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First published online 16 August 2005
doi: 10.1242/jcs.02527

This Article Figures Only Full Text Full Text (PDF) An erratum has been published All Versions of this Article: jcs.02527v1 118/17/3959    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tsacoumango, A. Articles by Carlin, C. Search for Related Content PubMed PubMed Citation Articles by Tsacoumango, A. Articles by Carlin, C. Social Bookmarking                         What's this?

A novel dileucine lysosomal-sorting-signal mediates intracellular EGF-receptor retention independently of protein ubiquitylation

Amy Tsacoumango^1,*, Song Jae Kil^1,*, Liping Ma¹, Frank D. Sönnichsen^1,2,3 and Cathleen Carlin^1,3,4,

¹ Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4970, USA
² Cleveland Center for Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4970, USA
³ Case Western Reserve University Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4970, USA
⁴ The Rainbow Center for Childhood PKD at Rainbow Babies and Children's Hospital of Cleveland, 11100 Euclid Avenue, Cleveland, OH 44106, USA

Author for correspondence (e-mail: cathleen.carlin{at}case.edu)

Accepted 7 June 2005

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 -helical structural propensities but that this structure is not stable in water. A similar analysis carried out in hydrophobic media showed that the -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 ß or ß-like conformation. Taken together, these data show that 679-LL is an -helical stabilizing motif that regulates a predominant step during lysosomal sorting, involving intracellular retention under both sub-saturating and saturating conditions.

Key words: Adenovirus E3 protein, CBL, EGF receptor, Endosomes, NMR spectroscopy, Protein ubiquitylation

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