Interactions between intracellular domains as key determinants of the quaternary structure and function of receptor heteromers

G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A_2A, cannabinoid CB₁, and dopamine D₂ receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A_2A, CB₁, andD ₂) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB₁ receptor with A_2A andD₂ receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A _2A-CB₁-D₂ receptor heteromers. Analysis of MAPK signaling in striatal slices of CB₁ receptor KO mice and wild-type littermates supported the existence ofA₁-CB₁-D₂ receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer.