Bidirectional allosteric interactions between cannabinoid receptor 1 (CB) and dopamine receptor 2 long (D) heterotetramers.

Type 1 cannabinoid (CB) and dopamine 2 long form (D) receptors can physically interact to form heteromers that display unique pharmacology in vitro compared to homomeric complexes. Co-expression of CB and D and co-application of CB and D 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 CB/D/Gα protein complexes. Using bioluminescence resonance energy transfer 2 (BRET), we confirmed that, CB homodimers, D homodimers, and CB/D heteromers are formed. By using sequential resonance energy transfer 2 (SRET) combined with bimolecular fluorescence complementation (BiFC), we were able to demonstrate that CB/D form heterotetramers consisting of CB and D homodimers. We demonstrated that CB/D heterotetramers are coupled to at least two Gα proteins. The second aim of the study was to investigate allosteric effects of a D agonist (quinpirole) on CB receptor function and to investigate the effects of a CB agonist [arachidonyl-2-chloroethylamide (ACEA)] on D receptor function within CB/D heterotetramers. Treating cells co-expressing CB and D with both ACEA and quinpirole switched CB and D receptor coupling and signaling from Gα to Gα proteins, enhanced β-arrestin1 recruitment and receptor co-internalization. The concept of bidirectional allosteric interaction within CB/D heterotetramers has important implications for understanding the activity of receptor complexes in native tissues and under pathological conditions.