Post-translational modifications regulate signalling by Ror1
Language English Country Great Britain, England Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- B-Lymphocytes metabolism MeSH
- CHO Cells MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell metabolism MeSH
- Cricetulus MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Glycosylation MeSH
- HEK293 Cells MeSH
- Immunohistochemistry MeSH
- Microscopy, Confocal MeSH
- Cricetinae MeSH
- Humans MeSH
- Molecular Weight MeSH
- Protein Processing, Post-Translational * drug effects MeSH
- Flow Cytometry MeSH
- Pseudopodia metabolism MeSH
- Signal Transduction * drug effects MeSH
- Receptor Tyrosine Kinase-like Orphan Receptors chemistry genetics metabolism MeSH
- Transfection MeSH
- Protein Transport MeSH
- Ubiquitination MeSH
- Blotting, Western MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- ROR1 protein, human MeSH Browser
- Receptor Tyrosine Kinase-like Orphan Receptors MeSH
AIM: In this study, we analysed the post-translational modification of receptor tyrosine kinase-like orphan receptor (Ror1). Ror1 is highly upregulated in B cells of patients with chronic lymphocytic leukaemia (CLL). Molecularly, Ror1 acts as the Wnt receptor in the non-canonical Wnt pathway. METHODS: The level of Ror1 glycosylation in HEK293 cells and in primary human CLL cells was analysed by treatment of inhibitors interfering with different steps of glycosylation process and by direct treatment of cell lysates with N-glycosidase. Ror1 ubiquitination was determined by ubiquitination assay. Functional consequences of post-translational modifications were analysed by immunohistochemistry and by analysis of cell surface proteins. Differences in Ror1 glycosylation were confirmed by analysis of 14 samples of B cells from CLL patients. RESULTS: We demonstrate that Ror1 is extensively modified by N-linked glycosylation. Glycosylation produces several variants of Ror1 with electrophoretic migration of approx. 100, 115 and 130 kDa. Inhibition of glycosylation interferes with cell surface localization of the 130-kDa variant of Ror1 and prevents Ror1-induced formation of filopodia. Moreover, we show that 130-kDa Ror1 is mono-ubiquitinated. Furthermore, individual CLL patients show striking differences in the electrophoretic migration of Ror1, which correspond to the level of glycosylation. CONCLUSION: Our data show that Ror1 undergoes complex post-translational modifications by glycosylation and mono-ubiquitination. These modifications regulate Ror1 localization and signalling, and are highly variable among individual CLL patients. These may suggest that Ror1 signals only in a subset of CLL patients despite Ror1 levels are ubiquitously high in all CLL patients.
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Endoplasmic reticulum stress disrupts signaling via altered processing of transmembrane receptors
