Evidence exists that the adenosine receptor A(2A)R and the dopamine receptor D2R form constitutive heteromers in living cells. Mass spectrometry and pull-down data showed that an arginine-rich domain of the D2R third intracellular loop binds via electrostatic interactions to a specific motif of the A(2A)R C-terminal tail. It has been indicated that the phosphorylated serine 374 might represent an important residue in this motif. In the present study, it was found that a point mutation of serine 374 to alanine reduced the A(2)AR ability to interact with D2R. Also, this point mutation abolished the A(2A)R-mediated inhibition of both the D2R high affinity agonist binding and signaling. These results point to a key role of serine 374 in the A(2A)R-D2R interface. All together these results indicate that by targeting A(2A)R serine 374 it will be possible to allosterically modulate A(2A)R-D2R function, thus representing a new approach for therapeutically modulate D2R function. (C) 2010 Elsevier Inc. All rights reserved.