Eps15 Is a Component of Clathrin-coated Pits and Vesicles and Is Located at the Rim of Coated Pits

Tebar, Francesc; Sorkina, Tatiana; Sorkin, Alexander; Ericsson, Maria; Kirchhausen, Tomas

doi:10.1074/jbc.271.46.28727

Cited by 249 publications

(250 citation statements)

References 26 publications

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“…These results reveal that Eps15 associates only transiently with the clathrin baskets. Furthermore, these results are consistent with the finding that Eps15 is present at the rims of clathrincoated pits but does not associate with clathrin in mature coated vesicles (15).…”

Section: Eps15supporting

confidence: 82%

“…This is also consistent with immunoelectron microscopy localization of Eps15 to the rims of coated pits (15).…”

Section: Figsupporting

confidence: 76%

“…For example, Eps15 associates transiently with the growing edges of clathrin-coated pits, as identified by immunoelectron microscopy (15). Furthermore, clathrin-mediated endocytosis is inhibited in cells that overexpress Eps15 mutants lacking either the DPF-rich AP-2 binding domain (16) or the EH domains (17).…”

mentioning

confidence: 94%

“…Given that Eps15 is localized at the growing edges of clathrin-coated pits (15), it was hypothesized that Eps15 plays a role in the assembly of clathrin into coated pits and vesicles. We have evaluated this hypothesis by biochemically characterizing the effects of Eps15 on clathrin assembly.…”

mentioning

confidence: 99%

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Eps15 Homology Domain-NPF Motif Interactions Regulate Clathrin Coat Assembly during Synaptic Vesicle Recycling

Morgan

Prasad

Jin

et al. 2003

Journal of Biological Chemistry

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Although genetic and biochemical studies suggest a role for Eps15 homology domain containing proteins in clathrin-mediated endocytosis, the specific functions of these proteins have been elusive. Eps15 is found at the growing edges of clathrin-coated pits, leading to the hypothesis that it participates in the formation of coated vesicles. We have evaluated this hypothesis by examining the effect of Eps15 on clathrin assembly. We found that although Eps15 has no intrinsic ability to assemble clathrin, it potently stimulates the ability of the clathrin adaptor protein, AP180, to assemble clathrin at physiological pH. We have also defined the binding sites for Eps15 on squid AP180. These sites contain an NPF motif, and peptides derived from these binding sites inhibit the ability of Eps15 to stimulate clathrin assembly in vitro. Furthermore, when injected into squid giant presynaptic nerve terminals, these peptides inhibit the formation of clathrin-coated pits and coated vesicles during synaptic vesicle endocytosis. This is consistent with the hypothesis that Eps15 regulates clathrin coat assembly in vivo, and indicates that interactions between Eps15 homology domains and NPF motifs are involved in clathrin-coated vesicle formation during synaptic vesicle recycling.

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Section: Eps15supporting

confidence: 82%

“…This is also consistent with immunoelectron microscopy localization of Eps15 to the rims of coated pits (15).…”

Section: Figsupporting

confidence: 76%

mentioning

confidence: 94%

mentioning

confidence: 99%

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Eps15 Homology Domain-NPF Motif Interactions Regulate Clathrin Coat Assembly during Synaptic Vesicle Recycling

Morgan

Prasad

Jin

et al. 2003

Journal of Biological Chemistry

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“…The interaction involves a short segment located in the hinge region of the ␤ chain of AP-2 (5) and the terminal domain of clathrin. 1 Recently, it was suggested that Eps15 (6), a ϳ100-kDa molecule that forms complexes with AP-2 (7-9), is involved in the clathrin-mediated internalization pathway. Eps15-AP-2 complexes are found both in the cytosol and at the plasma membrane (1, 9, 10).…”

mentioning

confidence: 99%

Assembly of Clathrin Coats Disrupts the Association between Eps15 and AP-2 Adaptors

Cupers

Jadhav

Kirchhausen

1998

Journal of Biological Chemistry

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Eps15 is a phosphorylation substrate of the epidermal growth factor receptor kinase. In vivo, it is largely found in complex with AP-2, the plasma membrane clathrin adaptor protein complex. Although AP-2 is uniformly distributed across the surface of clathrin-coated pits and vesicles, Eps15 is preferentially found in the rims of endocytic clathrin-coated pits (1). This observation suggests that Eps15 may disengage from AP-2 during coat formation. Here we use two new anti-Eps15 antibodies to show that, contrary to our own earlier suggestion, coated vesicles isolated from brain do not contain detectable amounts of Eps15. Furthermore, when AP-2 complexes that are saturated with Eps15 are used for in vitro assembly of clathrin-AP-2 coats, normal structures are formed that contain the expected amounts of clathrin and AP-2, but the amount of Eps15 present is dramatically lower than that of AP-2. We propose that during coated pit formation, addition of clathrin to the growing edge at the rim of the pit releases Eps15 from AP-2.During the past decade, several groups have extensively studied the protein components and the mechanism responsible for the formation of clathrin-coated pits. Clathrin and its tetrameric adaptors AP-1 and AP-2 are among the best characterized structural elements of the coat (for recent reviews, see Refs. 2-4). The APs recruit clathrin, and they facilitate coat formation through its association with clathrin. The interaction involves a short segment located in the hinge region of the ␤ chain of AP-2 (5) and the terminal domain of clathrin. 1 Recently, it was suggested that Eps15 (6), a ϳ100-kDa molecule that forms complexes with AP-2 (7-9), is involved in the clathrin-mediated internalization pathway. Eps15-AP-2 complexes are found both in the cytosol and at the plasma membrane (1, 9, 10). In solution, the interaction between Eps15 and AP-2 involves a short segment toward the C terminus of Eps15 and the C-terminal ear domain of the ␣ chain of AP-2 (7, 8).Most of the Eps15 in the cell is bound to AP-2, whereas ϳ80% of AP-2 is free (1). The membrane-bound Eps15/AP-2 complexes are mainly found within clathrin-coated pits (1, 10), usually at the rims and growing edges of coated pits (1).The formation of coated pits at the plasma membrane requires AP-2 complexes and clathrin to be recruited to the membrane and a poorly understood nucleation event that initiates clathrin coat assembly. After nucleation, new clathrin and AP-2 complexes presumably add to the free edges of the pit, so that an AP-2 complex that is added to the pit early on will move progressively deeper into the pit. How is it, then, that Eps15 is concentrated at the edges of pits, regardless of the state of completion of the pit? We previously examined whether Eps15 is present in coated vesicles (1) and found a band of the approximately correct molecular weight in gels of purified bovine brain vesicle preparations. However, this band was not conclusively identified as Eps15, and in the absence of purified Eps15 quantitation was impossible...

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Dynamic regulation of cell adhesion molecules during axon outgrowth

Long

Lemmon

2000

J. Neurobiol.

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Eps15 Is a Component of Clathrin-coated Pits and Vesicles and Is Located at the Rim of Coated Pits

Cited by 249 publications

References 26 publications

Eps15 Homology Domain-NPF Motif Interactions Regulate Clathrin Coat Assembly during Synaptic Vesicle Recycling

Eps15 Homology Domain-NPF Motif Interactions Regulate Clathrin Coat Assembly during Synaptic Vesicle Recycling

Assembly of Clathrin Coats Disrupts the Association between Eps15 and AP-2 Adaptors

Dynamic regulation of cell adhesion molecules during axon outgrowth

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