Real-time analysis of agonist-induced activation of protease-activated receptor 1/Gαi1 protein complex measured by bioluminescence resonance energy transfer in living cells
Real-time analysis of agonist-induced activation of protease-activated receptor 1/Gαi1 protein complex measured by bioluminescence resonance energy transfer in living cells
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DOI：
10.1124/mol.106.030304
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发表时间：
2007-05-01
影响因子：
3.6
通讯作者：
Pin, Jean-Philippe
中科院分区：
文献类型：
--
作者：
Ayoub, Mohammed A.;Maurel, Damien;Pin, Jean-Philippe
G蛋白偶联受体通过激活异源三聚体G蛋白将细胞外信号传递到细胞中，这一过程通常伴随着受体脱敏。在真实的时间和活细胞中监测这样的过程将有助于更好地理解G蛋白激活是如何发生的。基于能量转移的方法[荧光共振能量转移（FRET）和生物发光共振能量转移（BRET）]最近被证明是监测活细胞中G蛋白偶联受体（GPCR）-G蛋白缔合的有力方法。在这里，我们使用BRET技术来监测蛋白酶激活受体1（PAR 1）和G α（i1）蛋白之间的偶联。可以在未活化的PAR 1和以生理水平表达的G α（i1）蛋白之间测量特异性组成型BRET信号。该信号对百日咳毒素（PTX）不敏感，可能反映了这两种蛋白质的预组装。BRET信号在受体活化后以PTX敏感的方式快速增加。BRET信号在几分钟后恢复到基础水平。BRET信号的脱敏伴随着β-抑制蛋白-1向受体的募集，与已知的PAR快速脱敏一致。激动剂诱导的BRET增加依赖于蛋白质中荧光团的插入位点。总而言之，我们的结果表明GPCR和G α蛋白之间的BRET可用于真实的实时监测活细胞中的受体激活。我们的数据还显示，PAR 1可以是一个预组装的复合物与G α（i1）蛋白，从这些合作伙伴之间的直接相互作用或从他们的共定位在特定的微结构域，受体激活可能导致在这种复合物内的重排。
G protein-coupled receptors transmit extracellular signals into the cells by activating heterotrimeric G proteins, a process that is often followed by receptor desensitization. Monitoring such a process in real time and in living cells will help better understand how G protein activation occurs. Energy transfer-based approaches [ fluorescence resonance energy transfer ( FRET) and bioluminescence resonance energy transfer (BRET)] were recently shown to be powerful methods to monitor the G protein-coupled receptors (GPCRs)-G protein association in living cells. Here, we used a BRET technique to monitor the coupling between the protease-activated receptor 1 (PAR1) and G alpha(i1) protein. A specific constitutive BRET signal can be measured between nonactivated PAR1 and the G alpha(i1) protein expressed at a physiological level. This signal is insensitive to pertussis toxin (PTX) and probably reflects the preassembly of these two proteins. The BRET signal rapidly increases upon receptor activation in a PTX-sensitive manner. The BRET signal then returns to the basal level after few minutes. The desensitization of the BRET signal is concomitant with beta-arrestin-1 recruitment to the receptor, consistent with the known rapid desensitization of PARs. The agonist-induced BRET increase was dependent on the insertion site of fluorophores in proteins. Taken together, our results show that BRET between GPCRs and G alpha proteins can be used to monitor the receptor activation in real time and in living cells. Our data also revealed that PAR1 can be part of a preassembled complex with G alpha(i1) protein, resulting either from a direct interaction between these partners or from their colocalization in specific microdomains, and that receptor activation probably results in rearrangements within such complexes.