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 number of membrane proteins. Previously, we have shown that the dileucine motif Leu(679), Leu(680) in the juxtamembrane domain of the human epidermal growth factor receptor is involved in the endosome-to-lysosome transport of ligand-receptor complexes. Substitution of alanine residues for Leu(679), Leu(680) led to a reduction in ligand-induced receptor degradation without affecting internalization. In the current study, we have further characterized ligand-dependent intracellular sorting of EGF receptors containing a L679A, L680A. Immunocytochemical studies reveal that although mutant receptors redistribute from the cell surface to transferrin receptor-positive endocytic vesicles similar to wild-type following ligand stimulation, their accumulation in Lamp-1-positive late endosomes/lysosomes is retarded compared to wild-type. Kinetic analysis of (125)I-EGF trafficking shows that reduced accumulation of internalized mutant receptors in Lamp-1-positive vesicles is due to rapid recycling of ligand-receptor complexes from early endocytic compartments. In addition, the fraction of intracellular (125)I-EGF that is transported to late endocytic compartments in cells with mutant receptors is not as efficiently degraded as it is in cells with wild-type receptors. Furthermore, wild-type receptors in endocytic vesicles isolated by Percoll gradient fractionation are more resistant to in vitro digestion with proteinase K than mutant receptors. We propose that mutant receptors interact inefficiently with lysosomal sorting machinery, leading to their increased recycling. Our results are consistent with a model in which the Leu(679), Leu(680) signal facilitates sequestration of ligand-receptor complexes into internal vesicles of multivesicular endosome-to-lysosome transport intermediates.
Kil
S J
SJ
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Carlin
C
C
D000818:Animals; D002460:Cell Line; D011992:Endosomes / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007930:Leucine; D008024:Ligands; D051379:Mice; D011485:Protein Binding; D015398:Signal Transduction
D008024:Ligands; D066246:ErbB Receptors; D007930:Leucine