EGF receptor residues Leu(679), Leu(680) mediate selective sorting of ligand-receptor complexes in early endosomal compartments

Dileucine-based motifs have been shown to regulate endosomal sorting of a n umber of membrane proteins. Previously, we have shown that the dileucine mo tif Leu(679), Leu(680) in the juxtamembrane domain of the human epidermal g rowth factor receptor is involved in the endosome-to-lysosome transport of ligand-receptor reduction in ligand-induced receptor degradation without af fecting internalization. In the current study, we have further characterize d ligand-dependent intracellular sorting of EGF receptors containing a L679 A, L680A. Immunocytochemical studies reveal that although mutant receptors redistribute from the cell surface to transferrin receptor-positive endocyt ic vesicles similar to wild-type following ligand stimulation, their accumu lation in Lamp-1-positive late endosomes/lysosomes is retarded compared to wild-type. Kinetic analysis of I-125-EGF trafficking shows that reduced acc umulation of internalized mutant receptors in Lamp-1-positive vesicles is d ue to rapid recycling of ligand-receptor complexes from early endocytic com partments. In addition, the fraction of intracellular I-125-EGF that is tra nsported to late endocytic compartments in cells with mutant receptors is n ot as efficiently degraded as it is in cells with wild-type receptors. Furt hermore, wild-type receptors in endocytic vesicles isolated by Percoll grad ient fractionation are more resistant to in vitro digestion with proteinase K than mutant receptors. We propose that mutant receptors interact ineffic iently with lysosomal sorting machinery, leading to their increased recycli ng. Our results are consistent with a model in which the Leu(679), Leu(680) signal facilitates sequestration of ligand-receptor complexes into interna l Vesicles of multivesicular endosome-to-lysosome transport intermediates. (C) 2000 Wiley-Liss, Inc.