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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

Item Type:Article
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
Creators Name:Ciruela, F. and Burgueno, J. and Casado, V.V. and Canals, M. and Marcellino, D. and Goldberg, S.R. and Bader, M. and Fuxe, K. and Agnati, L.F. and Lluis, C. and Franco, R. and Ferre, S. and Woods, A.S.
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 Argrich epitope from the I3 of the D2R ( 217RRRRKR222) and two adjacent Asp residues (DD 401-402) 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 370SAQ- EpSQGNT378). The present results are the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization, a new, expanding area of protein-protein interactions.
Keywords:Adenosine A2A Receptor, Amino Acid Sequence, Binding Sites, Cell Line, Dimerization, Dopamine D2 Receptors, Electrostatics, Epitopes, Mass Spectrometry, Protein Binding
Source:Analytical Chemistry
ISSN:0003-2700
Publisher:American Chemical Society
Volume:76
Number:18
Page Range:5354-5363
Date:1 January 2004
Official Publication:https://doi.org/10.1021/ac049295f
PubMed:View item in PubMed

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