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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.

Impaired IL10 signaling characterizes a subgroup of IBD patients, whereas the majority of children with severe IBD including EO forms normally produces and responds to IL10. Defective IL22 signaling may additionally impair intestinal epithelial clearance. Our data point out the complexity of IBD, which represent a group of distinct diseases with several pathogenetic abnormalities.

The role of Eps15 in clathrin-mediated endocytosis is supported by two observations. First, it interacts specifically and constitutively with the plasma membrane adaptor AP-2. Second, its NH 2 terminus shows significant homology to the NH 2 terminus of yeast End3p, necessary for endocytosis of ␣-factor. To gain further insight into the role of Eps15-AP-2 association, we have now delineated their sites of interactions. AP-2 binds to a domain of 72 amino acids (767-739) present in the COOH terminus of Eps15. This domain contains 4 of the 15 DPF repeats characteristic of the COOH-terminal domain of Eps15 and shares no homology with known proteins, including the related Eps15r protein. Precipitation of proteolytic fragments of AP-2 with Eps15-derived fusion proteins containing the binding site for AP-2 showed that Eps15 binds specifically to a 40-kDa fragment corresponding to the ear of ␣-adaptin, a result confirmed by precipitation of Eps15 by ␣-adaptin-derived fusion proteins. Our data indicate that this specific part of AP-2 binds to a cellular component and provide the tools for investigating the function(s) of the association between AP-2 and Eps15 .Eps15 is the prototype of a new family of signal transducers characterized by their ability to interact with a large number of proteins (1). It was initially described as a substrate of the epidermal growth factor (EGF) 1 and platelet-derived growth factor tyrosine kinase receptors endowed with transforming properties (2, 3). A novel protein, Eps15r, with 47% identity to Eps15 has recently been cloned using a probe derived from the region encoding the NH 2 -terminal domain of Eps15 (1). Both Eps15 and Eps15r are organized into three distinct structural domains. The amino terminus domain of Eps15 (amino acids 1-300) displays 70% identity to the amino terminus of Eps15r and is composed of three imperfect repeats of approximately 100 amino acids with candidate tyrosine phosphorylation sites and two EF-hand type calcium binding sites. Each repeat contains a domain of 70 amino acids, which is conserved not only in Eps15 and Eps15r, but also in several proteins in yeast and nematodes and is therefore designated EH for Eps15-Homology domain. The first domain of Eps15 interacts with several unidentified cytosolic proteins (1). The homology between the two proteins drops to 45% in the second domain, but the heptads required for coiled-coil structure are conserved. A possible function of these heptads in homo-or heterodimerization has been hypothesized (2, 3). Finally, there is little conservation between the COOH-terminal domains (amino acids 520 -896) of Eps15 and Eps15r, with two notable exceptions. First, multiple DPF motifs are present in both proteins. Second, the two proteins contain a proline-rich domain, PALPPK, which binds the Src homology 3-domain of the crk protooncogene (4).Besides its possible function in signal transduction, Eps15 may play a role in endocytosis. First, there is 62% homology between Eps15 EH domains and the NH 2 terminus of End3p, a protein requ...

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-epsl5 antibody, 6G4, coimmunoprecipitated proteins of molecular mass 102 kD. In human cells, these proteins were identified as the ~-and [3-adaptins of the AP-2 complex on the basis of their NH2-terminal sequence and their immunoreactivity with anti-a-and anti-13-adaptin antibodies but not with anti-~/-adaptin antibody. In addition, the anti-epsl5 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, ~2 and 0-2. Constitutive association of epsl5 with AP-2 was confirmed by two sets of experiments. First, eps15 was detected in immunoprecipitates of anti--a-and anti-13-adaptin antibodies. Second, et-and [3-but not ~/-adaptins were precipitated by a glutathione-S-transferase epsl5 fusion protein. The association of epsl5 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 epsl5 and the product of the gene END3, involved in clathfin-mediated endocytosis of the pheromone a factor in Saccharomyces cerevisiae, and suggest a possible role for epsl5 in clathrin-mediated endocytosis in mammals.

The capacity of non-pathogenic enteric bacteria to induce a pro-inflammatory response is under debate in terms of its effect on the symbiosis between the mammalian host and its commensal gut microflora.

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