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We recently identified receptor-mediated endocytosis 8 (RME-8), a DnaJ domain protein localized to endosomes. We now demonstrate that RME-8 depletion leads to decreased levels of epidermal growth factor receptor (EGFR) without influencing receptors that primarily recycle to the plasma membrane. Decreases in EGFR are detected at both surface and intracellular pools and result from increased rates of EGFR degradation. Interestingly, RME-8 depletion also decreases EGFR levels in breast cancer cell lines in which overexpression of the EGFR family member ErbB2 has been shown to protect EGFR from degradation. These data implicate RME-8 in sorting decisions influencing EGFR at the level of endosomes and point to RME-8 as a potential regulatory target in ErbB2-positive breast cancers.

PDZ domains mediate protein interactions primarily through either classical recognition of carboxyl-terminal motifs or PDZ/PDZ domain associations. Several studies have also described internal modes of PDZ recognition, most of which depend on beta-finger structures. Here, we describe a novel interaction between the PDZ domain of nNOS and Vac14, the activator of the PtdIns(3)P 5-kinase PIKfyve. Binding assays using various Vac14 deletion constructs revealed a beta-finger independent interaction that is based on a novel internal motif. Mutational analyses reveal essential residues within the motif allowing us to define a new type of PDZ domain interaction.

Aftiphilin was identified through a database search for proteins containing binding motifs for the gamma-ear domain of clathrin adaptor protein 1 (AP-1). Here, we demonstrate that aftiphilin is expressed predominantly in brain where it is enriched on clathrin-coated vesicles. In addition to eight gamma-ear-binding motifs, aftiphilin contains two WXXF-acidic motifs that mediate binding to the alpha-ear of clathrin adaptor protein 2 (AP-2) and three FXXFXXF/L motifs that mediate binding to the alpha- and beta2-ear. We demonstrate that aftiphilin uses these motifs for interactions with AP-1 and AP-2 and that it immunoprecipitates these APs but not AP-3 or AP-4 from brain extracts. Aftiphilin demonstrates a brefeldin A sensitive localization to the trans-Golgi network in hippocampal neurons where it co-localizes with AP-1. Aftiphilin is also found at synapses where it co-localizes with synaptophysin and AP-2. Our data suggest a role for aftiphilin in clathrin-mediated trafficking in neurons.

Enthoprotin, a newly identified component of clathrin-coated vesicles, interacts with the trans-Golgi network (TGN) clathrin adapters AP-1 and GGA2. Here we perform a multi-faceted analysis of the site in enthoprotin that is responsible for the binding to the gamma-adaptin ear (gamma-ear) domain of AP-1. Alanine scan mutagenesis and nuclear magnetic resonance (NMR) studies reveal the full extent of the site as well as critical residues for this interaction. NMR studies of the gamma-ear in complex with a synthetic peptide from enthoprotin provide structural details of the binding site for TGN accessory proteins within the gamma-ear.

Amphiphysin I and II are nerve terminal-enriched proteins thought to function in synaptic vesicle endocytosis. In addition to a C-terminal SH3 domain, the proteins contain a highly conserved putative SH3 binding site and numerous consensus phosphorylation sites. We now demonstrate that amphiphysin I but not amphiphysin II is a phosphoprotein which undergoes dephosphorylation during nerve terminal depolarization. Further, both amphiphysin I and II interact with the SH3 domain of endophilin, a synaptically enriched protein implicated in synaptic vesicle endocytosis. The interaction is direct and mediated through a 43 amino acid region of amphiphysin containing the putative SH3 binding site. These data further support a role for amphiphysin I, II and endophilin in synaptic vesicle endocytosis.