BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein, is required for correct glycosylation and function of integrin β1

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Abstract
Glycosylation of β1 integrin (β1) in the Golgi complex has been related to its function in multiple cell processes, e.g., invasiveness, matrix adhesion, and migration. Brefeldin A-inhibited guanine nucleotide-exchange proteins (BIG) 1 and BIG2 activate human ADP-ribosylation factors (ARF) 1 and ARF3 by catalyzing the replacement of ARF-bound GDP with GTP to regulate Golgi vesicular transport. We show here a requirement for BIG1 (but not BIG2) in glycosylation and function of β1. In HepG2 cells treated for 48 or 72 h with BIG1, but not BIG2, siRNA, both the amount and electrophoretic mobility of the initially 130-kDa β1 were increased. BIG1 content had risen by 48 h after removal of BIG1 siRNA, and the faster-migrating, aberrant 130-kDa β1 was not seen. Peptide N-glycosidase F, but not endoglycosidase H, digestion converted all β1 to an ≈85-kDa (core protein) form. By electron microscopy, Golgi membranes in BIG1-depleted cells were less sharply defined than those in mock or BIG2 siRNA-treated cells, with more vesicle-like structures at the transface. Amounts of active RhoA-GTP also were decreased in such cells and restored by overexpression of HA-BIG1. Aberrant β1 was present on the cell surface, but its function in cell spreading, adhesion, and migration was impaired. By immunofluorescence microscopy, BIG1 siRNA-treated cells showed less spreading and concentration of β1 at the cell surface. These results indicate a previously unrecognized role for BIG1 in the glycosylation of β1 by Golgi enzymes, which is critical for its function in developmental and other vital cell processes.