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First published online 29 April 2008
doi: 10.1242/jcs.024455

Journal of Cell Science 121, 1727-1738 (2008)
Published by The Company of Biologists 2008
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Ubiquitin-independent binding of Hrs mediates endosomal sorting of the interleukin-2 receptor β-chain

Yuki Yamashita1,*, Katsuhiko Kojima1,*, Tomonori Tsukahara1, Hideyuki Agawa1, Koichiro Yamada1, Yuji Amano1, Naoki Kurotori1, Nobuyuki Tanaka2, Kazuo Sugamura3 and Toshikazu Takeshita1,{ddagger}

1 Department of Microbiology and Immunology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan
2 Division of Immunology, Miyagi Cancer Center Research Institute, Natori, Miyagi, 981-1293, Japan
3 Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan


Figure 1
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  		Fig. 1. Hrs associates with IL-2Rβ lacking the UIM domain. (A) HEK293T cells were cotransfected with 2 µg wild-type Flag-IL-2Rβ, Flag-IL-2R{gamma} or the empty vector and 2 µg wild-type Hrs. Lysates of the HEK293T cells (2x106) were immunoprecipitated with an anti-Flag monoclonal antibody and immunoblotted with an anti-Hrs monoclonal antibody. Total lysate: aliquots (1.25%) of lysates from the indicated cells (2x106) were immunoblotted with an anti-Hrs antibody. (B) Lysates of HEK293T and HEK293Tβ4 cells (5x107) were immunoprecipitated with an anti-IL-2Rβ monoclonal antibody (TU11) and immunoblotted with an anti-Hrs antibody. Total lysate: aliquots (0.05%) of lysates from the indicated cells (5x107) were immunoblotted with an anti-Hrs antibody. (C) HEK293T cells were cotransfected with 2 µg wild-type Flag-IL-2Rβ and 2 µg wild-type Hrs, Hrsd257-277 (dUIM) mutant or the empty vector. Lysates of the HEK293T cells (2x106) were immunoprecipitated with TU11 and immunoblotted with an anti-Hrs antibody. The levels of IL-2Rβ and IL-2R{gamma}c in the precipitates were examined by immunoblotting with an anti-Flag antibody, whereas the levels of Hrs in the total lysates of transfected HEK293T cells were examined by immunoblotting with an anti-Hrs antibody. Total lysate: aliquots (1.25%) of lysates from the indicated cells (2x106) were immunoblotted with an anti-Hrs antibody. WT, wild-type; IP, immunoprecipitation; IB, immunoblotting.

 

Figure 2
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  		Fig. 2. The C-terminal region of Hrs is required for IL-2Rβ binding. (A,C) Structures of wild-type Hrs and its deletion (d) mutants. The Vps27-Hrs-STAM (VHS), Fab1-YGL023-Vps27-EEA1 (FYVE), coiled-coil and clathrin-binding domain (CBD) are shown at the top. The ubiquitin-interacting motif (UIM), PSAP sequence, proline (Pro)-rich region and proline or glutamine (Pro/Gln)-rich region are also indicated. (B,D) Lysates from HEK293T cells (2x106) cotransfected with 2 µg IL-2Rβ and 2 µg wild-type Hrs or Hrs mutants were immunoprecipitated with TU11 and immunoblotted with an anti-Hrs monoclonal antibody. The levels of IL-2β and Hrs were examined by immunoblotting with an anti-IL-2Rβ antibody (C-20) and anti-Hrs antibody, respectively, as controls. Total lysate: aliquots (1.25%) of lysates from the indicated cells (2x106) were immunoblotted with an anti-Hrs antibody. Asterisks indicate endogenous Hrs. WT, wild-type; IP, immunoprecipitation; IB, immunoblotting.

