Although interactions between the μ2 subunit of the clathrin adaptor protein complex AP-2 and tyrosinebased internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of μ2-receptor interactions, we constructed an epitope-tagged μ2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of μ2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant μ2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous μ2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a μ2-dependent and -independent manner.

A chimera of the epidermal growth factor receptor (EGFR) and green fluorescent protein (GFP) has been engineered by fusing GFP to the carboxyl terminus of EGFR. Data are provided to demonstrate that the GFP moiety does not affect the expected functioning of EGFR. EGFR-GFP becomes phosphorylated at tyrosine residues in response to EGF and is capable of phosphorylating endogenous substrates and initiating signaling cascades. EGF-dependent association of the chimeric receptor with the clathrin adaptor protein AP-2, involved in endocytosis, and with Shc adaptor protein, which binds in close proximity to the fusion point, is not affected by the GFP moiety. Receptor down-regulation and internalization occur at rates similar to those in cells expressing wild-type EGFR. Western blot analysis reveals that lysosomal degradation of EGFR-GFP proceeds from the extracellular domain and that GFP is not preferentially cleaved. Time-dependent co-localization of EGFR-GFP and Texas Red-conjugated EGF in living cells using digital deconvolution microscopy demonstrates the trafficking of ligand-receptor complexes through the early and multivesicular endosomes followed by segregation of the ligand and receptor at the late stages of endocytosis. Time-lapse optical analysis of the early stages of endocytosis reveals localization of EGFR-GFP in the tubular-vesicular endosomal compartments. Rapid dynamics of membrane movement and fusion within these compartments were observed. This approach and the fidelity of the biochemical properties of the EGFR-GFP demonstrate that real-time visualization of trafficking and protein interactions of tyrosine kinase receptors in the presence or absence of the ligand are feasible.

Eps15 homology (EH) domains are eukaryotic signaling modules that recognize proteins containing Asn-Pro-Phe (NPF) sequences. The structure of the central EH domain of Eps15 has been solved by heteronuclear magnetic resonance spectroscopy. The fold consists of a pair of EF hand motifs, the second of which binds tightly to calcium. The NPF peptide is bound in a hydrophobic pocket between two alpha helices, and binding is mediated by a critical aromatic interaction as revealed by structure-based mutagenesis. The fold is predicted to be highly conserved among 30 identified EH domains and provides a structural basis for defining EH-mediated events in protein trafficking and growth factor signaling.

Eps15 is a member of an emerging family of proteins containing a novel protein/protein interaction domain, the EH domain, of as yet unknown function. Recent findings of Eps15 association with clathrin adaptor complex AP-2 and its localization in clathrin-coated pits have implicated Eps15 in the regulation of vesicle trafficking. Here we show that Eps15 exists in several multimeric states in vivo. When purified recombinant Eps15 or lysates of NIH 3T3 cells were treated with cross-linking reagents, covalent dimers of Eps15 and larger covalent multimers were detected in high yield. Large Eps15 oligomers co-immunoprecipitated with AP-2 at an efficiency higher than that of Eps15 dimers. Furthermore, cross-linking of the membrane-bound fraction of Eps15 in mildly permeabilized cells was as efficient as that of the cytosolic fraction. Size-exclusion column chromatography of recombinantly produced Eps15 and of total cell lysates was performed to examine the equilibrium ratio of the monomers versus the aggregated forms of Eps15. These experiments showed that essentially all the Eps15 was aggregated, whereas monomers of Eps15 could be obtained only under strong denaturing conditions. To map the region of Eps15 responsible for dimerization, fusion proteins corresponding to the three structural domains of Eps15 were prepared. Cross-linking analysis revealed that the central portion of Eps15, which possesses a coiled-coil region (residues 321-520), serves as the interacting interface. The possibility that hetero-oligomeric complexes of Eps15 dimers and AP-2 function during the recruitment of proteins into coated pits is discussed.The Eps15 protein was originally discovered as a phosphorylation substrate of the epidermal growth factor receptor kinase (1). Subsequent studies revealed that Eps15 is constitutively associated with the plasma membrane clathrin adaptor protein complex AP-2 (2). Immunomorphological analysis demonstrated that membrane-bound Eps15 is mainly associated with the plasma membrane clathrin-coated pits and vesicles (3). Moreover, Eps15 is distributed asymmetrically within the coat, mostly at the rim of the pits and at the neck of the budding vesicles. All these findings suggested that Eps15 might play a role in clathrin-dependent endocytosis.Further evidence suggesting a role for Eps15 in protein trafficking comes from the analysis of the primary structure of Eps15 and its homology to other proteins. The predicted amino acid sequence of Eps15 identifies at least three structural domains (1, 4). The amino-terminal region is composed of three relatively conserved repeats of ϳ70 amino acids referred to as EH domains (for Eps15 homology). The central domain of Eps15 presents the characteristic heptad repeats of coiled-coil proteins. The carboxyl-terminal domain displays several AspPro-Phe (DPF) 1 repeats, proline-rich sequences capable of interacting with SH3 domains (5), and the binding site for the ␣-subunit of AP-2 (6, 7). Importantly, an EH domain is found in End3p, a protein required for endocytosis of ␣-f...