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

Background: Fluorescence resonance energy transfer (FRET) is a powerful tec hnique for measuring molecular interactions at Angstrom distances. We prese nt a new method for FRET that utilizes the unique spectral properties of va riants of the green fluorescent protein (GFP) for large-scale analysis by f low cytometry. Methods: The proteins of interest are fused in frame separately to the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP). FRET be tween these differentially tagged fusion proteins is analyzed using a dual- laser FACSVantage cytometer. Results: We show that homotypic interactions between individual receptor ch ains of tumor necrosis factor receptor (TNTFR) family members can be detect ed as FRET from CFP-tagged receptor chains to YFP-tagged receptor chains. N oncovalent molecular complexation can be detected as FRET between fusions o f CFP and YFP to either the intracellular or extracellular regions of the r eceptor chains. The specificity of the assay is demonstrated by the absence of FRET between heterologous receptor pairs that do not biochemically asso ciate with each other. Interaction between a TNFR-like receptor (Fas/CD95/A po-1) and a downstream cytoplasmic signaling component (FADD) can also be d emonstrated by flow cytometric FRET analysis. Conclusions: The utility of spectral variants of GFP in flow cytometric FRE T 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 a s proteins within the cells. Unlike biochemical methods, FRET allows the qu antitative determination of noncovalent molecular associations at Angstrom ngstrom level in living cells. Moreover, flow cytometry allows quantitative analyses to be carried out on a cell-by-cell basis on large number of cell s. Cytometry 44: 361-368, 2001. Published 2001 Witey-Liss, Inc.dagger