Preassembly of interleukin 2 (IL-2) receptor subunits on resting Kit 225 K6 T cells and their modulation by IL-2, IL-7, and IL-15: A fluorescence resonance energy transfer study

Assembly and mutual proximities of alpha, beta, and gamma(c) subunits of the interleukin 2 receptors (IL-2R) in plasma membranes of Kit 225 K6 T lymphoma cells were investigated by fluorescence resonance energy transfer (FRET) using fluorescein isothiocyanate-and Cy3-conjugated monoclonal antibodies (mAbs) that were directed against the IL-2R alpha, IL-2R beta, and gamma(c) subunits of IL-2R. The cell-surface distribution of subunits was analyzed at the nanometer scale (2-10 nm) by FRET on a cell-by-cell basis, The cells were probed in resting phase and after coculture with saturating concentrations of IL-2, IL-7, and IL-15, FRET data from donor-and acceptor-labeled IL-2R beta-alpha, gamma-alpha, and gamma-beta pairs demonstrated close proximity of all subunits to each other in the plasma membrane of resting T cells, These mutual proximities do not appear to represent m4b-induced microaggregation, because FRET measurements with Fab fragments of the mAbs gave similar results, The relative proximities were meaningfully modulated by binding of IL-2, IL-7, and IL-15, Based on FRET analysis the topology of the three subunits at the surface of resting cells can be best described by a ''triangular model'' in the absence of added interleukins. IL-2 strengthens the bridges between the subunits, making the triangle more compact, IL-7 and IL-15 act in the opposite direction by opening the triangle possibly because they associate their private specific alpha receptors with the beta and/or gamma(c) subunits of the IL-2R complex, These data suggest that IL-2R subunits are already colocalized in resting T cells and do not require cytokine-induced redistribution, This colocalization is significantly modulated by binding of relevant interleukins in a cytokine-specific manner.