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. 2001 May;12(5):1293-301.
doi: 10.1091/mbc.12.5.1293.

Involvement of the ubiquitin/proteasome system in sorting of the interleukin 2 receptor beta chain to late endocytic compartments

A Rocca  1 , C Lamaze, A Subtil, A Dautry-Varsat
Affiliations
Free PMC article

Involvement of the ubiquitin/proteasome system in sorting of the interleukin 2 receptor beta chain to late endocytic compartments

A Rocca et al. Mol Biol Cell. 2001 May.
Free PMC article

Abstract

Down-regulation of cell surface growth factor receptors plays a key role in the tight control of cellular responses. Recent reports suggest that the ubiquitin system, in addition to participating in degradation by the proteasome of cytosolic and nuclear proteins, might also be involved in the down-regulation of various membrane receptors. We have previously characterized a signal in the cytosolic part of the interleukin 2 receptor beta chain (IL2Rbeta) responsible for its targeting to late endosomes/lysosomes. In this report, the role of the ubiquitin/proteasome system on the intracellular fate of IL2Rbeta was investigated. Inactivation of the cellular ubiquitination machinery in ts20 cells, which express a thermolabile ubiquitin-activating enzyme E1, leads to a significant decrease in the degradation rate of IL2Rbeta, with little effect on its internalization. In addition, we show that a fraction of IL2Rbeta can be monoubiquitinated. Furthermore, mutation of the lysine residues of the cytosolic region of a chimeric receptor carrying the IL2Rbeta targeting signal resulted in a decreased degradation rate. When cells expressing IL2Rbeta were treated either by proteasome or lysosome inhibitors, a significant decrease in receptor degradation was observed. Our data show that ubiquitination is required for the sorting of IL2Rbeta toward degradation. They also indicate that impairment of proteasome function might more generally affect intracellular routing.

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Figures

Figure 1
Figure 1
Cell surface half-life (A) and endocytosis (B) of IL2Rβ on stably transfected ts20 mutant cells. Adherent ts20β cells were harvested using a 10-min incubation with PBS containing 5 mM EDTA at 33°C and, after one wash, preincubated for 1 h at permissive (30°C) or nonpermissive (42°C) temperature before and during the assays. (A) Cell surface expression of IL2Rβ on cells treated for different times with of 50 μM of cycloheximide was assayed by flow cytometry by using mAb 341 as described in MATERIALS AND METHODS. (B) For internalization assays, after 1-h preincubation at 30°C or 42°C, cells were labeled with the antibody at 4°C for 1 h, washed once at 4°C, and incubated for the indicated times at permissive or nonpermissive temperature. Disappearance of anti-IL2Rβ mAb from the cell surface upon internalization was assessed by cytofluorimetry and the percentage of internalized antibody was calculated. The y-axis is the ratio of the antibody internalized to the amount of antibody bound to the cell surface at each time point. The slope of this plot is the internalization rate constant (Wiley and Cunningham, 1982). The results presented in A and B are the means ± SE of at least three independent experiments.
Figure 2
Figure 2
Western blot analysis of lysates of IARC301.5 cells preincubated for 3 h with control medium (lanes a, c, and e), or 200 μM chloroquine (lanes b and d). Immunoprecipitation (IP) was performed using anti-IL2Rβ (lanes a and b), a rabbit polyclonal anti-ubiquitin antibody (lanes c and d) or an irrelevant antibody (lane e) and analyzed by immunoblotting (Blot) by using anti-IL2Rβ (341 mAb). The upper arrow indicates a ubiquitinated form of the IL2Rβ chain migrating at 78 kDa.
Figure 3
Figure 3
Effect of mutating the cytosolic lysine residues of the chimeric receptor αYβ18-27 on its cell surface half-life (A) and endocytosis (B). Experiments were performed as described in Figure 1, except that the incubations were performed at 37°C. The half-life values (A) or the percentage of internalized antibody (B) are shown. The results are the means ± SE of at least three independent experiments. The sequence of the cytosolic tail of the αYβ18-27 chimera is shown with its two lysine residues boxed.
Figure 4
Figure 4
Lysates of YT cells preincubated for 3 h with control medium (lanes a and c) or with 10 μM MG132 (lane b) were immunoprecipitated with an anti-IL2Rβ antibody (lanes a and b) or with a mouse monoclonal anti-ubiquitin antibody (lane c) and analyzed by immunoblotting with anti-IL2Rβ antibody. The upper arrow indicates a ubiquitinated form of the IL2Rβ chain migrating at 78 kDa.
Figure 5
Figure 5
Cell surface half-life (A) and internalization rate (B) of IL2Rβ on IARC301.5 cells treated with or without the proteasome inhibitor lactacystin. Cells were preincubated for 3 h at 37°C before and during the assays with 12.5 μM lactacystin or with control medium and the experiments were performed as described in Figure 1. The results presented in A and B are the means ± SE of at least three independent experiments.
Figure 6
Figure 6
Cell surface half-life (A) and endocytosis (B) of the chimeric receptor αYβ18-27 in cells preincubated with or without (control) proteasome inhibitor (lactacystin), chloroquine, or the lysosomal proteases inhibitor leupeptin. Cells were incubated with 25 μM lactacystin, 200 μM chloroquine, or 100 μM leupeptin for 3 h at 37°C and experiments performed as described in Figure 1. Cell surface expression of the chimera on cells treated for different times with 50 μM cycloheximide was assayed by flow cytometry (A) with mAb 2A3A1H. The kinetics of endocytosis was measured and results expressed as internalization rate constants (B). The results presented in A and B are the means ± SE of at least three independent experiments.
Figure 7
Figure 7
Intracellular localization of internalized receptors in K562 cells transfected with αYβ18-27 and incubated for 4 h at 37°C with or without 25 μM lactacystin or in cells expressing the ΔLys mutant. Cells were incubated for 30 min with 50 μM cycloheximide in the presence of 100 nM Tf-Cy5. After fixation and permeabilization, cells were first incubated with anti-chimera antibody (7G7B6) and with anti-Lamp-1 antibody (H4A3). The cells were then incubated with secondary labeled-antibody (anti-IgG2a-Texas Red and anti-IgG1-FITC) and analyzed by confocal microscopy. Merged images are shown where chimeric receptors are stained in green and the immunodetected intracellular markers (TfR or Lamp-1) in red. The yellow color indicates colocalization. (A) Control cells. (B) Cells expressing the ΔLys mutant. (C) Lactacystin-treated cells. Bar, 5 μM. (D) colocalization of the chimeric receptors with TfR or Lamp-1 was quantified as described in MATERIALS AND METHODS. (E) Colocalization of Lamp-1 with TfR in K562 αYβ18-27 cells incubated with or without lactacystin.-32767.
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