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

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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., Burgueno, J., Casado, V.V., Canals, M., Marcellino, D., Goldberg, S.R., Bader, M., Fuxe, K., Agnati, L.F., Lluis, C., Franco, R., 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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