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 - PubMed (original) (raw)

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

S Damjanovich et al. Proc Natl Acad Sci U S A. 1997.

Abstract

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 mAb-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.

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Figures

Figure 1

Figure 1

Representative flow cytometric histograms of energy transfer efficiencies measured between FITC (F)- and Cy3-conjugated mAbs bound to IL-2R α, β, and γ subunits on Kit 225 K6 T cells. Averages and error estimates (E ± Δ_E_) of mean values of flow cytometric energy transfer histograms for the indicated donor–acceptor pairs were calculated from data of three to five independent measurements: (A) 1.5 ± 0.1% for F-anti-Tac + Cy3-anti-Tac, 18.2 ± 3.5% for F-anti-Tac + Cy3–7G7; (B) 25.4 ± 5.9% for F-Mikβ3 + Cy3–7G7; (C) 20.6 ± 5.1% for F-TUGh4 + Cy3-anti-Tac; and (D) 12.4 ± 5.0% for F-TUGh4 + Cy3-Mikβ1. Displacement from a mean value of 0 indicates energy transfer and nonrandom proximity of the epitopes.

Figure 2

Figure 2

Effect of IL-2, IL-7, and IL-15 on the accessibility of epitopes to FITC-conjugated mAbs 7G7/B6 (against IL-2Rα), Mikβ3 (against IL-2Rβ), and TUGh4 (against IL-2Rγ) added as single agents on Kit 225 K6 wild-type T cells. Interleukins were added to cell cultures 6 hr before harvesting at the following concentrations: IL-2 (solid bars), 20 units/ml; IL-7 (shaded bars) and IL-15 (open bars), both 1 ng/ml (20 pM). The mean values of fluorescence histograms collected on cells treated with the interleukins were expressed as percent change from control values. Bars represent mean ± SEM of four to six independent measurements.

Figure 3

Figure 3

Effect of IL-2, IL-7, and IL-15 on energy transfer efficiencies measured between FITC (F)- and Cy3-conjugated mAbs specific to the IL-2R α, β, and γ subunits on Kit 225 K6 T cells. The cells were treated with interleukins as described in the legend to Fig. 2 by IL-2 (solid bars), IL-7 (shaded bars), and IL-15 (open bars). The mean values of the fluorescence energy transfer histograms collected from cytokine-treated cells were expressed as percent change from the control values. Bars represent mean ± SEM of four to six independent measurements. Greek letters with arrows indicate the subunits labeled with the mAbs and the direction of energy transfer. The subscripts 1 and 2 on the letter α designate the two epitopes recognized by mAbs anti-Tac and 7G7, respectively, on the same IL-2Rα subunit.

Figure 4

Figure 4

Schematic representation of lateral organization of the subunits of the IL-2 receptor complex on resting Kit 225 K6 T cells and the complex’s modulation by addition of IL-2, IL-7, and IL-15. FRET data suggested that IL-2R α, β, and γ subunits are preassembled, forming heterotrimers on the surface of resting cells (Middle). The proximity between the β and γ subunits was not altered significantly with any of the interleukins. Whereas IL-2 promoted a stronger contact of the α subunit with the β and γ chains (Top), IL-7 loosened it (Bottom Left). IL-15 induced a closer proximity of β and α subunits, whereas the contact between the γ and α subunits became weaker, thereby leading to a somewhat linearized configuration of the IL-2R complex (Bottom Right).

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