Clathrin-independent endocytosis of ubiquitinated cargos - PubMed (original) (raw)

Clathrin-independent endocytosis of ubiquitinated cargos

Sara Sigismund et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Plasma membrane receptors can be endocytosed through clathrin-dependent and clathrin-independent pathways. Here, we show that the epidermal growth factor (EGF) receptor (EGFR), when stimulated with low doses of EGF, is internalized almost exclusively through the clathrin pathway, and it is not ubiquitinated. At higher concentrations of ligand, however, a substantial fraction of the receptor is endocytosed through a clathrin-independent, lipid raft-dependent route, as the receptor becomes ubiquitinated. An ubiquitination-impaired EGFR mutant was internalized through the clathrin pathway, whereas an EGFR/ubiquitin chimera, that can signal solely through its ubiquitin (Ub) moiety, was internalized exclusively by the non-clathrin pathway. Non-clathrin internalization of ubiquitinated EGFR depends on its interaction with proteins harboring the Ub-interacting motif, as shown through the ablation of three Ub-interacting motif-containing proteins, eps15, eps15R, and epsin. Thus, eps15s and epsin perform an important function in coupling ubiquitinated cargo to clathrin-independent internalization.

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Figures

Fig. 6.

Fig. 6.

Effects of multiple KDs on internalization. (A)(Top) Lysates from HeLa KD stable clones (+) for eps15/eps15R, epsin, or eps15/eps15R/epsin (triple KD) or from control clones obtained with mismatched oligos (–) were immunoblotted as indicated. (Middle) EGFR levels were comparable in all clones. (Bottom) The clathrin levels in the clathrin KDs. (B)(Upper) The indicated HeLa KD clones were transfected with a HA-EGFR/Ubmut chimera (see Supporting Materials and Methods) to circumvent the presence of endogenous EGFR. Internalization was assessed with an anti-HA antibody. Data are expressed as the percentage of cells in which internalization was inhibited. (Lower) A HeLa triple KD clone expressing HA-EGFR/Ubmut was transfected with the indicated eps15mut constructs (see Supporting Materials and Methods). Internalization assays and bar graph are as in Upper. Actual images of these experiments are in Fig. 11. (C) HeLa clones harboring the indicated KDs were incubated with 1.5 ng/ml 125I-EGF. The rate of internalization is expressed as internalized/surface-bound radioactivity. *, P = 0.0164; **, P = 0.003; linear regression analysis. (D) The indicated HeLa KD clones were treated with filipin (+) or mock treated (–), followed by incubation with 1.5 or 20 ng/ml 125I-EGF for 6 min at 37°C. The rate of internalization is expressed as internalized/surface-bound radioactivity.

Fig. 1.

Fig. 1.

Internalization of EGFR at different doses of EGF. (A) Immunoelectron microscopy of EGFRwt in HeLa cells in coated pits (CP), coated vesicles (CV), caveolae (CAV), or caveosome-like structures (CVS). (Bar, 111 nm.) (B) Lysates (1 mg) from HeLa cells were immunoprecipitated (IP) or directly loaded (50 μg) and immunoblotted (IB) with the indicated antibodies.

Fig. 2.

Fig. 2.

Internalization of EGFR/Ubmut. (A) NR6 cells expressing EGFRwt or EGFR/Ubmut were treated (15 min) with rhodamine-EGF (red) and stained for either clathrin or caveolin (green). (Left) Boxes enclose areas magnified in Right (magnification, ×4). Similar results were obtained when the internalization assay was performed with the 13A9 anti-EGFR antibodies in the presence or absence of EGF (data not shown). (B) Immunoelectron microscopy on NR6 expressing EGFR/Ubmut or EGFR/FLAG [a Ub-less control chimera (4)]. EGFR/Ubmut is exclusively detected in caveolae (20 cell profiles analyzed). (Bar, 83 nm.)

Fig. 4.

Fig. 4.

UIM proteins and EGFR/Ubmut. (A) φ-NX cells were transfected as indicated above the gels. Lysates (5 mg) were immunoprecipitated (IP) and immunoblotted (IB) as indicated. (B) Immunoelectron microscopy of NR6 expressing EGFR/Ubmut. (Bar, 83 nm.) Arrowheads point to caveolar membranes. Note that in cells expressing the EGFR/FLAG control chimera, eps15 and epsin were never detected in caveolae (data not shown). (C) CHO cells were cotransfected with EGFR/Ubmut and either GFP or GFP-UIM (amino acids 842–897 of eps15) and incubated with rhodamine-EGF (red, Left) or rhodamine-transferrin (red, Center). Merged images are shown. (Right) A quantitative assessment of EGF internalization (3 ng/ml 125I-EGF for 15 min at 37°C).

Fig. 3.

Fig. 3.

Ubiquitination and internalization. (A) Lysates (5 mg) from CHO cells expressing EGFRwt or Y1045F were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. (B) CHO cells were cotransfected with the indicated GFP-tagged and various EGFR-based constructs and incubated with rhodamine-EGF. The bar graphs represent the percentage of cells in which EGF internalization was inhibited (normalized to control GFP-transfected cells). (C) NR6 cells expressing the indicated constructs were treated with the indicated drugs. Internalization was monitored with the anti-EGFR 13A9 antibody in the presence of high EGF. (D) NR6 cells expressing the indicated constructs were treated with filipin and then incubated with 20 ng/ml 125I-EGF for 15 min at 37°C. The rate of internalization is expressed as internalized/surface-bound radioactivity. Actual images of the experiments described for B and C are in Fig. 8.

Fig. 5.

Fig. 5.

UIM proteins bind to ubiquitinated EGFR. (A) Lysates (5 mg) from HeLa cells were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. (B) CHO cells were transiently cotransfected with either EGFRwt or Y1045F and individually transfected with epsin, eps15, and hepatocyte growth factor receptor substrate (Hrs). Lysates (2 mg) were immunoprecipitated/immunoblotted as indicated. (C) HeLa cells were transfected with the indicated FLAG-tagged constructs encoding wild-type eps15 or an eps15-UIM-deleted mutant [eps15Δ859–897 (29)], or wild-type epsin, or an epsin-UIM-deleted mutant [epsinΔ190–192 (29)]. FLAG is an empty control vector. Lysates (2 mg) were immunoprecipitated/immunoblotted as indicated.

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