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Ubc9 interacts with Lu/BCAM adhesion glycoproteins and regulates their stability at the membrane of polarized MDCK cells
- Source :
- Biochemical Journal, Biochemical Journal, Portland Press, 2006, 402 (2), pp.311-319. ⟨10.1042/BJ20060861⟩
- Publication Year :
- 2006
- Publisher :
- HAL CCSD, 2006.
-
Abstract
- Lu (Lutheran) blood group and BCAM (basal cell adhesion molecule) antigens both reside on two gp (glycoprotein) isoforms, Lu and Lu(v13), that differ by the size of their cytoplasmic tail. They are receptors of laminin-10/11 and are expressed in RBCs (red blood cells), epithelial cells of multiple tissues and vascular endothelial cells. To gain more insights into the biological function of Lu/BCAM gps, we looked for potential partners of their cytoplasmic tail. We isolated Ubc9 (ubiquitin-conjugating enzyme 9) protein by screening a human kidney library using the yeast two-hybrid system. Lu/Ubc9 interaction was validated by GST (glutathione S-transferase) pull-down and co-immunoprecipitation experiments. Endogenous Ubc9 formed a complex with endogenous or recombinant Lu gp in A498 and MDCK (Madin–Darby canine kidney) epithelial cells respectively. Replacement of Lys585 by alanine in the Lu gp abolished in vitro and ex vivo interactions of Lu gp with Ubc9 protein. Lu K585A mutant transfected in MDCK cells exhibited a normal basolateral membrane expression but was overexpressed at the surface of polarized MDCK cells as compared with wild-type Lu. Pulse–chase experiments showed extended half-life of Lu K585A gp at the plasma membrane, suggesting an impaired endocytosis of this mutant leading to protein accumulation at the membrane. Furthermore, we showed that the ability of MDCK-Lu K585A cells to spread on immobilized laminin was dramatically decreased. Our results support a physiological role for the direct interaction between Lu gp and Ubc9 protein and reveal a role for this enzyme in regulating the stability of Lu gp at the cell membrane.
- Subjects :
- Cytoplasm
Molecular Sequence Data
Saccharomyces cerevisiae
Endocytosis
Biochemistry
Cell Line
Cell membrane
03 medical and health sciences
0302 clinical medicine
BCAM
Dogs
Laminin
Two-Hybrid System Techniques
medicine
Cell Adhesion
Animals
Humans
Amino Acid Sequence
Molecular Biology
Cell Shape
030304 developmental biology
Epithelial polarity
chemistry.chemical_classification
0303 health sciences
Binding Sites
biology
Lysine
Cell Membrane
Cell Polarity
Life Sciences
Cell Biology
Transfection
Molecular biology
Red blood cell
medicine.anatomical_structure
chemistry
Gene Expression Regulation
030220 oncology & carcinogenesis
Mutation
Ubiquitin-Conjugating Enzymes
biology.protein
Small Ubiquitin-Related Modifier Proteins
Glycoprotein
Cell Adhesion Molecules
Protein Binding
Research Article
Subjects
Details
- Language :
- English
- ISSN :
- 02646021 and 14708728
- Database :
- OpenAIRE
- Journal :
- Biochemical Journal, Biochemical Journal, Portland Press, 2006, 402 (2), pp.311-319. ⟨10.1042/BJ20060861⟩
- Accession number :
- edsair.doi.dedup.....26e49d022fef228fe0eaa36c260f7c89