Ligand binding causes the EGF receptor (EGFR) to become ubiquitinated by Cbl upon association with the adaptor protein Grb2. We have investigated the role of ubiquitin and Grb2 in ligand-induced endocytosis of the EGFR. Incubation of cells with EGF on ice caused translocation of Grb2 and Cbl from the cytosol to the rim of coated pits. Grb2 with point mutations in both SH3 domains inhibited recruitment of the EGFR to clathrin-coated pits, in a Ras-independent manner. On overexpression of the Cbl-binding protein Sprouty, ubiquitination of the EGFR was inhibited, the EGFR was recruited only to the rim of coated pits, and endocytosis of the EGFR was inhibited. Conjugation-defective ubiquitin similarly inhibited recruitment of EGF-EGFR to clathrin-coated pits. Even though this does not prove that cargo must be ubiquitinated, this indicates the importance of interaction of ubiquitinated protein(s) with proteins harboring ubiquitininteracting domains. We propose that Grb2 mediates transient anchoring of the EGFR to an Eps15-containing molecular complex at the rim of coated pits and that Cbl-induced ubiquitination of the EGFR allows relocation of EGFR from the rim to the center of clathrin-coated pits.

†These authors contributed equally to this work.Epsin consists of an epsin NH 2 -terminal homology domain that promotes interaction with phospholipids, several AP-2-binding sites, two clathrin-binding sequences and several Eps15 homology domain-binding motifs. Epsin additionally possesses ubiquitin-interacting motifs (UIMs) and has been demonstrated to bind ubiquitinated cargo. We therefore investigated whether epsin promoted clathrin-mediated endocytosis of the ubiquitinated EGF receptor (EGFR). By immunoprecipitation, we found that epsin 1 interacted with ubiquitinated EGFR and that functional UIMs were essential for complex formation. Furthermore, RNA interference-mediated knockdown of epsin 1 was found to inhibit internalization of the EGFR, while having no effect on endocytosis of the transferrin receptor. Additionally, upon knockdown of epsin 1, translocation of the EGFR to central parts of clathrin-coated pits was inhibited. This supports the contention that epsin 1 promotes endocytosis of the ubiquitinated EGFR.

*Corresponding author: Espen Stang, Espen.Stang@medisin.uio.no † These authors contributed equally to this work Caveolae-dependent endocytosis has recently been proposed in the uptake of EGF receptor (EGFR) at high concentrations of ligand. Consistently, upon incubation of HEp2 and HeLa cells with methyl-b-cyclodextrin, we observed a small inhibitory effect on endocytosis of ligated EGFR in HEp2 cells. However, immunoelectron microscopy showed the same relative amount of bound EGF localizing to caveolae on incubation with high and low concentrations of EGF, not supporting rapid recruitment of EGFR to caveolae. Live-cell microscopy furthermore demonstrated that incubating HEp2 cells with high concentrations of EGF did not increase the mobility of caveolae. By RNA-interference-mediated knockdown of clathrin heavy chain in HEp2 and HeLa cells, we found that endocytosis of EGFR was efficiently inhibited both at high and low concentrations of EGF. Our results show that caveolae are not involved in endocytosis of EGFbound EGFR to any significant degree and that high concentrations of EGF do not further mobilize caveolae.

The significance of charged residues for the target-cell binding, potency and specificity of pediocin-like bacteriocins has been studied by site-directed mutagenesis of sakacin P. Most of the charged residues are located in the N-terminal half, which is thought to mediate the initial binding of these bacteriocins to target cells through electrostatic interaction. All the mutated peptides in which the net positive charge was reduced by one (by replacing a charged residue with threonine) exhibited reduced binding to target cells and a 2-15-fold reduction in potency. The least deleterious of these mutations was the removal of the positive charge in position 8 (H8T). This mutation was, in fact, less deleterious than the conservative His to Lys mutation, indicating that the positive charge in position 8 per se is not of major importance. Somewhat more deleterious was the removal of positive charges at the N-and C-terminal ends (K1T, K43T). Most deleterious was the elimination of the positive charge at positions 11 and (but to a lesser extent) 12, demonstrating the importance of the cationic patch in the middle of the N-terminal half of pediocin-like bacteriocins. Mutated peptides in which the net positive charge was increased by one were also constructed. Some of these exhibited increased cell binding and a potency that was the same as (44K, i.e. an extra positive charge at the Cterminus), or somewhat greater (T20K) than, that of sakacin P, whereas others (0K, i.e. an extra positive charge at the N-terminus) had reduced potency. Sakacin P contains only one negatively charged residue (Asp17). This negative charge and its orientation in space were crucial for activity, since the Asp to Asn mutation and (especially) the conservative Asp to Glu mutation were deleterious. Mutations that made the peptide less cationic had, overall, less effect on the potency toward the Carnobacterium piscicola strain than on the potency toward the three other strains tested, whereas the opposite was the case for mutations that made the peptide more cationic. Thus, charged residues in the N-terminal half may -apparently via the initial electrostatic binding of the bacteriocin to target cells -influence the target-cell specificity.Keywords : antimicrobial peptides, mutagenesis, lactic acid bacteria INTRODUCTIONMany Gram-positive bacteria produce antimicrobial peptides, generally termed bacteriocins. These peptides are usually cationic, less than 50 amino acid residues long, contain an amphiphilic or hydrophobic region, and often kill their target cells by permeabilizing the cell membrane (Nes et al., 1996(Nes et al., , 2002. Antimicrobial peptides with these characteristics are also produced by plants and a wide variety of animals, including humans, and are thus widely distributed in nature M. Kazazic, J. Nissen-Meyer and G. Fimland phospholipid-containing membranes and\or acidic cell walls, whereas their amphiphilic\hydrophobic character enables membrane permeabilization. The pediocin-like bacteriocins constitute a dominant and well-studie...

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