Detergent-free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles
Detergent-free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles
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DOI：
10.1021/acs.biochem.5b01040
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发表时间：
2016-01-12
期刊：
影响因子：
2.9
通讯作者：
Baneres, Jean-Louis
中科院分区：
文献类型：
--
作者：
Logez, Christel;Damian, Marjorie;Baneres, Jean-Louis
G蛋白偶联受体（GPCRs）是一种在信号转导中起关键作用的膜蛋白。了解它们的动态是绝对必要的，以获得一个清晰的图片信号如何进行。然而，在洗涤剂中分离的GPCR的分子表征在这些化合物对受体功能和稳定性的有害作用上绊倒。我们在这里探索了苯乙烯-马来酸聚合物直接从脂质环境中溶解受体的潜力。为此，我们使用了两个GPCR，褪黑激素和生长激素释放肽受体，嵌入两个膜系统的复杂性增加，脂质体和膜巴斯德毕赤酵母。在这两种情况下，苯乙烯-马来酸聚合物都能够提取尺寸明确的膜贴片。SMA稳定的脂质盘中的GPCR不仅识别它们的配体，而且还传递信号，如它们以激动剂依赖性方式激活其同源G蛋白和募集抑制蛋白的能力所证明的。此外，纯化的受体在脂盘经历所有特定的变化与配体介导的激活相关的构象，表现在荧光构象报告和化合物从不同的药理学类的生长激素释放肽受体的情况下。总之，这些数据突出了苯乙烯-马来酸稳定的脂质盘在良好控制的膜样环境中用于分析GPCR介导的信号传导的分子基础的潜力。
G protein-coupled receptors (GPCRs) are integral membrane proteins that play a pivotal role in signal transduction. Understanding their dynamics is absolutely required to get a clear picture of how signaling proceeds. Molecular characterization of GPCRs isolated in detergents nevertheless stumbles over the deleterious effect of these compounds on receptor function and stability. We explored here the potential of a styrene-maleic acid polymer to solubilize receptors directly from their lipid environment. To this end, we used two GPCRs, the melatonin and ghrelin receptors, embedded in two membrane systems of increasing complexity, liposomes and membranes from Pichia pastoris. The styrene-maleic acid polymer was able, in both cases, to extract membrane patches of a well-defined size. GPCRs in SMA-stabilized lipid discs not only recognized their ligand but also transmitted a signal, as evidenced by their ability to activate their cognate G proteins and recruit arrestins in an agonist-dependent manner. Besides, the purified receptor in lipid discs undergoes all specific changes in conformation associated with ligand-mediated activation, as demonstrated in the case of the ghrelin receptor with fluorescent conformational reporters and compounds from distinct pharmacological classes. Altogether, these data highlight the potential of styrene-maleic stabilized lipid discs for analyzing the molecular bases of GPCR-mediated signaling in a well-controlled membrane-like environment.