The Tyrosine Kinase Substrate eps15 Is Constitutively Associated with the Plasma Membrane Adaptor AP-2

• Published in: The Journal of cell biology Vol. 131; no. 6; pp. 1831 - 1838
• Main Authors: Benmerah, Alexandre, Gagnon, Jean, Bègue, Bernadette, Mégarbané, Bruno, Dautry-Varsat, Alice, Cerf-Bensussan, Nadine
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 01.12.1995; The Rockefeller University Press
• Subjects: 3T3 Cells - enzymology; Adaptor Protein Complex 1; Adaptor Protein Complex 2; Adaptor Protein Complex 3; Adaptor Protein Complex alpha Subunits; Adaptor Protein Complex beta Subunits; Adaptor Protein Complex mu Subunits; Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Amino acids; Animals; Antibodies; Antibodies, Monoclonal; Antibody Specificity; Antiserum; B lymphocytes; Calcium-Binding Proteins - immunology; Calcium-Binding Proteins - metabolism; Cell lines; Cells; Cellular Biology; Cellular immunity; Clathrin; Development Biology; Epithelial cells; Genes; Genetics; Glutathione Transferase - metabolism; Human health and pathology; Humans; Intracellular Signaling Peptides and Proteins; Life Sciences; Membrane Proteins - analysis; Membrane Proteins - immunology; Membranes; Mice; Molecular Weight; Nerve Tissue Proteins - immunology; Nerve Tissue Proteins - metabolism; Peptides - analysis; Peptides - immunology; Phosphoproteins - immunology; Phosphoproteins - metabolism; Precipitin Tests; Protein-Tyrosine Kinases - metabolism; Proteins; Receptors; Recombinant Fusion Proteins - metabolism; Signal Transduction - physiology; T lymphocytes; T-Lymphocytes - cytology; T-Lymphocytes - enzymology; Urology and Nephrology; Yeast
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.131.6.1831

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 α- and β-adaptins of the AP-2 complex on the basis of their NH2-terminal sequence and their immunoreactivity with anti-α- and anti-β-adaptin antibodies but not with anti-γ-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, μ2 and σ2. Constitutive association of eps15 with AP-2 was confirmed by two sets of experiments. First, eps15 was detected in immunoprecipitates of anti-α- and anti-β-adaptin antibodies. Second, α- and β- but not γ-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 α factor in Saccharomyces cerevisiae, and suggest a possible role for eps15 in clathrin-mediated endocytosis in mammals.