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

Open Access
Publisher Website
Google Scholar
Abstract
Background Fluorescence resonance energy transfer (FRET) is a powerful technique for measuring molecular interactions at Ångstrom 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 Ångstrom 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. Cytometry 44:361–368, 2001. Published 2001 Wiley‐Liss, Inc.