title: BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein, is required for correct glycosylation and function of integrin beta1.
abstract: Glycosylation of beta1 integrin (beta1) 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 beta1. 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 beta1 were increased. BIG1 content had risen by 48 h after removal of BIG1 siRNA, and the faster-migrating, aberrant 130-kDa beta1 was not seen. Peptide N-glycosidase F, but not endoglycosidase H, digestion converted all beta1 to an approximately 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 beta1 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 beta1 at the cell surface. These results indicate a previously unrecognized role for BIG1 in the glycosylation of beta1 by Golgi enzymes, which is critical for its function in developmental and other vital cell processes.
authors.affiliation: Pulmonary-Critical Care Medicine Branch, Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. shenx2@nhlbi.nih.gov
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Shen
authors.forename: Myoung-Soon
authors.initials: MS
authors.lastname: Hong
authors.forename: Joel
authors.initials: J
authors.lastname: Moss
authors.forename: Martha
authors.initials: M
authors.lastname: Vaughan
mesh_terms: D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D024042:Collagen Type I / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism / Q000502:physiology*; D034741:RNA, Small Interfering
chemical_list: C119087:ARFGEF1 protein, human; D024042:Collagen Type I; D020662:Guanine Nucleotide Exchange Factors; D019012:Integrin beta1; D034741:RNA, Small Interfering