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Abstract
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.
Highlights
Interactions between growth factors and their cognate receptors convey signals from the plasma membrane to the interior of the cell (Schlessinger, 2000)
Proteins that are sequestered in multivesicular bodies (MVBs) internal vesicles are degraded, in contrast to those retained on MVB limiting membranes that recycle back to the plasma membrane (Authier and Chauvet, 1999; Hopkins, 1983)
The following antibodies were used: anti-adenovirus-E1A-specific antibody (Oncogene Science, Cambridge, MA), E3-13.7-specific peptide antibody produced in rabbits by this laboratory (Hoffman et al, 1990), rabbit anti-CBL antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-human-epidermal growth factor receptor (EGFR) monoclonal antibody (Novus Biologicals, Inc., Littleton, CO), rabbit EGFR antiserum (Research Diagnostics, Inc., Flanders, N.J.), rabbit anti-phosphoEGFR (Tyr1045) antiserum (Cell Signaling Technology, Inc., Beverly, MA), mouse human-specific anti-IL2R␣ monoclonal antibody (American Type Culture Collection, Rockville, MD), recombinant HRP-conjugated anti-phosphotyrosine antibody (Transduction Laboratories, Lexington, KY), and mouse anti-ubiquitin monoclonal antibody (Berkeley Antibody Co., Richmond, CA)
Summary
Interactions between growth factors and their cognate receptors convey signals from the plasma membrane to the interior of the cell (Schlessinger, 2000). The endocytic machinery controls the cell’s capacity to continue signaling by determining whether internalized receptors recycle to the cell surface, or are sent to lysosomes for degradation (Sorkin, 2000). The involvement of endocytosis in cell-signaling is striking in the ERBB family of receptor tyrosine kinases, which includes the epidermal growth factor receptor (EGFR) (Carpenter, 2000; Wiley and Burke, 2001). Ligandactivated EGFRs are recruited to clathrin-coated pits that pinch off from the plasma membrane and subsequently fuse with early endosomes (Wiley, 2003). The majority of endosomal EGFRs are sorted into the internal vesicles of multivesicular bodies (MVBs) (Miller et al, 1986). The importance of understanding the relationship between signaling and endocytosis is underscored by the prominence of endocytic sorting abnormalities in human cancers linked to ERBB receptors (Blume-Jensen and Hunter, 2001; Kim and Muller, 1999; Neve et al, 2001)
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