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. 2011 Apr 29;286(17):15458-72.
doi: 10.1074/jbc.M110.191924. Epub 2011 Mar 1.

Hrs recognizes a hydrophobic amino acid cluster in cytokine receptors during ubiquitin-independent endosomal sorting

Yuji Amano  1 , Yuki Yamashita, Katsuhiko Kojima, Kazuhisa Yoshino, Nobuyuki Tanaka, Kazuo Sugamura, Toshikazu Takeshita
Affiliations

Hrs recognizes a hydrophobic amino acid cluster in cytokine receptors during ubiquitin-independent endosomal sorting

Yuji Amano et al. J Biol Chem. .

Abstract

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of the ESCRT-0 protein complex that captures ubiquitylated cargo proteins and sorts them to the lysosomal pathway. Although Hrs acts as a key transporter for ubiquitin-dependent endosomal sorting, we previously reported that Hrs is also involved in ubiquitin-independent endosomal sorting of interleukin-2 receptor β (IL-2Rβ). Here, we show direct interactions between bacterially expressed Hrs and interleukin-4 receptor α (IL-4Rα), indicating that their binding is not required for ubiquitylation of the receptors, similar to the case for IL-2Rβ. Examinations of the Hrs binding regions of the receptors reveal that a hydrophobic amino acid cluster in both IL-2Rβ and IL-4Rα is essential for the binding. Whereas the wild-type receptors are delivered to LAMP1-positive late endosomes, mutant receptors lacking the hydrophobic amino acid cluster are sorted to lysobisphosphatidic acid-positive late endosomes rather than LAMP1-positive late endosomes. We also show that the degradation of these mutant receptors is attenuated. Accordingly, Hrs functions during ubiquitin-independent endosomal sorting of the receptors by recognizing the hydrophobic amino acid cluster. These findings suggest the existence of a group of cargo proteins that have this hydrophobic amino acid cluster as a ubiquitin-independent sorting signal.

