Improved methodical approach for quantitative BRET analysis of G Protein Coupled Receptor dimerization.
Improved methodical approach for quantitative BRET analysis of G Protein Coupled Receptor dimerization.
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DOI：
10.1371/journal.pone.0109503
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Hunyady L
中科院分区：
文献类型：
--
作者：
Szalai B;Hoffmann P;Prokop S;Erdélyi L;Várnai P;Hunyady L
G蛋白偶联受体（GPCR）可以形成二聚体或更高顺序的低聚物，其过程会显著影响这些受体的生理和药理功能。定量生物发光共振能量转移（qBRET）测量是证明假定的GPCR二聚体的原聚物之间直接物理相互作用的金标准。为了正确解释这些实验，能量供体Renilla荧光素酶标记受体的表达必须保持恒定，这在表达系统中很难实现。为了分析非恒定供体表达对qBRET曲线的影响，我们进行了蒙特卡罗模拟。我们的研究结果表明，即使供体和受体标记受体之间的相互作用是非特异性的，供体表达的减少也会导致饱和的qBRET曲线，从而导致对二聚化状态的错误解释。我们在此提出了一种分析qBRET数据的新方法，将BRET比率绘制为不同供体受体表达水平下受体标记受体表达的函数。利用这种方法，当经典qBRET实验结果不明确时，我们能够区分二聚化和非特异性相互作用。利用雷帕霉素诱导的异源二聚化体系对模拟结果进行了实验验证。我们使用这种新方法研究了各种gpcr的二聚化，我们的数据证实了V2血管加压素和CaSR钙敏感受体的二聚化，而我们的数据反对这些受体与其他gpcr的异二聚化，包括I型和II型血管紧张素，β2肾上腺素能和CB1大麻素受体。
G Protein Coupled Receptors (GPCR) can form dimers or higher ordered oligomers, the process of which can remarkably influence the physiological and pharmacological function of these receptors. Quantitative Bioluminescence Resonance Energy Transfer (qBRET) measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V2 vasopressin and CaSR calcium sensing receptors, whereas our data argue against the heterodimerization of these receptors with other studied GPCRs, including type I and II angiotensin, β2 adrenergic and CB1 cannabinoid receptors.
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影响因子：
14.8
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
通讯作者：
Durroux T
影响因子：
46.9
作者：
Nagai, T;Ibata, K;Miyawaki, A
通讯作者：
Miyawaki, A
影响因子：
4.8
作者：
Jiang, YF;Minet, E;Bai, M
通讯作者：
Bai, M
影响因子：
4.8
作者：
Balla, Andras;Toth, Daniel J.;Hunyady, Laszlo
通讯作者：
Hunyady, Laszlo
DOI：
10.1073/pnas.0906695106
发表时间：
2010-02-02
影响因子：
11.1
作者：
Rivero-Muller, Adolfo;Chou, Yen-Yin;Huhtaniemi, Ilpo
通讯作者：
Huhtaniemi, Ilpo