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

Abstract

Glycosylation of ␤1 integrin (␤1) 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 ␤1. 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 ␤1 were increased. BIG1 content had risen by 48 h after removal of BIG1 siRNA, and the faster-migrating, aberrant 130-kDa ␤1 was not seen. Peptide Nglycosidase F, but not endoglycosidase H, digestion converted all ␤1 to an Ϸ85-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 vesiclelike structures at the transface. Amounts of active RhoA-GTP also were decreased in such cells and restored by overexpression of HA-BIG1. Aberrant ␤1 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 ␤1 at the cell surface. These results indicate a previously unrecognized role for BIG1 in the glycosylation of ␤1 by Golgi enzymes, which is critical for its function in developmental and other vital cell processes.adhesion ͉ ADP-ribosylation factor ͉ migration I ntegrins are heterodimeric transmembrane glycoproteins comprising diverse ␣-and ␤-subunits. Most of the integrins involved in focal adhesion (FA) formation are members of the ␤ 1 or ␤ 3 families. The ␤1 integrin (␤1) subunit pairs with at least 12 different ␣-subunits to form transmembrane adhesion receptors for extracellular matrix (ECM) proteins, e.g., collagen, fibronectin, and laminin (1). Integrin binding of ECM initiates intracellular signaling, including RhoA-dependent events, such as development of lamellipodia, actin stress fibers, and FAs that act in cell spreading and adhesion (2).The function of ␤1 depends on its accurate glycosylation catalyzed by enzymes located in morphologically and biochemically distinct cisternae of the Golgi system. Mature ␤1 is transported to the cell surface, where it mechanically links plasma membrane adhesion complexes to the actin cytoskeleton for bidirectional transmembrane as well as intracellular signaling. Numerous processes, e.g., embryogenesis, wound healing, and tumorigenesis, are regulated in this way (3, 4); factors that can affect ␤1 glycosylation and transport include Ki-Ras (5), transforming growth factor-␤ (6), and HEMCAM/gicerin (7). Treatment of HeLa cells with ceramide or brefeldin A caused fragmentation of the Golgi complex and inhibition of ␤1 glycosylation (8). Accumulated data indicate the importance of ␤1 N-glycosylation in cell a...

Enhancement of β-catenin activity by BIG1 plus BIG2 via Arf activation and cAMP signals

“…Phosphorylation of β-catenin clearly influenced its intracellular distribution (1,3), and reversible O-glycosylation may affect its nuclear accumulation and transcriptional action (64)(65)(66). Correct N-glycosylation and trafficking of integrin β1 required BIG1 (30) and BIG2 (28), respectively, but their roles in O-glycosylation are unknown. We speculate that accumulation of β-catenin at Golgi structures after BIG1 or BIG2 depletion contributes to inhibition of Wnt signaling by delaying β-catenin transit to and action in nuclei.…”

“…Microscopically, BIG1 was seen mainly at the trans-Golgi network (TGN), sometimes partially overlapping BIG2, which was associated also with recycling endosomes (24-26), e.g., in moving proteins and lipids among TGN, endosomes, and cell surface (24,(27)(28)(29). Although actions of BIG1 and BIG2 at the TGN were described as "redundant" (27), each protein clearly has specific roles in moving proteins and lipids that are not shared with the other (24,30,31). Overexpression in cultured cells of mutant BIG2 lacking Arf GEF activity markedly altered intracellular distributions of E-cadherin and β-catenin (32).…”

Effects of brefeldin A-inhibited guanine nucleotide-exchange (BIG) 1 and KANK1 proteins on cell polarity and directed migration during wound healing

“…BIG1 is often found at trans-Golgi membranes (16,17); after BIG1 depletion, membranes of the HepG2 cell Golgi trans face appeared less smooth by electron microscopy with more vesicle-like structures (18). Boal and Stephens also reported that Golgi structure was altered after BIG1 depletion (19).…”

Cytohesin-2/ARNO, through Its Interaction with Focal Adhesion Adaptor Protein Paxillin, Regulates Preadipocyte Migration via the Downstream Activation of Arf6

“…Alternatively, the well characterized brefeldin A-sensitive GEFs may be upstream regulators of Arf1 in this signaling pathway. In fact, BIG1 mediates migration of HepG2 cells (40), and BIG2 promotes neural progenitor cell migration in the cerebral cortex (41). It will be interesting to study how the GEFs for Arf1 and/or Arf6 cooperatively regulate 3T3-L1 cell migration.…”