Time-resolved FRET between GPCR ligands reveals oligomers in native tissues.
Time-resolved FRET between GPCR ligands reveals oligomers in native tissues.
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DOI：
10.1038/nchembio.396
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发表时间：
2010-08
影响因子：
14.8
通讯作者：
Durroux T
中科院分区：
文献类型：
--
作者：
Albizu L;Cottet M;Kralikova M;Stoev S;Seyer R;Brabet I;Roux T;Bazin H;Bourrier E;Lamarque L;Breton C;Rives ML;Newman A;Javitch J;Trinquet E;Manning M;Pin JP;Mouillac B;Durroux T
G蛋白偶联受体（GPCR）寡聚体已被提出在细胞信号传导中发挥关键作用，但由于受体之间没有直接相互作用的报道，因此它们在天然环境中的存在的确认仍然难以捉摸。为了证明它们在天然组织中的存在，我们开发了一种时间分辨的FRET策略，该策略基于用选择性荧光配体标记受体。在细胞系中表达的四种不同受体中观察到特异性FRET信号，与这些转染细胞中的二聚体或寡聚体性质一致。更值得注意的是，用荧光激动剂和拮抗剂组测量的FRET信号之间的比较与活化的GPCR二聚体中两个原聚体的不对称关系一致。最后，我们将该策略应用于天然组织，并成功地证明了催产素受体二聚体和/或寡聚体在乳腺中的存在。
G protein–coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these transfected cells. More notably, the comparison between FRET signals measured with sets of fluorescent agonists and antagonists was consistent with an asymmetric relationship of the two protomers in an activated GPCR dimer. Finally, we applied the strategy to native tissues and succeeded in demonstrating the presence of oxytocin receptor dimers and/or oligomers in mammary gland.
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