Bidirectional allosteric interactions between cannabinoid receptor 1 (CB1) and dopamine receptor 2 long (D2L) 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 D-2 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/Ga 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 Ga 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 Gai to Gas proteins, enhanced beta-arrestin1 recruitment and receptor co-internalization. The concept of bidirectional allosteric interaction within CB1/D-2 heterotetramers has important implications for understanding the activity of receptor complexes in native tissues and under pathological conditions.