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An endosomally localized isoform of Eps15 interacts with Hrs to mediate degradation of epidermal growth factor receptor.

Roxrud I, Raiborg C, Pedersen NM, Stang E, Stenmark H - J. Cell Biol. (2008)

Bottom Line: Eps15b overexpression, similarly to Hrs overexpression, inhibits ligand-mediated degradation of EGFR, whereas Eps15 is without effect.Similarly, depletion of Eps15b but not Eps15 delays degradation and promotes recycling of EGFR.These results indicate that Eps15b is an endosomally localized isoform of Eps15 that is present in the Hrs complex via direct Hrs interaction and important for the sorting function of this complex.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Biomedicine, University of Oslo and the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

ABSTRACT
Down-regulation of activated and ubiquitinated growth factor (GF) receptors by endocytosis and subsequent lysosomal degradation ensures attenuation of GF signaling. The ubiquitin-binding adaptor protein Eps15 (epidermal growth factor receptor [EGFR] pathway substrate 15) functions in endocytosis of such receptors. Here, we identify an Eps15 isoform, Eps15b, and demonstrate its expression in human cells and conservation across vertebrate species. Although both Eps15 and Eps15b interact with the endosomal sorting protein Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in vitro, we find that Hrs specifically binds Eps15b in vivo (whereas adaptor protein 2 preferentially interacts with Eps15). Although Eps15 mainly localizes to clathrin-coated pits at the plasma membrane, Eps15b localizes to Hrs-positive microdomains on endosomes. Eps15b overexpression, similarly to Hrs overexpression, inhibits ligand-mediated degradation of EGFR, whereas Eps15 is without effect. Similarly, depletion of Eps15b but not Eps15 delays degradation and promotes recycling of EGFR. These results indicate that Eps15b is an endosomally localized isoform of Eps15 that is present in the Hrs complex via direct Hrs interaction and important for the sorting function of this complex.

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An Eps15-related protein is present in bilayered coats on endosomes. (A) HEp-2 cells incubated with EGF on ice and chased for 15 min at 37°C were prepared for immunoelectron microscopy and labeled using an antibody recognizing the C terminus of Eps15. (B) HeLa cells incubated with EGF on ice and chased for 15 min at 37°C were prepared for immunoelectron microscopy and labeled for Eps15 (15 nm gold) followed by labeling for Hrs (10 nm gold, arrows). The labeling for Eps15 localized to bilayered coats on endosome-like compartments as well as to the rim of coated pits at the plasma membrane (inset in A). The two micrographs shown in A are from the same cell. Coats are indicated by arrowheads. Bars, 100 nm.
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fig1: An Eps15-related protein is present in bilayered coats on endosomes. (A) HEp-2 cells incubated with EGF on ice and chased for 15 min at 37°C were prepared for immunoelectron microscopy and labeled using an antibody recognizing the C terminus of Eps15. (B) HeLa cells incubated with EGF on ice and chased for 15 min at 37°C were prepared for immunoelectron microscopy and labeled for Eps15 (15 nm gold) followed by labeling for Hrs (10 nm gold, arrows). The labeling for Eps15 localized to bilayered coats on endosome-like compartments as well as to the rim of coated pits at the plasma membrane (inset in A). The two micrographs shown in A are from the same cell. Coats are indicated by arrowheads. Bars, 100 nm.

Mentions: Previous studies have shown that Hrs resides in a characteristic “bilayered” clathrin coat on early endosomes, where it is involved in retention and lysosomal sorting of ubiquitinated membrane proteins (Raiborg et al., 2002; Sachse et al., 2002). Clathrin appears to retain Hrs molecules in restricted microdomains, thereby increasing their local concentration beyond a critical value required for cargo sorting through low-affinity interactions (Raiborg et al., 2006). Confocal microscopy has shown that an anti-Eps15 immunoreactive protein colocalizes with Hrs on endosomes (Bache et al., 2003) but the resolution of the light microscope is insufficient to establish whether Eps15 is present in the bilayered clathrin coat. To address this issue, we studied HEp-2 and HeLa cells by immunoelectron microscopy using an antibody against the C-terminal part of Eps15 followed by protein A conjugated to colloidal gold. The endosomal labeling with anti-Eps15 was low and we rarely detected more than one immunogold particle per endosome. However, the labeling was highly specific, and we consistently detected anti-Eps15 labeling within the bilayered coat of endosomes (Fig. 1 A). Double labeling with anti-Eps15 and anti-Hrs showed that the bilayered Eps15-positive coat contained Hrs as well (Fig. 1 B). As reported previously (Raiborg et al., 2002, Sachse et al., 2002), this coat was morphologically distinct from clathrin-coated pits at the plasma membrane, in which we could also detect Eps15 labeling (Fig. 1 A, inset). These results indicate that an Eps15-related protein is found in the bilayered clathrin coats on endosomes.


