Differential requirements for AP-2 in clathrin-mediated endocytosis

Conner, S. D.; Schmid, Sandra L.

doi:10.1083/jcb.200304069

Cited by 179 publications

(170 citation statements)

References 30 publications

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“…Consistently, AP-2 was found not to be required for direct interaction with the EGFR for clathrin-dependent internalization of the EGFR (Huang et al, 1999;Nesterov et al, 1999). Furthermore, it was recently reported that the EGFR was efficiently endocytosed even in the absence of AP-2 upon RNA interference knock-down (Conner and Schmid, 2003;Motley et al, 2003). This could be consistent with the possibility that ubiquinated EGFR interacts directly with an endocytosis adaptor harboring a ubiquitin-interacting domain.…”

Section: Discussionsupporting

confidence: 72%

Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

Stang

Blystad

Kazazic

et al. 2004

MBoC

146

170

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Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitininteracting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.

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Section: Discussionsupporting

confidence: 72%

Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

Stang

Blystad

Kazazic

et al. 2004

MBoC

146

170

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“…We have recently shown that these adaptors can function even in the absence of any detectable AP-2. When we knock down AP-2 in HeLa cells using siRNAs, clathrin-mediated endocytosis of the transferrin receptor goes down to background levels, but clathrin-mediated endocytosis of the EGF receptor and of an LDL receptor chimera (as assayed by binding the ligand to the cell surface at 4°C and then warming the cells to 37°C) still proceed normally, even though there are 12-fold fewer clathrin-coated pits at the plasma membrane see also Conner and Schmid, 2003;Hinrichsen et al, 2003;Huang et al, 2004). Thus, it now appears that AP-2 is just one, albeit very abundant, adaptor for clathrin-mediated endocytosis.…”

Section: Introductionmentioning

confidence: 99%

EpsinR Is an Adaptor for the SNARE Protein Vti1b

Hirst

Miller

Taylor

et al. 2004

MBoC

124

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EpsinR is a clathrin-coated vesicle (CCV)-associated protein that binds to vti1b, suggesting that it may be a vti1b-selective adaptor. Depletion of epsinR to undetectable levels in HeLa cells using siRNA causes vti1b to redistribute from the perinuclear region to the cell periphery, but vti1a also redistributes in epsinR-depleted cells, and both vti isoforms redistribute in AP-1-depleted cells. As a more direct assay for epsinR function, we isolated CCVs from control and siRNA-treated cells and then looked for differences in cargo content. In clathrin-depleted cells, both coat and cargo proteins are greatly reduced in this preparation. Knocking down epsinR causes a ϳ50% reduction in the amount of AP-1 copurifying with CCVs and vice versa, indicating that the two proteins are dependent on each other for maximum incorporation into the coat. In addition, vti1b, but not vti1a, is reduced by >70% in CCVs from both epsinR-and AP-1-depleted cells. Because AP-1 knockdown reduces the amount of epsinR in CCVs, it is possible that its effect on vti1b may be indirect. These findings provide in vivo evidence that epsinR is an adaptor for vti1b, and they also show that CCV isolation can be used as an assay for adaptor function.

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“…However, increasing evidence has shown that not all cargo molecules are internalized through an interaction with AP-2 (3,4). Both LDL and EGF are among the known examples of AP-2-independent cargos involved in clathrin-mediated endocytosis (5,6). Various cargo-specific adaptor proteins have been proposed to function as alternative adaptors for clathrinmediated endocytosis, but the exact adaptor proteins used by most of the AP-2-independent cargos are still unknown (3,4).…”

mentioning

confidence: 99%

Epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus

Chen

Zhuang²

2008

Proc. Natl. Acad. Sci. U.S.A.

178

175

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During clathrin-mediated endocytosis, adaptor proteins recognize specific internalization signals on cargo receptors, either recruiting cargos into clathrin-coated pits (CCPs) or initiating clathrin-coat assembly around the cargo molecules. Here, we identify epsin 1, a clathrin-, ubiquitin-, and phospholipid-interacting protein, as a cargo-specific adaptor for influenza virus entry through the clathrin-mediated pathway. Using live-cell imaging to monitor the entry of individual virus particles, we observed recruitment of epsin 1 to the binding sites of influenza viruses in synchrony with the assembly of CCPs. Epsin 1 knockdown by siRNA significantly inhibited the clathrin-mediated endocytosis of the influenza virus and caused the majority of the virus particles to enter through a clathrin-independent pathway. The same treatment did not affect the entry of several classical ligands for clathrin-mediated endocytosis, including transferrin, LDL, and EGF. Overexpression of the dominant-negative epsin 1 mutant lacking the ubiquitin-interaction motifs nearly completely blocked the clathrin-mediated entry of the influenza virus without affecting transferrin uptake. These results suggest that epsin 1 functions as a cargo-specific adaptor for the clathrin-mediated entry of the influenza virus.

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Differential requirements for AP-2 in clathrin-mediated endocytosis

Cited by 179 publications

References 30 publications

Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

EpsinR Is an Adaptor for the SNARE Protein Vti1b

Epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus

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