Homodimerization of adenosine A2A receptors:: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer
Homodimerization of adenosine A2A receptors:: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer
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DOI：
10.1046/j.1471-4159.2003.02200.x
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发表时间：
2004-02-01
影响因子：
4.7
通讯作者：
Franco, R
中科院分区：
文献类型：
--
作者：
Canals, M;Burgueño, J;Franco, R
结果表明，腺苷A(2A)受体(A(2A)R)形成同源二聚体，同源二聚体而不是单体是细胞表面的功能物种。用荧光共振能量转移(FRET)和生物发光共振能量转移(BRET)技术证实了在HEK293细胞中A(2A)R的同源二聚，A(2A)R是在纹状体中丰富表达的七聚膜受体。时间分辨FRET结果表明细胞表面存在同源二聚体。虽然激动剂激活受体导致受体簇的形成，但不影响A(2A)R-A(2A)R二聚化的程度。免疫印迹法检测到细胞提取液中存在单体和二聚体。然而，蛋白质的细胞表面生物素化作用表明，超过90%的细胞表面受体处于二聚体形式。因此，同源二聚体似乎是存在于质膜上的受体的功能形式。通过免疫印迹分析，A(2A)受体的一个缺失突变版本，缺少其C-末端结构域，也能够形成单体和二聚体。这表明C-末端的尾部不参与二聚化。这是相关的，因为A(2A)R的C端尾巴参与了A(2A)R和多巴胺D2受体形成的异构体。A(2A)R-A(2A)R同源二聚体的BRET比值高于A(2A)R和多巴胺D2受体形成的异源二聚体。由于A(2A)R和多巴胺D2受体确实相互作用，这些结果表明A(2A)R同源二聚体是细胞表面的功能物种，它们与A(2A)R/D2受体异二聚体共存。
The results presented in this paper show that adenosine A(2A) receptor (A(2A)R) form homodimers and that homodimers but not monomers are the functional species at the cell surface. Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques have been used to demonstrate in transfected HEK293 cells homodimerization of A(2A)R, which are heptaspanning membrane receptors with enriched expression in striatum. The existence of homodimers at the cell surface was demonstrated by time-resolved FRET. Although agonist activation of the receptor leads to the formation of receptor clusters, it did not affect the degree of A(2A)R-A(2A)R dimerization. Both monomers and dimers were detected by immunoblotting in cell extracts. However, cell surface biotinylation of proteins has made evident that more than 90% of the cell surface receptor is in its dimeric form. Thus, it seems that homodimers are the functional form of the receptor present on the plasma membrane. A deletion mutant version of the A(2A) receptor, lacking its C-terminal domain, was also able to form both monomeric and dimeric species when cell extracts from transfected cells were analyzed by immunoblotting. This suggests that the C-terminal tail does not participate in the dimerization. This is relevant as the C-terminal tail of A(2A)R is involved in heteromers formed by A(2A)R and dopamine D2 receptors. BRET ratios corresponding to A(2A)R-A(2A)R homodimers were higher than those encountered for heterodimers formed by A(2A)R and dopamine D2 receptors. As A(2A)R and dopamine D2 receptors do indeed interact, these results indicate that A(2A)R homodimers are the functional species at the cell surface and that they coexist with A(2A)R/D2 receptor heterodimers.