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 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.
Chan
F K
FK
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.
Siegel
R M
RM
Zacharias
D
D
Swofford
R
R
Holmes
K L
KL
Tsien
R Y
RY
Lenardo
M J
MJ
D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis*; D004735:Energy Transfer; D053200:Fas-Associated Death Domain Protein; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis*; D011956:Receptors, Cell Surface / Q000032:analysis*; D018124:Receptors, Tumor Necrosis Factor / Q000032:analysis*; D015398:Signal Transduction / Q000502:physiology; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis / Q000379:methods
D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; D002352:Carrier Proteins; D053200:Fas-Associated Death Domain Protein; D008164:Luminescent Proteins; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins