In-Cell Detection of Conformational Substates of a G Protein-Coupled Receptor Quaternary Structure: Modulation of Substate Probability by Cognate Ligand Binding
In-Cell Detection of Conformational Substates of a G Protein-Coupled Receptor Quaternary Structure: Modulation of Substate Probability by Cognate Ligand Binding
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G 蛋白偶联受体四级结构构象亚态的细胞内检测：通过同源配体结合调节亚态概率
DOI：
10.1021/acs.jpcb.0c06081
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Raicu, Valerică
中科院分区：
文献类型：
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作者：
Paprocki, Joel;Biener, Gabriel;Stoneman, Michael;Raicu, Valerică
近年来，虽然G蛋白偶联受体（GPCR）与同源和异质低聚体相关的概念得到了越来越多的认可，但研究人员对GPCR低聚化的功能相关性仍缺乏共识。一种技术，Förster共振能量转移（FRET）光谱法，允许通过分析能量的效率分布，从光激发荧光标签作为能量的供体转移到荧光标签作为能量的受体，并居住在相同的低聚物中，来确定活细胞中蛋白质的低聚物（或四元）结构。在本研究中，我们显著提高了FRET光谱的分辨率，以检测活细胞中GPCR低聚物的四元结构的细微差异。然后，我们用这种方法研究了无菌2 α-因子受体（Ste2）的低聚物的构象亚态，这是一种在酵母（saccharomyces cerevisiae）中的D类GPCR。在缺乏同源配体α-因子信息素的情况下，Ste2在相对较低的表达水平（11 - 140分子/μm2）下形成四聚体。显著改进的FRET光谱技术使我们能够检测到多种不同的Ste2低聚物的四元构象亚态，并评估α-因子配体如何改变这些亚态的比例。确定GPCR的四级结构底态的能力为阐明GPCR寡聚化的功能相关性提供了精细的手段。
While the notion that G protein-coupled receptors (GPCRs) associate into homo- and hetero-oligomers has gained more recognition in recent years, a lack of consensus remains among researchers regarding the functional relevance of GPCR oligomerization. A technique, Förster resonance energy transfer (FRET) spectrometry, allows for the determination of the oligomeric (or quaternary) structure of proteins in living cells via analysis of efficiency distributions of energy transferred from optically excited fluorescent tags acting as donors of energy to fluorescent tags acting as acceptors of energy and residing within the same oligomer. In this study, we significantly improved the resolution of FRET spectrometry to detect subtle differences in quaternary structures of GPCR oligomers within living cells. We then used this approach to study the conformational substates of oligomers of the sterile 2 α-factor receptor (Ste2), a class D GPCR found in the yeastSaccharomyces cerevisiaeof mating typea. Ste2 has previously been shown to form tetramers at relatively low expression levels (11 to 140 molecules/μm2) in the absence of its cognate ligand, the α-factor pheromone. The significantly improved FRET spectrometry technique allowed us to detect multiple distinct quaternary conformational substates of Ste2 oligomers, and to assess how the α-factor ligand altered the proportion of such substates. The ability to determine quaternary structure substates of GPCRs provides exquisite means to elucidate functional relevance of GPCR oligomerization.