Glycobiology Advance Access published online on February 18, 2009
Glycobiology, doi:10.1093/glycob/cwp023
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Knockdown of GnT-Va expression inhibits ligand-induced down-regulation of the epidermal growth factor receptor and intracellular signaling by inhibiting receptor endocytosis
1 Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, Athens, GA,30602, USA
2 Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510 USA
Address correspondence to: Michael Pierce, Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center, 315 Riverbend Rd. University of Georgia, Athens, Georgia 30602; Tel: 1-706-542-1701; Fax: 1-706-542-1759; e-mail: hawkeye{at}uga.edu
Received on December 20, 2008; accepted on February 12, 2009
Changes in the expression of N-glycan branching glycosyltransferases can alter cell surface receptor functions, involving their levels of cell surface retention, rates of internalization into the endosomal compartment, and subsequent intracellular signaling. To study in detail the regulation of signaling of the EGF receptor (EGFR) by GlcNAcβ(1,6)Man branching, we utilized specific siRNA to selectively knock-down GnT-Va expression in the highly invasive human breast carcinoma line MDA-MB231, which resulted in attenuation of its invasiveness-related phenotypes. Compared to control cells, ligand-induced down regulation of EGFR was significantly inhibited in GnT-Va-suppressed cells. This effect could be reversed by re-expression of GnT-Va, indicating that changes in ligand-induced receptor down-regulation were dependent on GnT-Va activity. Knockdown of GnT-Va had no significant effect on c-Cbl mediated receptor ubiquitination and degradation, but did cause inhibition of receptor internalization, showing that altered signaling and delayed ligand-induced down-regulation of EGFR expression resulted from decreased EGFR endocytosis. Similar results were obtained with HT1080 fibrosarcoma cells treated with GnT-Va siRNA. Inhibited receptor internalization caused by expression of GnT-Va siRNA appeared to be independent of galectin binding since decreased EGFR internalization in the knockdown cells was not affected by treatment of the cells with lactose, a galectin inhibitor. Our results show that decreased GnT-Va activity due to siRNA expression in human carcinoma cells inhibits ligand-induced EGFR internalization, consequently resulting in delayed downstream signal transduction and inhibition of the EGF-induced, invasiveness-related phenotypes.
Key words: GnT-V / N-glycan / EGFR / endocytosis