 

Figure 3
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  		Fig. 3. Hrs-binding region in the cytoplasmic tail of IL-2Rβ. (A) Structure of wild-type IL-2Rβ and its deletion (d) mutants. The signal sequence, Flag tag, WSxWS motif and transmembrane region (TM) are indicated. (B) HEK293T cells were cotransfected with 2 µg Hrs and 2 µg wild-type IL-2Rβ or its mutants. Lysates of HEK293T cells (2x106) were immunoprecipitated with TU11 and immunoblotted with an anti-Hrs monoclonal antibody. The levels of IL-2Rβ and Hrs were examined by immunoblotting with an anti-Flag monoclonal antibody and anti-Hrs antibody, respectively. Total lysate: aliquots (1.25%) of lysates from the indicated cells (2x106) were immunoblotted with an anti-Hrs antibody or anti-Flag antibody. WT, wild-type; IP, immunoprecipitation; IB, immunoblotting.

 

Figure 4
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  		Fig. 4. Hrs directly associates with IL-2Rβ in a ubiquitin-independent manner. (A) Glutathione-Sepharose beads containing immobilized GST or GST-Hrs were incubated with lysates of HEK293T cells (2x106) transfected with Flag-IL-2Rβ, Flag-IL-2Rβd268-348 or Flag-IL-2Rβd349-410. Bound proteins were separated by SDS-PAGE and immunoblotted with an anti-IL-2Rβ antibody (C-20). The level of wild-type IL-2Rβ and its mutants was examined by immunoblotting with an anti-IL-2Rβ antibody (C-20) using total lysates of transfected HEK293T cells. Expression of the GST fusion proteins was detected by immunoblotting with an anti-GST monoclonal antibody. (B) HEK293T cells were cotransfected with 2 µg HA-ubiquitin (HA-UB) or the empty vector and 2 µg Flag-IL-2Rβ, Flag-IL-2Rβd268-348 or Flag-IL-2Rβd349-410. Lysates of the HEK293T cells (2x106) were immunoprecipitated with TU11 and immunoblotted with an anti-HA antibody. The level of IL-2Rβ in the precipitates was examined by immunoblotting with an anti-Flag monoclonal antibody. (C) Glutathione-Sepharose beads containing immobilized GST or GST-Hrs were incubated with the His-tagged cytoplasmic tail fragment of IL-2Rβ (IL-2Rβ269-551His). The associated proteins were analyzed by immunoblotting with an anti-IL-2Rβ antibody (C-20). The input (2%) of IL-2Rβ269-551His was examined by immunoblotting with an anti-IL-2Rβ antibody (C-20). The levels of the GST fusion proteins were detected by western blotting with an anti-GST antibody. Total lysate: aliquots (1.25%) of lysates from the indicated cells (2x106) immunoblotted with an anti-Hrs antibody or anti-IL-2Rβ antibody. WT, wild-type; IP, immunoprecipitation; IB, immunoblotting.

 

Figure 5
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  		Fig. 5. Constitutive internalization of IL-2Rβ. (A) HEK293Tβ4 cells grown on coverslips were fixed and then incubated with the following combinations of antibodies: anti-EEA1 monoclonal antibody and anti-IL-2Rβ antibody (C20); anti-LAMP1 monoclonal antibody and anti-IL-2Rβ antibody (C20); and anti-Hrs monoclonal antibody and anti-IL-2Rβ antibody (C20). Subsequently, the cells were incubated with fluorescently labeled secondary antibodies. Scale bars: 5 µm. (B) Internalization of IL-2Rβ in HEK293Tβ4 cells. The radioactivity of cell-surface-bound acid-removable fractions (a) and intracellular acid-unremovable fractions (b) was counted. 125I-TU11 binding to parental HEK293T cells was 7.4% of that of HEK293Tβ4 cells. The values represent the mean ± s.e.m. of triplicate determinations. Cells were incubated with (IL-2+) or without (IL-2–) IL-2. (C) Kinetics of the endosomal localization of IL-2Rβ. Cells grown on coverslips were incubated with TU11 at 0°C, followed by treatment with a chemical crosslinker. The cells were incubated at 37°C, fixed at the indicated times and incubated with an anti-Hrs monoclonal antibody. Scale bars: 5 µm.

