Combining mass spectrometry and pull-down techniques for the study of receptor heteromerization. Direct epitope-epitope electrostatic interactions between adenosine A(2A) and dopamine D-2 receptors

Previous results from FRET and BRET experiments and computational analysis (docking simulations) have suggested that a portion of the third intracellular loop (13) of the human dopamine D-2 receptor (D2R) and the C-tail from the human adenosine A(2A) receptor (A(2A)R) are involved in A(2A)R-D2R heteromerization. The results of the present studies, using pull-down and mass spectrometry experiments, suggest that A(2A)R-D2R heteromerization depends on an electrostatic interaction between an Arg-rich epitope from the I3 of the D2R (217RRRRKR222) and two adjacent Asp residues (DD401-402) or a phosphorylated Ser (S-374) residue in the C-tail of the A(2A)R. A GST-fusion protein containing the C-terminal domain of the A(2A)R (GST-A2A(CT)) was able to pull down the whole D2R solubilized from D2R-tranfected HEK-293 cells. Second, a peptide corresponding to the Arg-rich 13 region of the D2R (215VLRRRRKRVN224) and bound to Sepharose was able to pull down both GST-A2A(CT) and the whole A(2A)R solubilized from A(2A)R-tranfected HEK-293 cells. Finally, mass spectometry and pull-down data showed that the Arg-rich D2R epitope binds to two different epitopes from the C-terminal part of the A(2A)R, containing the two adjacent Asp residues or the phosphorylated Ser residue ((388)HELKGVCPEPPGLDDPLAQDGAVGS(412) and (370)SAQ-EpSQGNT(378)). The present results are the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization, a new, expanding area of protein-protein interactions.