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 relatively recent 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 the FRET-spectrometry approach to detect small differences between the interprotomeric distances among GPCR oligomers with subtle differences in quaternary structures. We then used this approach to study the conformational substates of oligomers of sterile 2 α-factor receptor (Ste2), a class D GPCR found in the yeast Saccharomyces cerevisiae of mating type a. Ste2 has previously been shown to form tetrameric oligomers at relatively low expression levels (between 11 and 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.

中文翻译：

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GPCR四元结构构象亚态的细胞内检测：通过同源配体结合对亚态概率的调节

尽管近年来人们越来越认识到G蛋白偶联受体（GPCR）与同型和异型寡聚体相关的观点，但研究人员对GPCR寡聚化的功能相关性仍缺乏共识。相对较新的技术，福斯特共振能量转移（FRET）光谱法，可以通过分析从作为能量供体的光激发荧光标签转移的能量的效率分布来确定活细胞中蛋白质的低聚（或四级）结构。荧光标签充当能量的接受者并存在于同一低聚物中。在这个研究中，我们显着提高了FRET光谱方法的分辨率，以检测GPCR寡聚物之间的protopromeric距离之间的细微差别，并在四级结构上有细微差别。然后，我们使用这种方法研究了无菌2α因子受体（Ste2）（酵母中发现的D类GPCR）的寡聚体的构象亚状态酿酒酵母交配型的一个。STE2先前已经显示，以形成在相对低的表达水平的四聚体低聚物（11个140分子/微米之间²在不存在其同源配体，α因子信息素的）。显着改进的FRET光谱技术使我们能够检测Ste2低聚物的多个不同的季构象亚状态，并评估α-因子配体如何改变此类亚状态的比例。确定GPCR的四级结构亚状态的能力提供了阐明GPCR寡聚功能相关性的精妙方法。