Granier Sébastien, Kim Samuel, Fung Juan José, Bokoch Michael P, Parnot Charles
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA.
Methods Mol Biol. 2009;552:253-68. doi: 10.1007/978-1-60327-317-6_18.
The C-termini of G protein-coupled receptors (GPCRs) interact with specific kinases and arrestins in an agonist-dependent manner suggesting that conformational changes induced by ligand binding within the transmembrane domains are transmitted to the C-terminus. Förster resonance energy transfer (FRET) can be used to monitor changes in distance between two protein domains if each site can be specifically and efficiently labeled with a donor or acceptor fluorophore. In order to probe GPCR conformational changes, we have developed a FRET technique that uses site-specific donor and acceptor fluorophores introduced by two orthogonal labeling chemistries. Using this strategy, we examined ligand-induced changes in the distance between two labeled sites in the beta(2) adrenoceptor (beta(2)-AR), a well-characterized GPCR model system. The donor fluorophore, LumioGreen, is chelated by a CCPGCC motif [Fluorescein Arsenical Helix or Hairpin binder (FlAsH) site] introduced through mutagenesis. The acceptor fluorophore, Alexa Fluor 568, is attached to a single reactive cysteine (C265). FRET analyses revealed that the average distance between the intracellular end of transmembrane helix (TM) six and the C-terminus of the beta(2)-AR is 62 A. This relatively large distance suggests that the C-terminus is extended and unstructured. Nevertheless, ligand-specific conformational changes were observed (1). The results provide new insight into the structure of the beta(2)-AR C-terminus and ligand-induced conformational changes that may be relevant to arrestin interactions. The FRET labeling technique described herein can be applied to many GPCRs (and other membrane proteins) and is suitable for conformational studies of domains other than the C-terminus.
G蛋白偶联受体（GPCRs）的C末端以激动剂依赖的方式与特定激酶和抑制蛋白相互作用，这表明跨膜结构域内配体结合诱导的构象变化会传递到C末端。如果每个位点都能用供体或受体荧光团进行特异性和高效标记，那么福斯特共振能量转移（FRET）可用于监测两个蛋白质结构域之间距离的变化。为了探究GPCR的构象变化，我们开发了一种FRET技术，该技术使用通过两种正交标记化学方法引入的位点特异性供体和受体荧光团。使用这种策略，我们研究了β2肾上腺素能受体（β2-AR）（一种特征明确的GPCR模型系统）中两个标记位点之间距离的配体诱导变化。供体荧光团LumioGreen通过诱变引入的CCPGCC基序[荧光素砷螺旋或发夹结合剂（FlAsH）位点]螯合。受体荧光团Alexa Fluor 568连接到单个反应性半胱氨酸（C265）上。FRET分析表明，跨膜螺旋（TM）6的细胞内末端与β2-AR的C末端之间的平均距离为62埃。这个相对较大的距离表明C末端是伸展且无结构的。然而，观察到了配体特异性的构象变化（1）。这些结果为β2-AR C末端的结构以及可能与抑制蛋白相互作用相关的配体诱导构象变化提供了新的见解。本文所述的FRET标记技术可应用于许多GPCR（以及其他膜蛋白），适用于C末端以外结构域的构象研究。
