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.

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.