BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein, is required for correct glycosylation and function of integrin beta1.
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.
Shen
Xiaoyan
X
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
Hong
Myoung-Soon
MS
Moss
Joel
J
Vaughan
Martha
M
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
C119087:ARFGEF1 protein, human; D024042:Collagen Type I; D020662:Guanine Nucleotide Exchange Factors; D019012:Integrin beta1; D034741:RNA, Small Interfering