Characterization of the A2AR–D2R interface: Focus on the role of the C-terminal tail and the transmembrane helices

A single serine point mutation (S374A) in the adenosine A2A receptor (A2AR) C-terminal tail reduces A2AR–D2R heteromerization and prevents its allosteric modulation of the dopamine D2 receptor (D2R). By means of site directed mutagenesis of the A2AR and synthetic transmembrane (TM) α-helix peptides of the D2R we further explored the role of electrostatic interactions and TM helix interactions of the A2AR–D2R heteromer interface. We found evidence that the TM domains IV and V of the D2R play a major role in the A2AR–D2R heteromer interface since the incubation with peptides corresponding to these domains significantly reduced the ability of A2AR and D2R to heteromerize. In addition, the incubation with TM-IV or TM-V blocked the allosteric modulation normally found in A2AR–D2R heteromers. The mutation of two negatively charged aspartates in the A2AR C-terminal tail (D401A/D402A) in combination with the S374A mutation drastically reduced the physical A2AR–D2R interaction and lost the ability of antagonistic allosteric modulation over the A2AR–D2R interface, suggesting further evidence for the existence of an electrostatic interaction between the C-terminal tail of A2AR and the intracellular loop 3 (IL3) of D2R. On the other hand, molecular dynamic model and bioinformatic analysis propose that specific AAR, AQE, and VLS protriplets as an important motive in the A2AR–D2LR heteromer interface together with D2LR TM segments IV/V interacting with A2AR TM-IV/V or TM-I/VII.

Research highlights
► TM domains IV and V of the D2R play a major role in the A2AR–D2R heteromer interface.
► Allosteric modulation found in A2AR–D2R heteromers is blocked by incubation with D2R TM-IV or TM-V.
► Mutation of (D401A/D402A/S374A) reduced A2AR–D2R interaction and lost the ability of antagonistic allosteric modulation over the A2AR–D2R interface.
► Bioinformatic analysis proposes that specific AAR, AQE, and VLS protriplets as an important motive in the A2AR–D2LR heteromer interface.
► Molecular dynamic modeling implies D2LR TM-IV/V interacting with A2AR TM-IV/V and D2LR TM-IV/V interacting with A2AR TM-I/VII.