An endosomally localized isoform of Eps15 interacts with Hrs to mediate degradation of epidermal growth factor receptor.

Roxrud I, Raiborg C, Pedersen NM, Stang E, Stenmark H - J. Cell Biol. (2008)

An Eps15-related protein is present in bilayered coats on endosomes. (A) HEp-2 cells incubated with EGF on ice and chased for 15 min at 37°C were prepared for immunoelectron microscopy and labeled using an antibody recognizing the C terminus of Eps15. (B) HeLa cells incubated with EGF on ice and chased for 15 min at 37°C were prepared for immunoelectron microscopy and labeled for Eps15 (15 nm gold) followed by labeling for Hrs (10 nm gold, arrows). The labeling for Eps15 localized to bilayered coats on endosome-like compartments as well as to the rim of coated pits at the plasma membrane (inset in A). The two micrographs shown in A are from the same cell. Coats are indicated by arrowheads. Bars, 100 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2373575&req=5

fig1: An Eps15-related protein is present in bilayered coats on endosomes. (A) HEp-2 cells incubated with EGF on ice and chased for 15 min at 37°C were prepared for immunoelectron microscopy and labeled using an antibody recognizing the C terminus of Eps15. (B) HeLa cells incubated with EGF on ice and chased for 15 min at 37°C were prepared for immunoelectron microscopy and labeled for Eps15 (15 nm gold) followed by labeling for Hrs (10 nm gold, arrows). The labeling for Eps15 localized to bilayered coats on endosome-like compartments as well as to the rim of coated pits at the plasma membrane (inset in A). The two micrographs shown in A are from the same cell. Coats are indicated by arrowheads. Bars, 100 nm.
Mentions: Previous studies have shown that Hrs resides in a characteristic “bilayered” clathrin coat on early endosomes, where it is involved in retention and lysosomal sorting of ubiquitinated membrane proteins (Raiborg et al., 2002; Sachse et al., 2002). Clathrin appears to retain Hrs molecules in restricted microdomains, thereby increasing their local concentration beyond a critical value required for cargo sorting through low-affinity interactions (Raiborg et al., 2006). Confocal microscopy has shown that an anti-Eps15 immunoreactive protein colocalizes with Hrs on endosomes (Bache et al., 2003) but the resolution of the light microscope is insufficient to establish whether Eps15 is present in the bilayered clathrin coat. To address this issue, we studied HEp-2 and HeLa cells by immunoelectron microscopy using an antibody against the C-terminal part of Eps15 followed by protein A conjugated to colloidal gold. The endosomal labeling with anti-Eps15 was low and we rarely detected more than one immunogold particle per endosome. However, the labeling was highly specific, and we consistently detected anti-Eps15 labeling within the bilayered coat of endosomes (Fig. 1 A). Double labeling with anti-Eps15 and anti-Hrs showed that the bilayered Eps15-positive coat contained Hrs as well (Fig. 1 B). As reported previously (Raiborg et al., 2002, Sachse et al., 2002), this coat was morphologically distinct from clathrin-coated pits at the plasma membrane, in which we could also detect Eps15 labeling (Fig. 1 A, inset). These results indicate that an Eps15-related protein is found in the bilayered clathrin coats on endosomes.

Bottom Line: Eps15b overexpression, similarly to Hrs overexpression, inhibits ligand-mediated degradation of EGFR, whereas Eps15 is without effect.Similarly, depletion of Eps15b but not Eps15 delays degradation and promotes recycling of EGFR.These results indicate that Eps15b is an endosomally localized isoform of Eps15 that is present in the Hrs complex via direct Hrs interaction and important for the sorting function of this complex.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Biomedicine, University of Oslo and the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

ABSTRACT
Down-regulation of activated and ubiquitinated growth factor (GF) receptors by endocytosis and subsequent lysosomal degradation ensures attenuation of GF signaling. The ubiquitin-binding adaptor protein Eps15 (epidermal growth factor receptor [EGFR] pathway substrate 15) functions in endocytosis of such receptors. Here, we identify an Eps15 isoform, Eps15b, and demonstrate its expression in human cells and conservation across vertebrate species. Although both Eps15 and Eps15b interact with the endosomal sorting protein Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in vitro, we find that Hrs specifically binds Eps15b in vivo (whereas adaptor protein 2 preferentially interacts with Eps15). Although Eps15 mainly localizes to clathrin-coated pits at the plasma membrane, Eps15b localizes to Hrs-positive microdomains on endosomes. Eps15b overexpression, similarly to Hrs overexpression, inhibits ligand-mediated degradation of EGFR, whereas Eps15 is without effect. Similarly, depletion of Eps15b but not Eps15 delays degradation and promotes recycling of EGFR. These results indicate that Eps15b is an endosomally localized isoform of Eps15 that is present in the Hrs complex via direct Hrs interaction and important for the sorting function of this complex.

Show MeSH
Related in: MedlinePlus