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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

2004; American Chemical Society; Volume: 76; Issue: 18 Linguagem: Inglês

10.1021/ac049295f

1520-6882

Francisco Ciruela, Javier Burgueño, Vicent Casadó, Meritxell Canals, Daniel Marcellino, Steven R. Goldberg, Michael Bäder, Kjell Fuxé, Luigi F. Agnati, Carmen Lluís, Rafael Franco, Sergi Ferré, Amina S. Woods,

Monoclonal and Polyclonal Antibodies Research

Previous results from FRET and BRET experiments and computational analysis (docking simulations) have suggested that a portion of the third intracellular loop (I3) of the human dopamine D2 receptor (D2R) and the C-tail from the human adenosine A2A receptor (A2AR) are involved in A2AR−D2R heteromerization. The results of the present studies, using pull-down and mass spectrometry experiments, suggest that A2AR−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 (S374) residue in the C-tail of the A2AR. A GST-fusion protein containing the C-terminal domain of the A2AR (GST-A2ACT) was able to pull down the whole D2R solubilized from D2R-tranfected HEK-293 cells. Second, a peptide corresponding to the Arg-rich I3 region of the D2R (215VLRRRRKRVN224) and bound to Sepharose was able to pull down both GST-A2ACT and the whole A2AR solubilized from A2AR-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 A2AR, containing the two adjacent Asp residues or the phosphorylated Ser residue (388HELKGVCPEPPGLDDPLAQDGAVGS412 and 370SAQEpSQGNT378). The present results are the first example of epitope−epitope electrostatic interaction underlying receptor heteromerization, a new, expanding area of protein−protein interactions.