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

Abstract:

A single serine point mutation (S374A) in the adenosine A<inf>2A</inf> receptor (A<inf>2A</inf>R) C-terminal tail reduces A<inf>2A</inf>R-D<inf>2</inf>R heteromerization and prevents its allosteric modulation of the dopamine D<inf>2</inf> receptor (D<inf>2</inf>R). By means of site directed mutagenesis of the A<inf>2A</inf>R and synthetic transmembrane (TM) α-helix peptides of the D<inf>2</inf>R we further explored the role of electrostatic interactions and TM helix interactions of the A<inf>2A</inf>R-D<inf>2</inf>R heteromer interface. We found evidence that the TM domains IV and V of the D<inf>2</inf>R play a major role in the A<inf>2A</inf>R-D<inf>2</inf>R heteromer interface since the incubation with peptides corresponding to these domains significantly reduced the ability of A<inf>2A</inf>R and D<inf>2</inf>R to heteromerize. In addition, the incubation with TM-IV or TM-V blocked the allosteric modulation normally found in A<inf>2A</inf>R-D<inf>2</inf>R heteromers. The mutation of two negatively charged aspartates in the A<inf>2A</inf>R C-terminal tail (D401A/D402A) in combination with the S374A mutation drastically reduced the physical A<inf>2A</inf>R-D<inf>2</inf>R interaction and lost the ability of antagonistic allosteric modulation over the A<inf>2A</inf>R-D<inf>2</inf>R interface, suggesting further evidence for the existence of an electrostatic interaction between the C-terminal tail of A<inf>2A</inf>R and the intracellular loop 3 (IL3) of D<inf>2</inf>R. On the other hand, molecular dynamic model and bioinformatic analysis propose that specific AAR, AQE, and VLS protriplets as an important motive in the A<inf>2A</inf>R-D<inf>2L</inf>R heteromer interface together with D<inf>2L</inf>R TM segments IV/V interacting with A<inf>2A</inf>R TM-IV/V or TM-I/VII. © 2010 Elsevier Inc.