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.
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.
Ciruela
Francisco
F
Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, E-08028, Spain.
Burgueño
Javier
J
Casadó
Vicent
V
Canals
Meritxell
M
Marcellino
Daniel
D
Goldberg
Steven R
SR
Bader
Michael
M
Fuxe
Kjell
K
Agnati
Luigi F
LF
Lluis
Carmen
C
Franco
Rafael
R
Ferré
Sergi
S
Woods
Amina S
AS
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
D000939:Epitopes; D043705:Receptor, Adenosine A2A; D017448:Receptors, Dopamine D2