Analysis of various types of single-polypeptide-chain (sc) heterodimeric A₂AR/D₂R complexes and their allosteric receptor-receptor interactions.

Adenosine A2A receptor (A2AR) heteromerizes with dopamine D2 receptor (D2R). However, these class A G protein-coupled receptor (GPCR) dimers are not fully formed, but depend on the equilibrium between monomer and dimer. In order to stimulate the heteromerization, we have previously shown a successful design for a fusion receptor, single-polypeptide-chain (sc) heterodimeric A2AR/D2R complex. Here, using whole cell binding assay, six more different scA2AR/D2R constructs were examined. Not only in scA2AR/D2R 'liberated' with longer spacers between the two receptors, which confer the same configuration as the prototype, the A2AR-odr4TM-D2LR, but differ in size (Forms 1-3), but also in scA2AR/D2LR (Form 6) fused with a transmembrane (TM) of another type II TM protein, instead of odr4TM, neither of their fixed stoichiometry (the apparent ratios of A2AR to D2R binding sites) was 1, suggesting their compact folding. This suggests that type II TM, either odr4 or another, facilitates the equilibrial process of the dimer formation between A2AR and D2LR, resulting in the higher-order oligomer formation from monomer of scA2AR/D2LR itself. Also, in the reverse type scA2AR/D2LR, i.e., the D2LR-odr4TM-A2AR, counter agonist-independent binding cooperativity (cooperative folding) was found to occur (Forms 4 and 5). In this way, the scA2AR/D2LR system has unveiled the cellular phenomenon as a snapshot of the molecular behavior in A2AR/D2LR dimer. Thus, these results indicate that the various designed types of functional A2AR/D2R exist even in living cells and that this fusion expression system would be useful to analyze as a model of the interaction between class A GPCRs.