title: Combining mass spectrometry and pull-down techniques for the study of receptor heteromerization. Direct epitope-epitope electrostatic interactions between adenosine A2A and dopamine D2 receptors.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, E-08028, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Ciruela
authors.forename: Javier
authors.initials: J
authors.lastname: Burgueño
authors.forename: Vicent
authors.initials: V
authors.lastname: Casadó
authors.forename: Meritxell
authors.initials: M
authors.lastname: Canals
authors.forename: Daniel
authors.initials: D
authors.lastname: Marcellino
authors.forename: Steven R
authors.initials: SR
authors.lastname: Goldberg
authors.forename: Michael
authors.initials: M
authors.lastname: Bader
authors.forename: Kjell
authors.initials: K
authors.lastname: Fuxe
authors.forename: Luigi F
authors.initials: LF
authors.lastname: Agnati
authors.forename: Carmen
authors.initials: C
authors.lastname: Lluis
authors.forename: Rafael
authors.initials: R
authors.lastname: Franco
authors.forename: Sergi
authors.initials: S
authors.lastname: Ferré
authors.forename: Amina S
authors.initials: AS
authors.lastname: Woods
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D019281:Dimerization; D000939:Epitopes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D011485:Protein Binding; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000737:chemistry* / Q000378:metabolism*; D055672:Static Electricity
chemical_list: D000939:Epitopes; D043705:Receptor, Adenosine A2A; D017448:Receptors, Dopamine D2