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.

Ligand-independent interaction of the type I interferon receptor complex is necessary to observe its biological activity

“…To corroborate this, there should be evidence that the Type I IFN receptor complex can form one of two different structures from cell to cell in a population. We present data supporting this prediction elsewhere [30]. …”

“…Thus we replotted the data from Figs. 2F and 4C (except for cells expressing nonfluorescent RACK-1), and included data in which IFN-αR1/OFP and IFN-αR2c/ChFP was coexpressed with Jak1/TFP and Tyk2/CiFP [30]. The calculated FRET efficiencies of the original data between IFN-αR1 and IFN-αR2c were converted to the predicted FRET efficiency between EGFP and OFP to make the datasets more comparable [11].…”

Improving the spectral analysis of fluorescence resonance energy transfer in live cells: Application to interferon receptors and Janus kinases

“…This result is curious because this interaction does not survive immunoprecipitation while the weaker Jak2:IFN-γR2 interaction survives immunoprecipitation. Perhaps the membrane, that is dissolved during immunoprecipitation, plays a physical role in maintaining the receptor structure; data supporting this hypothesis is presented elsewhere [22]. Jak1 also plays a role in the association of IFN-γR1 and IFN-γR2.…”

“…In cells where FRET was present, the optimal observed FRET efficiency of 0.42 implies an inter-FP distance of 62 Å. We attempt to understand why FRET was observed only in some cells elsewhere [22]. This population was statistically significant from the other two populations, ( p = {0.22, 4× 10 –11 }, and {0.11, 0.0003} between it and the IL10R2:Tyk2 and IFN-γR2:Jak2 populations, respectively).…”

“…2A and 4B), we hypothesize that IFN-γR1 decreased the FRET efficiency between Jak2/ECFP and IFN-γR2/EYFP only in those few cells in which the levels of IFN-γR1 approach those of Jak2/ECFP and IFN-γR2/EYFP, and that IFN-γR1 somehow alters the configuration of Jak2 or of IFN-γR2. Proof of this requires analysis of more than two proteins simultaneously [22]. …”