AP-2/Eps15 interaction is required for receptor-mediated endocytosis.
We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2-binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2-binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2-binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane-coated pits.
Benmerah
A
A
Developpement Normal et Pathologique du Systeme Immunitaire, Institut National de la Sante et de la Recherche Medicale U 429, Hopital Necker-Enfants Malades, 75743 Paris Cedex 15, France.
Lamaze
C
C
Bègue
B
B
Schmid
S L
SL
Dautry-Varsat
A
A
Cerf-Bensussan
N
N
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D066246:ErbB Receptors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002135:Calcium-Binding Proteins; C486206:EPS15 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D066246:ErbB Receptors