title: Post-translational modifications regulate signalling by Ror1.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Faculty of Science, Institute of Experimental Biology, Masaryk University, Kotlářská, Brno, Czech Republic.
authors.forename: M
authors.initials: M
authors.lastname: Kaucká
authors.forename: P
authors.initials: P
authors.lastname: Krejčí
authors.forename: K
authors.initials: K
authors.lastname: Plevová
authors.forename: S
authors.initials: S
authors.lastname: Pavlová
authors.forename: J
authors.initials: J
authors.lastname: Procházková
authors.forename: P
authors.initials: P
authors.lastname: Janovská
authors.forename: J
authors.initials: J
authors.lastname: Valnohová
authors.forename: A
authors.initials: A
authors.lastname: Kozubík
authors.forename: S
authors.initials: S
authors.lastname: Pospíšilová
authors.forename: V
authors.initials: V
authors.lastname: Bryja
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000378:metabolism*; D018613:Microscopy, Confocal; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport; D011554:Pseudopodia / Q000378:metabolism; D057050:Receptor Tyrosine Kinase-like Orphan Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D054875:Ubiquitination
chemical_list: C078716:ROR1 protein, human; D057050:Receptor Tyrosine Kinase-like Orphan Receptors