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
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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DOI：
10.1021/ac049295f
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发表时间：
2004-09-15
影响因子：
7.4
通讯作者：
Woods, AS
中科院分区：
文献类型：
--
作者：
Ciruela, F;Burgueño, J;Woods, AS
先前的FRET和BRET实验和计算分析（对接模拟）的结果表明，人类多巴胺D-2受体（D2R）的第三胞内环（13）的一部分和人类腺苷a （2A）受体（a (2A)R）的c尾参与了a (2A)R-D2R异聚化。本研究利用下拉和质谱实验的结果表明，A(2A)R-D2R异聚化依赖于D2R的I3 （217RRRRKR222）富含arg的表位与相邻的两个Asp残基（DD401-402）或A(2A)R的c尾磷酸化Ser残基（S-374）之间的静电相互作用。含有A(2A)R c端结构域的gst融合蛋白（GST-A2A(CT)）能够从D2R转染的HEK-293细胞中溶解整个D2R。其次，与D2R （215VLRRRRKRVN224）富含arg的13区相对应并与Sepharose结合的肽能够降低GST-A2A（CT）和从a (2A)R转染的HEK-293细胞中溶解的整个a (2A)R。最后，质谱和下拉数据显示，富含arg的D2R表位与A(2A)R的c端部分的两个不同的表位结合，包含两个相邻的Asp残基或磷酸化的Ser残基(（388)HELKGVCPEPPGLDDPLAQDGAVGS（412）和（370）SAQ-EpSQGNT(378)）。目前的结果是受体异聚化下表位-表位静电相互作用的第一个例子，这是蛋白质-蛋白质相互作用的一个新的扩展领域。
Previous results from FRET and BRET experiments and computational analysis (docking simulations) have suggested that a portion of the third intracellular loop (13) of the human dopamine D-2 receptor (D2R) and the C-tail from the human adenosine A(2A) receptor (A(2A)R) are involved in A(2A)R-D2R heteromerization. The results of the present studies, using pull-down and mass spectrometry experiments, suggest that A(2A)R-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 (S-374) residue in the C-tail of the A(2A)R. A GST-fusion protein containing the C-terminal domain of the A(2A)R (GST-A2A(CT)) was able to pull down the whole D2R solubilized from D2R-tranfected HEK-293 cells. Second, a peptide corresponding to the Arg-rich 13 region of the D2R (215VLRRRRKRVN224) and bound to Sepharose was able to pull down both GST-A2A(CT) and the whole A(2A)R solubilized from A(2A)R-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 A(2A)R, containing the two adjacent Asp residues or the phosphorylated Ser residue ((388)HELKGVCPEPPGLDDPLAQDGAVGS(412) and (370)SAQ-EpSQGNT(378)). The present results are the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization, a new, expanding area of protein-protein interactions.