利用绿色荧光蛋白的光谱变体对细胞表面受体相互作用和信号传导进行荧光共振能量转移分析。

Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein.

作者信息

Chan F K, Siegel R M, Zacharias D, Swofford R, Holmes K L, Tsien R Y, Lenardo M J

机构信息

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.

出版信息

Cytometry. 2001 Aug 1;44(4):361-8. doi: 10.1002/1097-0320(20010801)44:4<361::aid-cyto1128>3.0.co;2-3.

DOI:10.1002/1097-0320(20010801)44:4<361::aid-cyto1128>3.0.co;2-3

PMID:11500853

Abstract

BACKGROUND

Fluorescence resonance energy transfer (FRET) is a powerful technique for measuring molecular interactions at Angstrom distances. We present a new method for FRET that utilizes the unique spectral properties of variants of the green fluorescent protein (GFP) for large-scale analysis by flow cytometry.

METHODS

The proteins of interest are fused in frame separately to the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP). FRET between these differentially tagged fusion proteins is analyzed using a dual-laser FACSVantage cytometer.

RESULTS

We show that homotypic interactions between individual receptor chains of tumor necrosis factor receptor (TNFR) family members can be detected as FRET from CFP-tagged receptor chains to YFP-tagged receptor chains. Noncovalent molecular complexation can be detected as FRET between fusions of CFP and YFP to either the intracellular or extracellular regions of the receptor chains. The specificity of the assay is demonstrated by the absence of FRET between heterologous receptor pairs that do not biochemically associate with each other. Interaction between a TNFR-like receptor (Fas/CD95/Apo-1) and a downstream cytoplasmic signaling component (FADD) can also be demonstrated by flow cytometric FRET analysis.

CONCLUSIONS

The utility of spectral variants of GFP in flow cytometric FRET analysis of membrane receptors is demonstrated. This method of analyzing FRET allows probing of noncovalent molecular interactions that involve both the intracellular and extracellular regions of membrane proteins as well as proteins within the cells. Unlike biochemical methods, FRET allows the quantitative determination of noncovalent molecular associations at Angstrom level in living cells. Moreover, flow cytometry allows quantitative analyses to be carried out on a cell-by-cell basis on large number of cells. Published 2001 Wiley-Liss, Inc.

摘要

背景

荧光共振能量转移（FRET）是一种用于测量埃级距离分子间相互作用的强大技术。我们提出了一种新的FRET方法，该方法利用绿色荧光蛋白（GFP）变体的独特光谱特性，通过流式细胞术进行大规模分析。

方法

将感兴趣的蛋白质分别与青色荧光蛋白（CFP）或黄色荧光蛋白（YFP）进行读框融合。使用双激光FACSVantage流式细胞仪分析这些差异标记的融合蛋白之间的FRET。

结果

我们发现肿瘤坏死因子受体（TNFR）家族成员的单个受体链之间的同型相互作用可作为从CFP标记的受体链到YFP标记的受体链的FRET被检测到。非共价分子络合可作为CFP和YFP与受体链的细胞内或细胞外区域融合之间的FRET被检测到。通过不发生生化相互作用的异源受体对之间不存在FRET，证明了该检测方法具有特异性。通过流式细胞术FRET分析也可证明TNFR样受体（Fas/CD95/Apo-1）与下游细胞质信号成分（FADD）之间的相互作用。

结论

证明了GFP光谱变体在膜受体流式细胞术FRET分析中的实用性。这种分析FRET的方法能够探测涉及膜蛋白细胞内和细胞外区域以及细胞内蛋白质的非共价分子相互作用。与生化方法不同，FRET能够在埃级水平定量测定活细胞中的非共价分子缔合。此外，流式细胞术允许对大量细胞逐个进行定量分析。2001年由Wiley-Liss公司出版。