Analytical use of multi-protein Fluorescence Resonance Energy Transfer to demonstrate membrane-facilitated interactions within cytokine receptor complexes.

Autor: Krause CD; 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., Izotova LS, Pestka S
Jazyk: angličtina
Zdroj: Cytokine [Cytokine] 2013 Oct; Vol. 64 (1), pp. 298-309. Date of Electronic Publication: 2013 Jun 13.
DOI: 10.1016/j.cyto.2013.05.008
Abstrakt: 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.
(Copyright © 2013 Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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