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Figures

FIGURE 1.
FIGURE 1.
The UIM domain of Hrs is dispensable for the interaction between Hrs and IL-4Rα. A, structures of wild-type Hrs and its mutants are shown. The Vps27-Hrs-STAM (VHS), Fab1-YGL023-Vps27-EEA1 (FYVE), ubiquitin interacting motif (UIM), proline (Pro)-rich region, coiled-coil, proline/glutamine (Pro/Gln)-rich region, and clathrin binding domain (CBD) are indicated. B, HEK293T cells (1 × 106) were cotransfected with 3 μg of wild-type FLAG-IL-2Rβ, wild-type FLAG-IL-4Rα, or empty vector and 3 μg of wild-type Hrs or its mutants. Aliquots (400 μg) of the cell lysates were immunoprecipitated with an anti-FLAG monoclonal antibody and immunoblotted with an anti-Hrs monoclonal antibody (top panel). The expression levels of Hrs and FLAG-tagged receptors in total lysates of the transfected HEK293T cells were examined by immunoblotting with an anti-Hrs monoclonal antibody and anti-FLAG monoclonal antibody, respectively. Total lysate, aliquots (10 μg) of the lysates were immunoblotted with an anti-Hrs antibody (middle panel) or anti-FLAG monoclonal antibody (lower panel). IP, immunoprecipitation; IB, immunoblotting.
FIGURE 2.
FIGURE 2.
A hydrophobic amino acid cluster in the cytoplasmic tail of IL-4Rα is required for Hrs binding. A, structures of wild-type IL-4Rα and its mutants are shown. The signal sequence, WSXWS (tryptophan, serine, any amino acid, tryptophan, serine) motif, transmembrane region (TM), box1 motif, and immunoreceptor tyrosine-based inhibitory motif (ITIM) are indicated. B, HEK293T cells (1 × 106) were cotransfected with 3 μg of FLAG-tagged wild-type IL-4Rα or its mutants and 3 μg of wild-type Hrs or empty vector. Aliquots (400 μg) of the cell lysates were immunoprecipitated with an anti-FLAG monoclonal antibody and immunoblotted with an anti-Hrs monoclonal antibody (top panel). The expression levels of Hrs and IL-4Rα were examined by immunoblotting with an anti-Hrs monoclonal antibody and anti-FLAG monoclonal antibody, respectively. Total lysate, aliquots (10 μg) of the lysates were immunoblotted with an anti-Hrs monoclonal antibody (middle panel) or anti-FLAG monoclonal antibody (lower panel). IP, immunoprecipitation; IB, immunoblotting. C, multiple alignment of the Hrs binding regions of human, bovine, swine, horse, rat, and mouse IL-4Rα is shown. The regions defined as acidic or hydrophobic clusters are boxed.
FIGURE 3.
FIGURE 3.
A hydrophobic amino acid cluster in the cytoplasmic tail of IL-2Rβ is required for Hrs binding. A, multiple alignment of the Hrs binding regions of human, chimpanzee, macaque, rat, and mouse IL-2Rβ is shown. The regions defined as acidic and hydrophobic clusters are boxed. B, shown are structures of wild-type IL-2Rβ and its mutants. The signal sequence, WSXWS motif, transmembrane region (TM), and Hrs binding region are indicated. C, HEK293T cells (1 × 106) were cotransfected with 3 μg of FLAG-tagged wild-type IL-2Rβ or its mutants and 3 μg of wild-type Hrs or empty vector. Aliquots (400 μg) of the cell lysates were immunoprecipitated with an anti-FLAG monoclonal antibody and immunoblotted with an anti-Hrs monoclonal antibody (top panel). The expression levels of Hrs and IL-2Rβ were examined by immunoblotting with an anti-Hrs monoclonal antibody and anti-FLAG monoclonal antibody, respectively. Total lysate, aliquots (10 μg) of the lysates were immunoblotted with an anti-Hrs monoclonal antibody (middle panel) or anti-FLAG monoclonal antibody (lower panel). IP, immunoprecipitation; IB, immunoblotting.
FIGURE 4.
FIGURE 4.
Hrs directly associates with the cytokine receptors IL-2Rβ and IL-4Rα by recognizing the hydrophobic amino acid cluster in a ubiquitin-independent manner. A, glutathione-Sepharose beads containing immobilized GST, GST-fused cytoplasmic tail fragment of IL-2Rβ (GST-IL-2RβcyWT or GST-IL-2RβcymH2), or GST-fused cytoplasmic tail fragment of IL-4Rα (GST-IL-4RαcyWT or GST-IL-4RαcymH) were incubated with His-tagged Hrs. The bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with an anti-His tag antibody. The input control of His-Hrs is shown in the right panel. B, structures of the IL-2Rβ and IL-4Rα mutants used in the receptor ubiquitylation assays are shown. C, HEK293T cells were cotransfected with 2 μg of HA-ubiquitin or empty vector and 2 μg of FLAG-IL-2Rβ, FLAG-IL-2Rβd268–348, FLAG-IL-2RβmH2, FLAG-IL-4Rα, or FLAG-IL-4RαmH. Aliquots (200 μg) of the cell lysates were immunoprecipitated with an anti-FLAG monoclonal antibody and immunoblotted with an anti-HA monoclonal antibody (top panel). The expression levels of the receptors (IL-2Rβ and IL-4Rα) and ubiquitylated total proteins were examined by immunoblotting with an anti-FLAG monoclonal antibody and anti-HA monoclonal antibody, respectively. Total lysate, aliquots (10 μg) of the lysates were immunoblotted with an anti-HA antibody (middle panel) or anti-FLAG antibody (lower panel). IP, immunoprecipitation; IB, immunoblotting.
FIGURE 5.
FIGURE 5.
