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 A(2A) receptor (A(2A)R) C-terminal tall reduces A(2A)R-D2R heteromerization and prevents its allosteric modulation of the dopamine D-2 receptor (D2R) By means of site directed mutagenesis of the A(2A)R and synthetic transmembrane (TM) alpha-helix peptides of the D2R we further explored the role of electrostatic interactions and TM helix interactions of the A(2A)R-D2R heteromer interface We found evidence that the TM domains IV and V of the D2R play a major role in the A(2A)R-D2R heteromer interface since the incubation with peptides corresponding to these domains significantly reduced the ability of A(2A)R and D2R to heteromenze In addition the incubation with TM-IV or TM-V blocked the allosteric modulation normally found in A(2A)R-D2R heteromers The mutation of two negatively charged aspartates in the A(2A)R C-terminal tail (D401A/D402A) in combination with the S374A mutation drastically reduced the physical A(2A)R-D2R interaction and lost the ability of antagonistic allosteric modulation over the A(2A)R-D2R Interface suggesting further evidence for the existence of an electrostatic interaction between the C-terminal tail of A(2A)R 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 A(2A)R-D2LR heteromer interface together with D2LR TM segments IV/V interacting with A(2A)R TM-IV/V or TM-I/VII (C)2010 Elsevier Inc All rights reserved