 

Figure 6
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  		Fig. 6. Internalization and degradation of IL-2Rβ in BAF-B03 and its transfectants. (A) IL-2Rβ expression on the surface of the pro-B cell line clones F7, S25, BAFβd349-410-3 and BAFβd349-410-4 was examined by flow cytometry. Cells were incubated with an anti-IL-2Rβ monoclonal antibody (TU11), followed by a FITC-conjugated secondary antibody. (B) Aliquots (1.25%) of total lysates from the indicated BAF-B03 clones (2x106 cells) were immunoblotted with an anti-IL-2Rβ antibody (C-20) or anti-β-actin antibody. IB, immunoblotting. (C) Internalization of IL-2Rβ in the transfectants. The radioactivity of cell surface-bound acid-removable fractions (a) and intracellular acid-unremovable fractions (b) was counted. (D) Degradation of IL-2Rβ in the transfectants. The radioactivity of culture supernatants (a), cell precipitate fractions (b) and TCA-soluble fractions of culture supernatants (c) was counted. 125I-TU11 binding to parental BAF-B03 cells was 3.3% of that of F7 cells. The values represent the mean ± s.e.m. of triplicate determinations. Cells were incubated with (IL-2+) or without (IL-2–) IL-2.

 

Figure 7
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  		Fig. 7. Effect of the Hrs-binding region in IL-2Rβ on late-endosomal localization. (A) IL-2Rβ expression on the surface of MEFβ, MEFβd268-348, MEFβd349-410-2 and HRSdβ cells was examined by flow cytometry as described for Fig. 6A. (B) Aliquots (1.25%) of total lysates from the indicated MEF clones (2x106 cells) were immunoblotted with an anti-IL-2Rβ antibody (C-20) or anti-β-actin antibody. IB, immunoblotting. (C) The indicated cells were grown on coverslips, fixed and double-labeled with an anti-IL-2Rβ antibody (C-20) and an anti-LAMP1 monoclonal antibody or anti-Hrs monoclonal antibody. HRSdβ cells were transfected with GFP-SARA-FYVE, an early endosome marker, and then fixed and labeled with an anti-IL-2Rβ antibody (C20). Red, green and yellow areas indicate IL-2Rβ staining, Hrs or GFP-SARA-FYVE staining and colocalization of the red and green staining, respectively. Scale bars: 5 µm.

 

Figure 8
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  		Fig. 8. Kinetics of the subcellular distribution of IL-2Rβ. (A) Kinetics of the IL-2Rβ subcellular localization. The indicated cells were grown on coverslips and treated as described in Fig. 5C. Fluorescence labeling was carried out for IL-2Rβ (red) and LAMP1 (green) in a or IL-2Rβ (green) and Hrs (red) in b. A large part of IL-2Rβd349-410 is not sorted to LAMP1-positive compartments (arrows) whereas some IL-2Rβd349-410 is delivered to LAMP1-positive compartments (arrowheads) at 120 minutes in MEFβd349-410 cells. Scale bars: 5 µm. (B) Degradation of IL-2Rβ in the transfectants. The radioactivity of culture supernatants (a), cell precipitate fractions (b) and TCA-soluble fractions of culture supernatants (c) was counted. 125I-TU11 binding to parental MEF cells was 4.7% of that of MEFβ cells. The values represent the mean ± s.e.m. of triplicate determinations.

 

Figure 9
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  		Fig. 9. Kinetics of IL-2Rβ sorting to transferrin receptor-positive compartments. Cells grown on coverslips were incubated with TU11 and an anti-transferrin receptor antibody at 0°C, followed by treatment with a chemical crosslinker. The cells were then incubated at 37°C, fixed at the indicated times and incubated with fluorescently labeled secondary antibodies. Red, green and yellow areas indicate IL-2Rβ staining, transferrin receptor staining and colocalization of the red and green staining, respectively. Scale bars: 5 µm.

 

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