Abstract
The ubiquitous eps15 protein was initially described as a substrate of the EGF receptor kinase. Its functions are not yet delineated and this work provides evidence for its possible role in endocytosis. A novel anti-eps15 antibody, 6G4, coimmunoprecipitated proteins of molecular mass 102 kD. In human cells, these proteins were identified as the alpha- and beta-adaptins of the AP-2 complex on the basis of their NH2-terminal sequence and their immunoreactivity with anti-alpha- and anti-beta-adaptin antibodies but not with anti-gamma-adaptin antibody. In addition, the anti-eps15 antibody coimmunoprecipitated metabolically labeled polypeptides with molecular mass of 50 and 17 kD, comparable to those of the two other components of the AP-2 complex, mu2 and sigma 2. Constitutive association of eps15 with AP-2 was confirmed by two sets of experiments. First, eps15 was detected in immunoprecipitates of anti-alpha- and anti-beta-adaptin antibodies. Second, alpha- and beta- but not gamma-adaptins were precipitated by a glutathione-S-transferase eps15 fusion protein. The association of eps15 with AP-2 was ubiquitous and conserved between species, since it was observed in human lymphocytes and epithelial cells and in murine NIH3T3 fibroblasts. Our results are in keeping with a recent study showing homology between the NH2-terminal domains of eps15 and the product of the gene END3, involved in clathrin-mediated endocytosis of the pheromone alpha factor in Saccharomyces cerevisiae, and suggest a possible role for eps15 in clathrin-mediated endocytosis in mammals.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3T3 Cells / enzymology
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Adaptor Protein Complex 1*
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Adaptor Protein Complex 2*
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Adaptor Protein Complex 3*
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Adaptor Protein Complex alpha Subunits
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Adaptor Protein Complex beta Subunits
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Adaptor Protein Complex mu Subunits*
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Adaptor Proteins, Signal Transducing
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Adaptor Proteins, Vesicular Transport
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Animals
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Antibodies, Monoclonal
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Antibody Specificity
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Calcium-Binding Proteins / immunology
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Calcium-Binding Proteins / metabolism*
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Clathrin
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Glutathione Transferase / metabolism
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Humans
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Intracellular Signaling Peptides and Proteins
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Membrane Proteins / analysis
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Membrane Proteins / immunology
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Mice
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Molecular Weight
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Nerve Tissue Proteins / immunology
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Nerve Tissue Proteins / metabolism*
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Peptides / analysis
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Peptides / immunology
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Phosphoproteins / immunology
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Phosphoproteins / metabolism*
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Precipitin Tests
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Protein-Tyrosine Kinases / metabolism*
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Recombinant Fusion Proteins / metabolism
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Signal Transduction / physiology
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T-Lymphocytes / cytology
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T-Lymphocytes / enzymology*
Substances
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AP1M2 protein, human
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AP3M2 protein, human
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Adaptor Protein Complex 1
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Adaptor Protein Complex 2
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Adaptor Protein Complex 3
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Adaptor Protein Complex alpha Subunits
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Adaptor Protein Complex beta Subunits
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Adaptor Protein Complex mu Subunits
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Adaptor Proteins, Signal Transducing
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Adaptor Proteins, Vesicular Transport
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Antibodies, Monoclonal
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Ap1m2 protein, mouse
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Ap3m2 protein, mouse
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Calcium-Binding Proteins
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Clathrin
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EPS15 protein, human
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Eps15 protein, mouse
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Intracellular Signaling Peptides and Proteins
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Membrane Proteins
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Nerve Tissue Proteins
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Peptides
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Phosphoproteins
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Recombinant Fusion Proteins
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adaptor protein complex 1, mu 2 subunit
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adaptor protein complex 2, mu 2 subunit
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Glutathione Transferase
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Protein-Tyrosine Kinases