Effects of the hydrophobic amino acid clusters in IL-2Rβ and IL-4Rα on their late-endosomal localizations. A, the IL-2Rβ and IL-4Rα expression levels on the surface of MEF transfectants were examined by flow cytometry. MEFβ, MEFβ-mH2, MEF-IL-4Rα, and MEF-IL-4Rα-mH cells were incubated with an anti-IL-2Rβ monoclonal antibody (TU11) or anti-IL-4Rα monoclonal antibody (MAB230) followed by a FITC-conjugated secondary antibody. B, the expression levels of IL-2Rβ and IL-4Rα in the indicated cells were examined by immunoblotting. Aliquots (15 μg) of the total lysates were immunoblotted with an anti-IL-2Rβ antibody (C20) or anti-IL-4Rα antibody (C20) (upper panels) or with an anti-β-actin antibody (lower panels). C and D, fluorescence images were observed using a confocal laser microscope. The indicated cells were grown on coverslips, fixed, and double-labeled with an anti-IL-2Rβ antibody (TU11) or anti-IL-4Rα antibody (MAB230) and an anti-LAMP1 monoclonal antibody or anti-Hrs antibody. Subsequently, the cells were incubated with fluorescently labeled secondary antibodies. Fairly large amounts of IL-2RβmH2 and IL-4RαmH are not sorted to LAMP1-positive compartments (arrows). Scale bars, 10 μm. IB, immunoblotting.
FIGURE 6.
FIGURE 6.
Kinetics of the endosomal localizations of IL-2Rβ and IL-4Rα. A and B, the indicated MEF transfectants were grown on coverslips, and the cell surface receptors were bound by an anti-IL-2Rβ antibody (TU11) or anti-IL-4Rα antibody (MAB230) at 0 °C followed by treatment with the chemical cross-linker DTSSP. The cells were cultured at 37 °C, fixed at the indicated times, and incubated with an anti-Hrs antibody or anti-LAMP1 monoclonal antibody. Fluorescence labeling was carried out for IL-2Rβ (red), IL-4Rα (red), Hrs (green), and LAMP1 (green). Large proportions of IL-2Rβ and IL-4Rα are delivered to LAMP1-positive compartments (arrowheads). In contrast, fairly large amounts of IL-2RβmH2 and IL-4RαmH are not delivered to LAMP1-positive compartments (arrows). Scale bars, 10 μm.
FIGURE 7.
FIGURE 7.
Internalization and degradation of IL-2Rβ and IL-4Rα in BAF-B03 transfectants. A and B, shown is internalization of IL-2Rβ and IL-4Rα in the transfectants. BAF transfectants were incubated with 125I-anti-IL-2Rβ antibody (TU11) or 125I-anti-IL-4Rα antibody (MAB230) at 0 °C. The cells were then cultured at 37 °C and harvested at the indicated times. The radioactivities of the cell surface-bound acid-removable fractions (a) and intracellular acid-unremovable fractions (b) were counted. C and D, degradation of IL-2Rβ and IL-4Rα in the transfectants is shown. BAF transfectants were incubated with 125I-anti-IL-2Rβ antibody or 125I-anti-IL-4Rα antibody at 0 °C followed by treatment with the chemical cross-linker DTSSP. The cells were then cultured at 37 °C and harvested at the indicated times. The radioactivities of the culture supernatants (a), cell precipitate fractions (b), and TCA-soluble fractions of the culture supernatants (c) were counted. The values represent the means ± S.E. of three separate experiments.
FIGURE 8.
FIGURE 8.
Internalization and degradation of 125I-IL-2 and 125I-IL-4 in BAF-B03 transfectants. A, internalization of 125I-IL-2 in the transfectants is shown. BAF transfectants were incubated with 350 pm 125I-IL-2 at 0 °C. The cells were then cultured at 37 °C and harvested at the indicated times. The radioactivities of the cell surface-bound acid-removable fractions (a) and intracellular acid-unremovable fractions (b) were counted. B, degradation of 125I-IL-2 in the transfectants is shown. BAF transfectants were incubated with 350 pm 125I-IL-2 and then cultured for the indicated times. The radioactivities of the culture supernatants (a), cell precipitate fractions (b), and TCA-soluble fractions of the culture supernatants (c) were counted. C, degradation of 125I-IL-4 in the transfectants is shown. BAF transfectants were incubated with 250 pm 125I-IL-4, treated with the cross-linker DTSSP, and then cultured for the indicated times. The radioactivities of the culture supernatants (a), cell precipitate fractions (b), and TCA-soluble fractions of the culture supernatants (c) were counted. The values represent the means ± S.E. of three separate experiments.
FIGURE 9.
FIGURE 9.
Localizations of IL-2Rβ and IL-4Rα mutants lacking the hydrophobic amino acid cluster to LBPA-positive compartments. MEF transfectants were grown on coverslips, fixed, and double-labeled with an anti-IL-2Rβ antibody (C20) or anti-IL-4Rα antibody (C20) and an anti-LAMP1 monoclonal antibody or anti-LBPA monoclonal antibody. Fluorescence images of the MEF transfectants (n ≥ 10 cells) were captured using a confocal laser microscope. The percentages of the IL-2Rβ-positive (A) and IL-4Rα-positive (B) pixel areas that were colocalized with LAMP1 (left) and LBPA (right) were analyzed. C, the IL-2Rβ-positive and IL-4Rα-positive pixel areas that were colocalized with LBPA (arrowheads) and not with LBPA (arrows) are indicated. Fluorescence labeling was carried out for IL-2Rβ (green), IL-4Rα (green), and LBPA (red). Scale bars, 10 μm.
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