The Ear of α-Adaptin Interacts with the COOH-terminal Domain of the Eps15 Protein

Abstract

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...

β3A-adaptin, a Subunit of the Adaptor-like Complex AP-3

“…Finally, the C region of ␤3A-adaptin might be analogous to the ear or appendage domains of ␤1-and ␤2-adaptins. The function of the ␤1-and ␤2-adaptin ear domains is still unclear, although the analogous domain of ␣-adaptin has been shown to bind regulatory molecules such as dynamin (44) and Eps15 (45). The domain organization of ␦-adaptin is also thought to resemble those of ␣-and ␥-adaptin.…”

Murine norovirus-1 cell entry is mediated through a non-clathrin-, non-caveolae-, dynamin- and cholesterol-dependent pathway

“…Eps15 is a molecular scaffold protein that associates with both the adaptin-2 (AP-2) adaptor protein complex (Benmerah et al, 1995(Benmerah et al, , 1996Iannolo et al, 1997) and epsin 1 (Chen et al, 1998), and is required for CME of transferrin. RAW264.7 cells were transfected to express a DN form of Eps15 that is known to inhibit CME (DNEps15 ED95/295; Benmerah et al, 1999), or a control form of Eps15 (DIIID2) that lacks one of the AP-2-binding sites and does not interfere with the clathrin pathway.…”

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

“…Epsin was first described as an Eps15 interacting protein . Eps15 is an AP-2 binding (Benmerah et al, 1996) protein with conserved N-terminal EH (Eps15 homology) domains through which Eps15 binds epsin . Eps15 also binds polyubiquitin and is suggested to act in partnership with epsin to sort polyubiquitinated cargo into clathrin-coated vesicles (Hawryluk et al, 2006).…”