title: Analytical use of multi-protein Fluorescence Resonance Energy Transfer to demonstrate membrane-facilitated interactions within cytokine receptor complexes.
abstract: Experiments measuring Fluorescence Resonance Energy Transfer (FRET) between cytokine receptor chains and their associated proteins led to hypotheses describing their organization in intact cells. These interactions occur within a larger protein complex or within a given nano-environment. To illustrate this complexity empirically, we developed a protocol to analyze FRET among more than two fluorescent proteins (multi-FRET). In multi-FRET, we model FRET among more than two fluorophores as the sum of all possible pairwise interactions within the complex. We validated our assumption by demonstrating that FRET among pairs within a fluorescent triplet resembled FRET between each pair measured in the absence of the third fluorophore. FRET between two receptor chains increases with increasing FRET between the ligand-binding chain (e.g., IFN-γR1, IL-10R1 and IFN-λR1) and an acylated fluorescent protein that preferentially resides within subsections of the plasma membrane. The interaction of IL-10R2 with IFN-λR1 or IL-10R1 results in decreased FRET between IL-10R2 and the acylated fluorescent protein. Finally, we analyzed FRET among four fluorescent proteins to demonstrate that as FRET between IFN-γR1 and IFN-γR2 or between IFN-αR1 and IFN-αR2c increases, FRET among other pairs of proteins changes within each complex.
authors.affiliation: Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School - The University of Medicine and Dentistry of New Jersey, 675 Hoes Lane West, Piscataway, NJ 08855, USA. Electronic address: christopher.d.krause@gmail.com.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Krause
authors.forename: Lara S
authors.initials: LS
authors.lastname: Izotova
authors.forename: Sidney
authors.initials: S
authors.lastname: Pestka
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D057809:HEK293 Cells; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes / Q000032:analysis; D011485:Protein Binding; D053633:Receptor, Interferon alpha-beta / Q000378:metabolism*; D017471:Receptors, Interferon / Q000378:metabolism*; D053701:Receptors, Interleukin-10 / Q000378:metabolism*; D000096743:Interferon gamma Receptor
chemical_list: D005456:Fluorescent Dyes; D007371:Interferon-gamma; D046912:Multiprotein Complexes; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon; D053701:Receptors, Interleukin-10; D000096743:Interferon gamma Receptor; C507964:IFNAR1 protein, human; C508033:IFNAR2 protein, human
keywords: CFS; ChFP; CiFP; Confocal fluorescence spectroscopy; EGFP; EYFP; FP; FRET; IFN; IFN-α; IFN-γ; IFN-λ; Interferon receptor; Lipid raft; Multi-protein; OFP; ORF; PEI; StFP; TFP; ToFP; confocal fluorescence spectroscopy; enhanced green fluorescent protein; enhanced yellow fluorescent protein; fluorescence resonance energy transfer; fluorescent protein; interferon; interferon-α; interferon-γ; interferon-λ; mCherry/cherry fluorescent protein; mCitrine/monomeric citrine fluorescent protein; mOrange/orange fluorescent protein; mStrawberry/strawberry fluorescent protein; mTeal/teal fluorescent protein; open reading frame; polyethyleneimine; tdTomato/tomato fluorescent protein