A novel dileucine lysosomal-sorting-signal mediates intracellular EGF-receptor retention independently of protein ubiquitylation.
One of the main goals of this study was to understand the relationship between an epidermal growth factor (EGF) receptor dileucine (LL)-motif (679-LL) required for lysosomal sorting and the protein ubiquitin ligase CBL. We show that receptors containing 679-AA (di-alanine) substitutions that are defective for ligand-induced degradation nevertheless bind CBL and undergo reversible protein ubiquitylation similar to wild-type receptors. We also demonstrate that 679-LL but not CBL is required for EGF receptor downregulation by an endosomal membrane protein encoded by human adenoviruses that uncouples internalization from post-endocytic sorting to lysosomes. 679-LL is necessary for endosomal retention as well as degradation by the adenovirus protein, and is also transferable to reporter molecules. Using NMR spectroscopy, we show that peptides with wild-type 679-LL or mutant 679-AA sequences both exhibit alpha-helical structural propensities but that this structure is not stable in water. A similar analysis carried out in hydrophobic media showed that the alpha-helical structure of the wild-type peptide is stabilized by specific interactions mediated by side-chains in both leucine residues. This structure distinguishes 679-LL from other dileucine-based sorting-signals with obligatory amino-terminal acidic residues that are recognized in the form of an extended beta or beta-like conformation. Taken together, these data show that 679-LL is an alpha-helical stabilizing motif that regulates a predominant step during lysosomal sorting, involving intracellular retention under both sub-saturating and saturating conditions.
Tsacoumangos
Amy
A
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4970, USA.
Tsacoumango
Amy
A
Kil
Song Jae
SJ
Ma
Liping
L
Sönnichsen
Frank D
FD
Carlin
Cathleen
C
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D025801:Ubiquitin / Q000378:metabolism*
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D066246:ErbB Receptors; D007930:Leucine