resonance energy transfer 2 (BRET²), we confirmed that, CB₁ homodimers, D_2L homodimers, and CB₁/D_2L heteromers are formed. By using sequential resonance energy transfer 2 (SRET²) combined with bimolecular fluorescence complementation (BiFC), we were able to demonstrate that CB₁/D_2L form heterotetramers consisting of CB₁ and D_2L homodimers. We demonstrated that CB₁/D_2L heterotetramers are coupled to at least two Gα proteins. The second aim of the study was to investigate allosteric effects of a D_2L agonist (quinpirole) on CB₁ receptor function and to investigate the effects of a CB₁ agonist [arachidonyl-2-chloroethylamide (ACEA)] on D_2L receptor function within CB₁/D_2L heterotetramers. Treating cells co-expressing CB₁ and D_2L with both ACEA and quinpirole switched CB₁ and D_2L receptor coupling and signaling from Gα_i to Gα_s proteins, enhanced β-arrestin1 recruitment and receptor co-internalization. The concept of bidirectional allosteric interaction within CB₁/D₂ heterotetramers has important implications for understanding the activity of receptor complexes in native tissues and under pathological conditions.